Ravi Majeti MD, PhD is Professor of Medicine, Chief of the Division of Hematology, and Member of the Institute for Stem Cell Biology and Regenerative Medicine at the Stanford University School of Medicine. He was an undergraduate at Harvard, earned his MD and PhD from UCSF, and trained in Internal Medicine at Brigham and Women’s Hospital in Boston. Dr. Majeti completed his Hematology Fellowship at Stanford, and is a board-certified hematologist. While at Stanford, he completed post-doctoral training in the laboratory of Irving Weissman, where he investigated acute myeloid leukemia (AML) stem cells and therapeutic targeting with anti-CD47 antibodies. With Dr. Weissman, he developed a humanized anti-CD47 antibody, initiated first-in-human clinical trials. Dr. Majeti directs an active NIH-funded laboratory that focuses on the molecular characterization and therapeutic targeting of leukemia stem cells in human hematologic disorders, particularly AML, and has published >90 peer-reviewed articles. He is a recipient of the Burroughs Wellcome Career Award for Medical Scientists, the New York Stem Cell Foundation Robertson Investigator Award, and the Leukemia and Lymphoma Society Scholar Award. Dr. Majeti is currently a member of the Committee on Scientific Affairs for the American Society of Hematology (ASH) and serves of the editorial board of Blood and eLife.

Clinical Focus

  • Hematology

Administrative Appointments

  • Chief, Division of Hematology (2017 - Present)
  • Assistant Director, Stanford Ludwig Center for Cancer Stem Cell Research (2014 - Present)
  • Co-Director, Lymphoma and Leukemia Program - Stanford Cancer Institute (2014 - Present)
  • Co-Director, Translational Research Program - Internal Medicine Residency (2013 - 2017)

Honors & Awards

  • Scholar Award, Leukemia and Lymphoma Society (2015)
  • Robertson Investigator Award, New York Stem Cell Foundation (2011)
  • Career Award for Medical Scientists, Burroughs Wellcome Fund (2008)

Boards, Advisory Committees, Professional Organizations

  • Member, American Society of Hematology - Committee on Myeloid Neoplasia (2013 - Present)

Professional Education

  • Board Certification: American Board of Internal Medicine, Hematology (2007)
  • Medical Education: University of California at San Francisco School of Medicine (2002) CA
  • Residency: Brigham and Women's Hospital Harvard Medical School (2004) MA
  • Fellowship: Stanford University Medical Center (2008) CA

Current Research and Scholarly Interests

Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow that is rapidly fatal within months if untreated. Even with aggressive treatment, including high dose chemotherapy and bone marrow transplantation, five-year overall survival rates range between 30-40%. A growing body of evidence indicates that not all cells in this cancer are the same, and that there is a rare population of leukemia stem cells (LSC) that are responsible for maintaining the disease. These findings have led to the idea that in order to cure this cancer, the LSC must be eliminated, while at the same time sparing the normal blood forming stem cells within the bone marrow.

The overall goal of our research is to identify molecular and genetic differences between human AML stem cells and their normal counterparts, and then to develop therapeutic strategies directed against these targets. We utilize bioinformatics, genomics, and functional methods to investigate genes and pathways preferentially expressed or activated in LSC. From this analysis, we have identified a number of factors, including several cell surface protein markers that are more highly expressed on AML LSC compared to their normal counterparts. We have focused on one of these markers, CD47, that contributes to leukemia development by blocking the ingestion and removal of leukemia cells by cells of the immune system. Most significantly, we determined that blocking monoclonal antibodies directed against CD47 targeted LSC and depleted leukemia in mouse pre-clinical models. We have now developed a clinical grade humanized anti-CD47 antibody that is in clinical trials at the Stanford Cancer Center.

Our research has also investigated the development of AML from normal blood forming, or hematopoietic, stem cells (HSC). Genomic studies have determined that most cases of AML are associated with an average of 5 mutations, raising the question of how these multiple mutations accumulate in a single lineage of cells. We hypothesized that since HSC are the only long-lived, self-propagating cells in the myeloid lineage, then the mutations must be serially acquired in clones of HSC. Using primary patient samples and single cell genomic methods, we found evidence of pre-leukemic HSC and mutations, confirming our hypothesis. Furthermore, we showed that these pre-leukemic HSC survive chemotherapy and may give rise to relapsed disease. Thus, these pre-leukemic mutations may be critical targets for curative therapies.

2020-21 Courses

Stanford Advisees

All Publications

  • IL-6 blockade reverses bone marrow failure induced by human acute myeloid leukemia. Science translational medicine Zhang, T. Y., Dutta, R., Benard, B., Zhao, F., Yin, R., Majeti, R. 2020; 12 (538)


    Most patients with acute myeloid leukemia (AML) die from complications arising from cytopenias resulting from bone marrow (BM) failure. The common presumption among physicians is that AML-induced BM failure is primarily due to overcrowding, yet BM failure is observed even with low burden of disease. Here, we use large clinical datasets to show the lack of correlation between BM blast burden and degree of cytopenias at the time of diagnosis. We develop a splenectomized xenograft model to demonstrate that transplantation of human primary AML into immunocompromised mice recapitulates the human disease course by induction of BM failure via depletion of mouse hematopoietic stem and progenitor populations. Using unbiased approaches, we show that AML-elaborated IL-6 acts to block erythroid differentiation at the proerythroblast stage and that blocking antibodies against human IL-6 can improve AML-induced anemia and prolong overall survival, suggesting a potential therapeutic approach.

    View details for DOI 10.1126/scitranslmed.aax5104

    View details for PubMedID 32269167

  • Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia. Nature cancer Zhang, H. n., Nakauchi, Y. n., Köhnke, T. n., Stafford, M. n., Bottomly, D. n., Thomas, R. n., Wilmot, B. n., McWeeney, S. K., Majeti, R. n., Tyner, J. W. 2020; 1 (8): 826–39


    Deregulation of the BCL2 gene family plays an important role in the pathogenesis of acute myeloid leukemia (AML). The BCL2 inhibitor, venetoclax, has received FDA approval for the treatment of AML. However, upfront and acquired drug resistance ensues due, in part, to the clinical and genetic heterogeneity of AML, highlighting the importance of identifying biomarkers to stratify patients onto the most effective therapies. By integrating clinical characteristics, exome and RNA sequencing, and inhibitor data from primary AML patient samples, we determined that myelomonocytic leukemia, upregulation of BCL2A1 and CLEC7A, as well as mutations of PTPN11 and KRAS conferred resistance to venetoclax and multiple venetoclax combinations. Venetoclax in combination with an MCL1 inhibitor AZD5991 induced synthetic lethality and circumvented venetoclax resistance.

    View details for DOI 10.1038/s43018-020-0103-x

    View details for PubMedID 33123685

    View details for PubMedCentralID PMC7591155

  • Single-cell mutational profiling enhances the clinical evaluation of AML MRD. Blood advances Ediriwickrema, A. n., Aleshin, A. n., Reiter, J. G., Corces, M. R., Köhnke, T. n., Stafford, M. n., Liedtke, M. n., Medeiros, B. C., Majeti, R. n. 2020; 4 (5): 943–52


    Although most patients with acute myeloid leukemia (AML) achieve clinical remission with induction chemotherapy, relapse rates remain high. Next-generation sequencing enables minimal/measurable residual disease (MRD) detection; however, clinical significance is limited due to difficulty differentiating between pre-leukemic clonal hematopoiesis and frankly malignant clones. Here, we investigated AML MRD using targeted single-cell sequencing (SCS) at diagnosis, remission, and relapse (n = 10 relapsed, n = 4 nonrelapsed), with a total of 310 737 single cells sequenced. Sequence variants were identified in 80% and 75% of remission samples for patients with and without relapse, respectively. Pre-leukemic clonal hematopoiesis clones were detected in both cohorts, and clones with multiple cooccurring mutations were observed in 50% and 0% of samples. Similar clonal richness was observed at diagnosis in both cohorts; however, decreasing clonal diversity at remission was significantly associated with longer relapse-free survival. These results show the power of SCS in investigating AML MRD and clonal evolution.

    View details for DOI 10.1182/bloodadvances.2019001181

    View details for PubMedID 32150611

  • Enasidenib drives human erythroid differentiation independently of isocitrate dehydrogenase 2. The Journal of clinical investigation Dutta, R. n., Zhang, T. Y., Köhnke, T. n., Thomas, D. n., Linde, M. n., Gars, E. n., Stafford, M. n., Kaur, S. n., Nakauchi, Y. n., Yin, R. n., Azizi, A. n., Narla, A. n., Majeti, R. n. 2020


    Cancer-related anemia is present in over 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and red blood cell (RBC) transfusion-independence in AML patients treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor, enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRIPSR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.

    View details for DOI 10.1172/JCI133344

    View details for PubMedID 31895700

  • CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma. The New England journal of medicine Advani, R., Flinn, I., Popplewell, L., Forero, A., Bartlett, N. L., Ghosh, N., Kline, J., Roschewski, M., LaCasce, A., Collins, G. P., Tran, T., Lynn, J., Chen, J. Y., Volkmer, J., Agoram, B., Huang, J., Majeti, R., Weissman, I. L., Takimoto, C. H., Chao, M. P., Smith, S. M. 2018; 379 (18): 1711–21


    BACKGROUND: The Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin's lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically.METHODS: We conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin's lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose.RESULTS: A total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing.CONCLUSIONS: The macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; number, NCT02953509 .).

    View details for PubMedID 30380386

  • Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease. Cell stem cell Chao, M. P., Gentles, A. J., Chatterjee, S., Lan, F., Reinisch, A., Corces, M. R., Xavy, S., Shen, J., Haag, D., Chanda, S., Sinha, R., Morganti, R. M., Nishimura, T., Ameen, M., Wu, H., Wernig, M., Wu, J. C., Majeti, R. 2017; 20 (3): 329-344 e7


    Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

    View details for DOI 10.1016/j.stem.2016.11.018

    View details for PubMedID 28089908

  • Biology and relevance of human acute myeloid leukemia stem cells. Blood Thomas, D., Majeti, R. 2017


    Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research.

    View details for DOI 10.1182/blood-2016-10-696054

    View details for PubMedID 28159741

  • Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. eLife Bak, R. O., Dever, D. P., Reinisch, A. n., Cruz Hernandez, D. n., Majeti, R. n., Porteus, M. H. 2017; 6


    Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.

    View details for PubMedID 28956530

  • Super-Enhancer Analysis Defines Novel Epigenomic Subtypes of Non-APL AML Including an RARα Dependency Targetable by SY-1425, a Potent and Selective RARα Agonist. Cancer discovery McKeown, M. R., Corces, M. R., Eaton, M. L., Fiore, C. n., Lee, E. n., Lopez, J. T., Chen, M. W., Smith, D. n., Chan, S. M., Koenig, J. L., Austgen, K. n., Guenther, M. G., Orlando, D. A., Lovén, J. n., Fritz, C. C., Majeti, R. n. 2017


    We characterized the enhancer landscape of 66 AML patients, identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their super-enhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong super-enhancer at the retinoic acid receptor alpha (RARA) gene locus. Presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of APL treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.

    View details for PubMedID 28729405

  • A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells NATURE MEDICINE Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D., Hong, W., Schallmoser, K., Strunk, D., Majeti, R. 2016; 22 (7): 812-821


    Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases.

    View details for DOI 10.1038/nm.4103

    View details for PubMedID 27213817

  • Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell stem cell Mazumdar, C., Shen, Y., Xavy, S., Zhao, F., Reinisch, A., Li, R., Corces, M. R., Flynn, R. A., Buenrostro, J. D., Chan, S. M., Thomas, D., Koenig, J. L., Hong, W., Chang, H. Y., Majeti, R. 2015; 17 (6): 675-688


    Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.

    View details for DOI 10.1016/j.stem.2015.09.017

    View details for PubMedID 26607380

  • Reprogramming of primary human Philadelphia chromosome-positive B cell acute lymphoblastic leukemia cells into nonleukemic macrophages PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McClellan, J. S., Dove, C., Gentles, A. J., Ryan, C. E., Majeti, R. 2015; 112 (13): 4074-4079


    BCR-ABL1(+) precursor B-cell acute lymphoblastic leukemia (BCR-ABL1(+) B-ALL) is an aggressive hematopoietic neoplasm characterized by a block in differentiation due in part to the somatic loss of transcription factors required for B-cell development. We hypothesized that overcoming this differentiation block by forcing cells to reprogram to the myeloid lineage would reduce the leukemogenicity of these cells. We found that primary human BCR-ABL1(+) B-ALL cells could be induced to reprogram into macrophage-like cells by exposure to myeloid differentiation-promoting cytokines in vitro or by transient expression of the myeloid transcription factor C/EBPα or PU.1. The resultant cells were clonally related to the primary leukemic blasts but resembled normal macrophages in appearance, immunophenotype, gene expression, and function. Most importantly, these macrophage-like cells were unable to establish disease in xenograft hosts, indicating that lineage reprogramming eliminates the leukemogenicity of BCR-ABL1(+) B-ALL cells, and suggesting a previously unidentified therapeutic strategy for this disease. Finally, we determined that myeloid reprogramming may occur to some degree in human patients by identifying primary CD14(+) monocytes/macrophages in BCR-ABL1(+) B-ALL patient samples that possess the BCR-ABL1(+) translocation and clonally recombined VDJ regions.

    View details for DOI 10.1073/pnas.1413383112

    View details for Web of Science ID 000351914500070

    View details for PubMedID 25775523

    View details for PubMedCentralID PMC4386392

  • Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia. Nature medicine Chan, S. M., Thomas, D., Corces-Zimmerman, M. R., Xavy, S., Rastogi, S., Hong, W., Zhao, F., Medeiros, B. C., Tyvoll, D. A., Majeti, R. 2015; 21 (2): 178-184


    Mutant isocitrate dehydrogenase (IDH) 1 and 2 proteins alter the epigenetic landscape in acute myeloid leukemia (AML) cells through production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). Here we performed a large-scale RNA interference (RNAi) screen to identify genes that are synthetic lethal to the IDH1(R132H) mutation in AML and identified the anti-apoptotic gene BCL-2. IDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to ABT-199, a highly specific BCL-2 inhibitor that is currently in clinical trials for hematologic malignancies, both ex vivo and in xenotransplant models. This sensitization effect was induced by (R)-2-HG-mediated inhibition of the activity of cytochrome c oxidase (COX) in the mitochondrial electron transport chain (ETC); suppression of COX activity lowered the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition. Our findings indicate that IDH1/2 mutation status may identify patients that are likely to respond to pharmacologic BCL-2 inhibition and form the rational basis for combining agents that disrupt ETC activity with ABT-199 in future clinical studies.

    View details for DOI 10.1038/nm.3788

    View details for PubMedID 25599133

  • Mutant WT1 is associated with DNA hypermethylation of PRC2 targets in AML and responds to EZH2 inhibition. Blood Sinha, S., Thomas, D., Yu, L., Gentles, A. J., Jung, N., Corces-Zimmerman, M. R., Chan, S. M., Reinisch, A., Feinberg, A. P., Dill, D. L., Majeti, R. 2015; 125 (2): 316-326


    Acute myeloid leukemia (AML) is associated with deregulation of DNA methylation; however, many cases do not bear mutations in known regulators of CpG methylation. We found that mutations in WT1, IDH2, and CEBPA were strongly linked to DNA hypermethylation in AML using a novel integrative analysis of TCGA data based on Boolean implications, if-then rules that identify all individual CpG sites that are hypermethylated in the presence of a mutation. Introduction of mutant WT1 (WT1mut) into wildtype AML cells induced DNA hypermethylation, confirming mutant WT1 to be causally associated with DNA hypermethylation. Methylated genes in WT1mut primary patient samples were highly enriched for polycomb repressor complex 2 (PRC2) targets, implicating PRC2 dysregulation in WT1mut leukemogenesis. We found that PRC2 target genes were aberrantly repressed in WT1mut AML, and that expression of mutant WT1 in CD34+ cord blood cells induced myeloid differentiation block. Treatment of WT1mut AML cells with shRNA or pharmacologic PRC2/EZH2 inhibitors promoted myeloid differentiation, suggesting EZH2 inhibitors may be active in this AML subtype. Our results highlight a strong association between mutant WT1 and DNA hypermethylation in AML, and demonstrate that Boolean implications can be used to decipher mutation-specific methylation patterns that may lead to therapeutic insights.

    View details for DOI 10.1182/blood-2014-03-566018

    View details for PubMedID 25398938

  • Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proceedings of the National Academy of Sciences of the United States of America Corces-Zimmerman, M. R., Hong, W., Weissman, I. L., Medeiros, B. C., Majeti, R. 2014; 111 (7): 2548-2553


    Cancer is widely characterized by the sequential acquisition of genetic lesions in a single lineage of cells. Our previous studies have shown that, in acute myeloid leukemia (AML), mutation acquisition occurs in functionally normal hematopoietic stem cells (HSCs). These preleukemic HSCs harbor some, but not all, of the mutations found in the leukemic cells. We report here the identification of patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, whereas mutations in "proliferative" genes occur late. Additionally, we analyze the persistence of preleukemic mutations in patients in remission and find CD34+ progenitor cells and various mature cells that harbor preleukemic mutations. These findings indicate that preleukemic HSCs can survive induction chemotherapy, identifying these cells as a reservoir for the reevolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease and show that some of these patterns are consistent with involvement of preleukemic HSCs. These findings provide key insights into the monitoring of minimal residual disease and the identification of therapeutic targets in human AML.

    View details for DOI 10.1073/pnas.1324297111

    View details for PubMedID 24550281

  • Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia SCIENCE TRANSLATIONAL MEDICINE Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Vyas, P., Weissman, I. L., Quake, S. R., Majeti, R. 2012; 4 (149)


    Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

    View details for DOI 10.1126/scitranslmed.3004315

    View details for Web of Science ID 000308491600005

    View details for PubMedID 22932223

  • Association of a Leukemic Stem Cell Gene Expression Signature With Clinical Outcomes in Acute Myeloid Leukemia JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Gentles, A. J., Plevritis, S. K., Majeti, R., Alizadeh, A. A. 2010; 304 (24): 2706-2715


    In many cancers, specific subpopulations of cells appear to be uniquely capable of initiating and maintaining tumors. The strongest support for this cancer stem cell model comes from transplantation assays in immunodeficient mice, which indicate that human acute myeloid leukemia (AML) is driven by self-renewing leukemic stem cells (LSCs). This model has significant implications for the development of novel therapies, but its clinical relevance has yet to be determined.To identify an LSC gene expression signature and test its association with clinical outcomes in AML.Retrospective study of global gene expression (microarray) profiles of LSC-enriched subpopulations from primary AML and normal patient samples, which were obtained at a US medical center between April 2005 and July 2007, and validation data sets of global transcriptional profiles of AML tumors from 4 independent cohorts (n = 1047).Identification of genes discriminating LSC-enriched populations from other subpopulations in AML tumors; and association of LSC-specific genes with overall, event-free, and relapse-free survival and with therapeutic response.Expression levels of 52 genes distinguished LSC-enriched populations from other subpopulations in cell-sorted AML samples. An LSC score summarizing expression of these genes in bulk primary AML tumor samples was associated with clinical outcomes in the 4 independent patient cohorts. High LSC scores were associated with worse overall, event-free, and relapse-free survival among patients with either normal karyotypes or chromosomal abnormalities. For the largest cohort of patients with normal karyotypes (n = 163), the LSC score was significantly associated with overall survival as a continuous variable (hazard ratio [HR], 1.15; 95% confidence interval [CI], 1.08-1.22; log-likelihood P <.001). The absolute risk of death by 3 years was 57% (95% CI, 43%-67%) for the low LSC score group compared with 78% (95% CI, 66%-86%) for the high LSC score group (HR, 1.9 [95% CI, 1.3-2.7]; log-rank P = .002). In another cohort with available data on event-free survival for 70 patients with normal karyotypes, the risk of an event by 3 years was 48% (95% CI, 27%-63%) in the low LSC score group vs 81% (95% CI, 60%-91%) in the high LSC score group (HR, 2.4 [95% CI, 1.3-4.5]; log-rank P = .006). In multivariate Cox regression including age, mutations in FLT3 and NPM1, and cytogenetic abnormalities, the HRs for LSC score in the 3 cohorts with data on all variables were 1.07 (95% CI, 1.01-1.13; P = .02), 1.10 (95% CI, 1.03-1.17; P = .005), and 1.17 (95% CI, 1.05-1.30; P = .005).High expression of an LSC gene signature is independently associated with adverse outcomes in patients with AML.

    View details for PubMedID 21177505

  • Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma CELL Chao, M. P., Alizadeh, A. A., Tang, C., Myklebust, J. H., Varghese, B., Gill, S., Jan, M., Cha, A. C., Chan, C. K., Tan, B. T., Park, C. Y., Zhao, F., Kohrt, H. E., Malumbres, R., Briones, J., Gascoyne, R. D., Lossos, I. S., Levy, R., Weissman, I. L., Majeti, R. 2010; 142 (5): 699-713


    Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

    View details for DOI 10.1016/j.cell.2010.07.044

    View details for PubMedID 20813259

  • CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells CELL Majeti, R., Chao, M. P., Alizadeh, A. A., Pang, W. W., Jaiswal, S., Gibbs, K. D., van Rooijen, N., Weissman, I. L. 2009; 138 (2): 286-299


    Acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells (LSC). We hypothesized that increased CD47 expression on human AML LSC contributes to pathogenesis by inhibiting their phagocytosis through the interaction of CD47 with an inhibitory receptor on phagocytes. We found that CD47 was more highly expressed on AML LSC than their normal counterparts, and that increased CD47 expression predicted worse overall survival in three independent cohorts of adult AML patients. Furthermore, blocking monoclonal antibodies directed against CD47 preferentially enabled phagocytosis of AML LSC and inhibited their engraftment in vivo. Finally, treatment of human AML LSC-engrafted mice with anti-CD47 antibody depleted AML and targeted AML LSC. In summary, increased CD47 expression is an independent, poor prognostic factor that can be targeted on human AML stem cells with blocking monoclonal antibodies capable of enabling phagocytosis of LSC.

    View details for DOI 10.1016/j.cell.2009.05.045

    View details for PubMedID 19632179

  • Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood CELL STEM CELL Majeti, R., Park, C. Y., Weissman, I. L. 2007; 1 (6): 635-645


    Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

    View details for DOI 10.1016/j.stem.2007.10.001

    View details for Web of Science ID 000251784300010

    View details for PubMedID 18371405

    View details for PubMedCentralID PMC2292126

  • Gene replacement of alpha-globin with beta-globin restores hemoglobin balance in beta-thalassemia-derived hematopoietic stem and progenitor cells. Nature medicine Cromer, M. K., Camarena, J., Martin, R. M., Lesch, B. J., Vakulskas, C. A., Bode, N. M., Kurgan, G., Collingwood, M. A., Rettig, G. R., Behlke, M. A., Lemgart, V. T., Zhang, Y., Goyal, A., Zhao, F., Ponce, E., Srifa, W., Bak, R. O., Uchida, N., Majeti, R., Sheehan, V. A., Tisdale, J. F., Dever, D. P., Porteus, M. H. 2021


    beta-Thalassemia pathology is due not only to loss of beta-globin (HBB), but also to erythrotoxic accumulation and aggregation of the beta-globin-binding partner, alpha-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in beta-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize beta-globin:alpha-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing beta-thalassemia.

    View details for DOI 10.1038/s41591-021-01284-y

    View details for PubMedID 33737751

  • The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells. Nature communications Sharma, R. n., Dever, D. P., Lee, C. M., Azizi, A. n., Pan, Y. n., Camarena, J. n., Köhnke, T. n., Bao, G. n., Porteus, M. H., Majeti, R. n. 2021; 12 (1): 472


    Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

    View details for DOI 10.1038/s41467-020-20792-y

    View details for PubMedID 33473139

  • CD34 expression does not correlate with immunophenotypic stem cell or progenitor content in human cord blood products. Blood advances Mantri, S., Reinisch, A., Dejene, B. T., Lyell, D. J., DiGiusto, D. L., Agarwal-Hashmi, R., Majeti, R., Weinberg, K. I., Porteus, M. H. 2020; 4 (21): 5357–61

    View details for DOI 10.1182/bloodadvances.2020002891

    View details for PubMedID 33136125

  • Targeting LSCs: Peeling Back the Curtain on the Metabolic Complexities of AML. Cell stem cell Zhang, T. Y., Majeti, R. 2020; 27 (5): 693–95


    Most patients with AML succumb to chemoresistant leukemia stem cells (LSCs), which persist and reinitiate disease years after clinical remission. In this issue of Cell Stem Cell, Jones etal. (2020) identify a therapeutically targetable mechanism of resistance to venetoclax in relapsed and refractory AML LSCs mediated by nicotinamide metabolism.

    View details for DOI 10.1016/j.stem.2020.10.007

    View details for PubMedID 33157042

  • Sufficiency for inducible Caspase-9 safety switch in human pluripotent stem cells and disease cells. Gene therapy Nishimura, T., Xu, H., Iwasaki, M., Karigane, D., Saavedra, B., Takahashi, Y., Suchy, F. P., Monobe, S., Martin, R. M., Ohtaka, M., Nakanishi, M., Burrows, S. R., Cleary, M. L., Majeti, R., Shibuya, A., Nakauchi, H. 2020


    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.

    View details for DOI 10.1038/s41434-020-0179-z

    View details for PubMedID 32704085

  • Reprogramming leukemia cells into antigen presenting cells as a novel cancer vaccination immunotherapy Linde, M. H., Dove, C. G., Gurev, S. F., Phan, P., Zhao, F., Gars, E. J., Marshall, P. L., Miller, L. P., Majeti, R. AMER ASSOC IMMUNOLOGISTS. 2020
  • A Dysregulated DNA Methylation Landscape Linked to Gene Expression in MLL-Rearranged AML. Epigenetics Koldobskiy, M. A., Abante, J., Jenkinson, G., Pujadas, E., Tetens, A., Zhao, F., Tryggvadottir, R., Idrizi, A., Reinisch, A., Majeti, R., Goutsias, J., Feinberg, A. P. 2020: 1–18


    Translocations of the KMT2A (MLL) gene define a biologically distinct and clinically aggressive subtype of acute myeloid leukaemia (AML), marked by a characteristic gene expression profile and few cooperating mutations. Although dysregulation of the epigenetic landscape in this leukaemia is particularly interesting given the low mutation frequency, its comprehensive analysis using whole genome bisulphite sequencing (WGBS) has not been previously performed. Here we investigated epigenetic dysregulation in nine MLL-rearranged (MLL-r) AML samples by comparing them to six normal myeloid controls, using a computational method that encapsulates mean DNA methylation measurements along with analyses of methylation stochasticity. We discovered a dramatically altered epigenetic profile in MLL-r AML, associated with genome-wide hypomethylation and a markedly increased DNA methylation entropy reflecting an increasingly disordered epigenome. Methylation discordance mapped to key genes and regulatory elements that included bivalent promoters and active enhancers. Genes associated with significant changes in methylation stochasticity recapitulated known MLL-r AML expression signatures, suggesting a role for the altered epigenetic landscape in the transcriptional programme initiated by MLL translocations. Accordingly, we established statistically significant associations between discordances in methylation stochasticity and gene expression in MLL-r AML, thus providing a link between the altered epigenetic landscape and the phenotype.

    View details for DOI 10.1080/15592294.2020.1734149

    View details for PubMedID 32114880

  • Multiomic single cell analysis of normal human bone marrow identifies a unique stem and progenitor population that expands in AML Proceedings of the Annual Meeting of the American Association for Cancer Research 2020 Ediriwickrema, A., Ramakrishnan, S., Nakamoto, M., Ghanekar, S., Luca, B., Newman, A., Gentles, A., Majeti, R. 2020
  • Venetoclax and hypomethylating agent therapy in high risk myelodysplastic syndromes: a retrospective evaluation of a real-world experience. Leukemia & lymphoma Azizi, A. n., Ediriwickrema, A. n., Dutta, R. n., Patel, S. A., Shomali, W. n., Medeiros, B. n., Iberri, D. n., Gotlib, J. n., Mannis, G. n., Greenberg, P. n., Majeti, R. n., Zhang, T. n. 2020: 1–8


    Treatment with hypomethylating agents (HMAs) azacitidine or decitabine is the current standard of care for high risk myelodysplastic syndromes (MDSs) but is associated with low rates of response. The limited number of treatment options for patients with high risk MDS highlights a need for new therapeutic options. Venetoclax is an inhibitor of the BCL-2 protein which, when combined with an HMA, has shown high response rates in unfit and previously untreated acute myeloid leukemia. We performed a retrospective study of high risk MDS patients receiving combination HMA plus venetoclax in order to determine their effectiveness in this context. We show that in our cohort, the combination results in high response rates but is associated with a high frequency of myelosuppression. These data highlight the efficacy of combination HMA plus venetoclax in high risk MDS, warranting further prospective evaluation in clinical trials.

    View details for DOI 10.1080/10428194.2020.1775214

    View details for PubMedID 32543932

  • Targeting macrophage checkpoint inhibitor SIRPa for anticancer therapy. JCI insight Liu, J. n., Xavy, S. n., Mihardja, S. n., Chen, S. n., Sompalli, K. n., Feng, D. n., Choi, T. S., Agoram, B. n., Majeti, R. n., Weissman, I. L., Volkmer, J. P. 2020


    The SIRPα-CD47 interaction provides a macrophage immune checkpoint pathway that plays a critical role in cancer immune evasion across multiple cancers. Here, we report the engineering of a humanized anti-SIRPα monoclonal antibody (1H9) for antibody target cancer therapy. 1H9 has broad activity across a wide range of SIRPα variants. Binding of 1H9 to SIRPα blocks its interaction with CD47, thereby promoting macrophage-mediated phagocytosis of cancer cells. Pre-clinical studies in vitro and in vivo demonstrate that 1H9 synergizes with other therapeutic antibodies to promote phagocytosis of tumor cells and inhibit tumor growth in both syngeneic and xenograft tumor models, leading to survival benefit. Thus, 1H9 can potentially act as a universal agent to enhance therapeutic efficacy when used in combination with most tumor-targeting antibodies. We report for the first time, a comparison of anti-SIRPα and anti-CD47 antibodies in CD47/SIRPα double humanized mice, and found that 1H9 exhibits a substantially reduced antigen-sink effect due to the limited tissue distribution of SIRPα expression. Toxicokinetic studies in non-human primates show that 1H9 is well tolerated with no treatment-related adverse effects noted. These data highlight the clinical potential of 1H9 as a pan-therapeutic with the desired properties when used in combination with tumor-targeting antibodies.

    View details for DOI 10.1172/jci.insight.134728

    View details for PubMedID 32427583

  • Single-cell multiomic analysis identifies regulatory programs in mixed-phenotype acute leukemia. Nature biotechnology Granja, J. M., Klemm, S., McGinnis, L. M., Kathiria, A. S., Mezger, A., Corces, M. R., Parks, B., Gars, E., Liedtke, M., Zheng, G. X., Chang, H. Y., Majeti, R., Greenleaf, W. J. 2019


    Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.

    View details for DOI 10.1038/s41587-019-0332-7

    View details for PubMedID 31792411

  • Single-cell mutational profiling of paired AML samples at diagnosis, remission and relapse: Implications for therapeutic resistance and MRD detection Aleshin, A., Durruthy-Durruthy, R., Corces, R., Liedtke, M., Eastburn, D., Majeti, R. AMER ASSOC CANCER RESEARCH. 2019
  • Barcoded Clonal Tracking of CRISPR-Cas9 and rAAV6-Mediated Gene Targeting in Human Hematopoietic Stem and Progenitor Cells Dever, D. P., Sharma, R., Lee, C. M., Aziz, A., Koehnke, T., Camarena, J., Pan, Y., Zhao, F., Bao, G., Majeti, R., Porteus, M. CELL PRESS. 2019: 5
  • First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers JOURNAL OF CLINICAL ONCOLOGY Sikic, B., Lakhani, N., Patnaik, A., Shah, S. A., Chandana, S. R., Rasco, D., Colevas, A., O'Rourke, T., Narayanan, S., Papadopoulos, K., Fisher, G. A., Villalobos, V., Prohaska, S. S., Howard, M., Beeram, M., Chao, M. P., Agoram, B., Chen, J. Y., Huang, J., Axt, M., Liu, J., Volkmer, J., Majeti, R., Weissman, I. L., Takimoto, C. H., Supan, D., Wakelee, H. A., Aoki, R., Pegram, M. D., Padda, S. K. 2019; 37 (12): 946-+
  • CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors CLINICAL CANCER RESEARCH Majzner, R. G., Theruvath, J. L., Nellan, A., Heitzeneder, S., Cui, Y., Mount, C. W., Rietberg, S. P., Linde, M. H., Xu, P., Rota, C., Sotillo, E., Labanieh, L., Lee, D. W., Orentas, R. J., Dimitrov, D. S., Zhu, Z., St Croix, B., Delaidelli, A., Sekunova, A., Bonvini, E., Mitra, S. S., Quezado, M. M., Majeti, R., Monje, M., Sorensen, P. B., Maris, J. M., Mackall, C. L. 2019; 25 (8): 2560–74
  • Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA ELIFE Xu, J., Nuno, K., Litzenburger, U. M., Qi, Y., Corces, M., Majeti, R., Chang, H. Y. 2019; 8
  • Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA. eLife Xu, J., Nuno, K., Litzenburger, U. M., Qi, Y., Corces, M. R., Majeti, R., Chang, H. Y. 2019; 8


    Simultaneous measurement of cell lineage and cell fates is a longstanding goal in biomedicine. Here we describe EMBLEM, a strategy to track cell lineage using endogenous mitochondrial DNA variants in ATAC-seq data. We show that somatic mutations in mitochondrial DNA can reconstruct cell lineage relationships at single cell resolution with high sensitivity and specificity. Using EMBLEM, we define the genetic and epigenomic clonal evolution of hematopoietic stem cells and their progenies in patients with acute myeloid leukemia. EMBLEM extends lineage tracing to any eukaryotic organism without genetic engineering.

    View details for PubMedID 30958261

  • Data mining for mutation-specific targets in acute myeloid leukemia LEUKEMIA Benard, B., Gentles, A. J., Kohnke, T., Majeti, R., Thomas, D. 2019; 33 (4): 826–43
  • No Matter How You Splice It, RBM39 Inhibition Targets Spliceosome Mutant AML. Cancer cell Thomas, R., Majeti, R. 2019; 35 (3): 337–39


    A report in this issue of Cancer Cell identifies the RNA-binding protein RBM39 as a potential target in spliceosome mutant AML that can be targeted by existing sulfonamide drugs. These results support a proposed clinical trial in patients with myeloid malignancies bearing spliceosome mutations relapsed or refractory to standard therapy.

    View details for PubMedID 30889374

  • First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology Sikic, B. I., Lakhani, N., Patnaik, A., Shah, S. A., Chandana, S. R., Rasco, D., Colevas, A. D., O'Rourke, T., Narayanan, S., Papadopoulos, K., Fisher, G. A., Villalobos, V., Prohaska, S. S., Howard, M., Beeram, M., Chao, M. P., Agoram, B., Chen, J. Y., Huang, J., Axt, M., Liu, J., Volkmer, J., Majeti, R., Weissman, I. L., Takimoto, C. H., Supan, D., Wakelee, H. A., Aoki, R., Pegram, M. D., Padda, S. K. 2019: JCO1802018


    PURPOSE: To evaluate the safety, pharmacokinetics, and pharmacodynamics of Hu5F9-G4 (5F9), a humanized IgG4 antibody that targets CD47 to enable phagocytosis.PATIENTS AND METHODS: Adult patients with solid tumors were treated in four cohorts: part A, to determine a priming dose; part B, to determine a weekly maintenance dose; part C, to study a loading dose in week 2; and a tumor biopsy cohort.RESULTS: Sixty-two patients were treated: 11 in part A, 14 in B, 22 in C, and 15 in the biopsy cohort. Part A used doses that ranged from 0.1 to 3 mg/kg. On the basis of tolerability and receptor occupancy studies that showed 100% CD47 saturation on RBCs, 1 mg/kg was selected as the priming dose. In subsequent groups, patients were treated with maintenance doses that ranged from 3 to 45 mg/kg, and most toxicities were mild to moderate. These included transient anemia (57% of patients), hemagglutination on peripheral blood smear (36%), fatigue (64%), headaches (50%), fever (45%), chills (45%), hyperbilirubinemia (34%), lymphopenia (34%), infusion-related reactions (34%), and arthralgias (18%). No maximum tolerated dose was reached with maintenance doses up to 45 mg/kg. At doses of 10 mg/kg or more, the CD47 antigen sink was saturated by 5F9, and a 5F9 half-life of approximately 13 days was observed. Strong antibody staining of tumor tissue was observed in a patient at 30 mg/kg. Two patients with ovarian/fallopian tube cancers had partial remissions for 5.2 and 9.2 months.CONCLUSION: 5F9 is well tolerated using a priming dose at 1 mg/kg on day 1 followed by maintenance doses of up to 45 mg/kg weekly.

    View details for PubMedID 30811285

  • Data mining for mutation-specific targets in acute myeloid leukemia. Leukemia Benard, B., Gentles, A. J., Kohnke, T., Majeti, R., Thomas, D. 2019


    Three mutation-specific targeted therapies have recently been approved by the FDA for the treatment of acute myeloid leukemia (AML): midostaurin for FLT3 mutations, enasidenib for relapsed or refractorycases with IDH2 mutations, and ivosidenib for cases with an IDH1 mutation. Together, these agents offer a mutation-directed treatment approach for up to 45% of de novo adult AML cases, a welcome deluge after a prolonged drought. At the same time, a number of computational tools have recently been developed that promise to further accelerate progress in mutation-specific therapy for AML and other cancers. Technical advances together with comprehensively annotated AML tissue banks have resulted in the availability of large and complex data sets for exploration by the end-user, including (i) microarray gene expression, (ii) exome sequencing, (iii) deep sequencing data of sub-clone heterogeneity, (iv) RNA sequencing of gene expression (bulk and single cell), (v) DNA methylation and chromatin, (vi) and germline quantitative trait loci. Yet few clinicians or experimental hematologists have the time or the training to access or analyze these repositories. This review summarizes the data sets and bioinformatic tools currently available to further the discovery of mutation-specific targets with an emphasis on web-based applications that are open, accessible, user-friendly, and do not require coding experience to navigate. We show examples of how available data can be mined to identify potential targets using synthetic lethality, drug repurposing, epigenetic sub-grouping, and proteomic networks while also highlighting strengths and limitations and the need for superior models for validation.

    View details for PubMedID 30728456

  • CAR T cells targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research Majzner, R. G., Theruvath, J. L., Nellan, A., Heitzeneder, S., Cui, Y., Mount, C. W., Rietberg, S. P., Linde, M. H., Xu, P., Rota, C., Sotillo, E., Labanieh, L., Lee, D. W., Orentas, R. J., Dimitrov, D. S., Zhu, Z., St Croix, B., Delaidelli, A., Sekunova, A., Bonvini, E., Mitra, S. S., Quezado, M. M., Majeti, R., Monje, M., Sorensen, P. H., Maris, J. M., Mackall, C. L. 2019


    PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T cell therapy of pediatric solid tumors, including those arising in the central nervous system.EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a monoclonal antibody that has been shown to preferentially bind tumor tissues and has been safely used in humans in early phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models.RESULTS: B7-H3 CAR T cells mediate significant anti-tumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3.CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies which should be tested in carefully designed clinical trials.

    View details for PubMedID 30655315

  • Induced pluripotent stem cell modeling of malignant hematopoiesis. Experimental hematology Chao, M. P., Majeti, R. 2019


    The ability to epigenetically reprogram differentiated somatic cells to pluripotency, the discovery of induced pluripotent stem cells (iPSCs), has unlocked fundamental biologic insights into numerous genetic diseases. These insights have resulted from the key property of iPSCs to differentiate into all cell lineages in an unlimited manner while maintaining the genetic identity of the originating cell. iPSCs have been utilized to investigate both monogenic and complex genetic disorders spanning hereditary and acquired diseases. Recently, iPSCs have been utilized to model human cancer, with a specific focus on modeling conditions of malignant hematopoiesis. In addition to serving as a genetic disease model in cancer, iPSCs can also be used as a tool to address a key question in interrogating the interaction between the cancer epigenome-genome. Specifically, how does reprogramming the epigenome impact cancer phenotype and specifically malignant hematopoiesis? This review will address this question and highlight the state of the field in generating iPSCs from hematologic malignancies, key biologic insights that can be uniquely generated from cancer-derived iPSCs, and their clinical applications. Lastly, challenges to expanding the use of iPSC modeling in blood cancers will be discussed.

    View details for PubMedID 30659850

  • The Phosphatidylethanolamine Biosynthesis Pathway Provides a New Target for Cancer Chemotherapy. Journal of hepatology Guan, Y. n., Chen, X. n., Wu, M. n., Zhu, W. n., Arslan, A. n., Takeda, S. n., Nguyen, M. H., Majeti, R. n., Thomas, D. n., Zheng, M. n., Peltz, G. n. 2019


    Since iPSC human develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development.Single cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidyl-ethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined.Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidyl-ethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro; and in a xenotransplantation model without causing significant side effets. This drug combination was also highly active against acute myeloid leukemic cells.Our data indicates that the phosphatidyl-ethanolamine biosynthesis is a targetable pathway for cancer; and that meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy.

    View details for DOI 10.1016/j.jhep.2019.11.007

    View details for PubMedID 31760071

  • Therapeutic Targeting of the Macrophage Immune Checkpoint CD47 in Myeloid Malignancies. Frontiers in oncology Chao, M. P., Takimoto, C. H., Feng, D. D., McKenna, K., Gip, P., Liu, J., Volkmer, J., Weissman, I. L., Majeti, R. 2019; 9: 1380


    In recent years, immunotherapies have been clinically investigated in AML and other myeloid malignancies. While most of these are focused on stimulating the adaptive immune system (including T cell checkpoint inhibitors), several key approaches targeting the innate immune system have been identified. Macrophages are a key cell type in the innate immune response with CD47 being identified as a dominant macrophage checkpoint. CD47 is a "do not eat me" signal, overexpressed in myeloid malignancies that leads to tumor evasion of phagocytosis by macrophages. Blockade of CD47 leads to engulfment of leukemic cells and therapeutic elimination. Pre-clinical data has demonstrated robust anti-cancer activity in multiple hematologic malignancies including AML and myelodysplastic syndrome (MDS). In addition, clinical studies have been underway with CD47 targeting agents in both AML and MDS as monotherapy and in combination. This review will describe the role of CD47 in myeloid malignancies and pre-clinical data supporting CD47 targeting. In addition, initial clinical data of CD47 targeting in AML/MDS will be reviewed, and including the first-in-class anti-CD47 antibody magrolimab.

    View details for DOI 10.3389/fonc.2019.01380

    View details for PubMedID 32038992

  • Mebendazole for Differentiation Therapy of Acute Myeloid Leukemia Identified by a Lineage Maturation Index. Scientific reports Li, Y. n., Thomas, D. n., Deutzmann, A. n., Majeti, R. n., Felsher, D. W., Dill, D. L. 2019; 9 (1): 16775


    Accurate assessment of changes in cellular differentiation status in response to drug treatments or genetic perturbations is crucial for understanding tumorigenesis and developing novel therapeutics for human cancer. We have developed a novel computational approach, the Lineage Maturation Index (LMI), to define the changes in differentiation state of hematopoietic malignancies based on their gene expression profiles. We have confirmed that the LMI approach can detect known changes of differentiation state in both normal and malignant hematopoietic cells. To discover novel differentiation therapies, we applied this approach to analyze the gene expression profiles of HL-60 leukemia cells treated with a small molecule drug library. Among multiple drugs that significantly increased the LMIs, we identified mebendazole, an anti-helminthic clinically used for decades with no known significant toxicity. We tested the differentiation activity of mebendazole using primary leukemia blast cells isolated from human acute myeloid leukemia (AML) patients. We determined that treatment with mebendazole induces dramatic differentiation of leukemia blast cells as shown by cellular morphology and cell surface markers. Furthermore, mebendazole treatment significantly extended the survival of leukemia-bearing mice in a xenograft model. These findings suggest that mebendazole may be utilized as a low toxicity therapeutic for human acute myeloid leukemia and confirm the LMI approach as a robust tool for the discovery of novel differentiation therapies for cancer.

    View details for DOI 10.1038/s41598-019-53290-3

    View details for PubMedID 31727951

  • Targeting Cancer Stemness in the Clinic: From Hype to Hope. Cell stem cell Saygin, C., Matei, D., Majeti, R., Reizes, O., Lathia, J. D. 2018


    Tumors are composed of non-homogeneous cell populations exhibiting varying degrees of genetic and functional heterogeneity. Cancer stem cells (CSCs) are capable of sustaining tumors by manipulating genetic and non-genetic factors to metastasize, resist treatment, and maintain the tumor microenvironment. Understanding the key traits and mechanisms of CSC survival provides opportunities to improve patient outcomes via improved prognostic models and therapeutics. Here, we review the clinical significance of CSCs and results of potential CSC-targeting therapies in various cancers. We discuss barriers to translating cues from pre-clinical models into clinical applications and propose new strategies for rational design of future anti-CSC trials.

    View details for PubMedID 30595497

  • Accumulation of JAK Activation-Loop Phosphorylation Promotes Type I JAK Inhibitor Withdrawal Syndrome in Myelofibrosis Tvorogov, D., Thomas, D., Liau, N. D., Dottore, M., Barry, E. F., Lathi, M., Kan, W. L., Hercus, T. R., Stomski, F., Hughes, T. P., Tergaonkar, V., Parker, M. W., Ross, D. M., Babon, J. J., Majeti, R., Lopez, A. F. AMER SOC HEMATOLOGY. 2018
  • Macrophage de novo NAD+ synthesis specifies immune function in aging and inflammation. Nature immunology Minhas, P. S., Liu, L., Moon, P. K., Joshi, A. U., Dove, C., Mhatre, S., Contrepois, K., Wang, Q., Lee, B. A., Coronado, M., Bernstein, D., Snyder, M. P., Migaud, M., Majeti, R., Mochly-Rosen, D., Rabinowitz, J. D., Andreasson, K. I. 2018


    Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD+ derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo NAD+ synthesis depleted NAD+, suppressed mitochondrial NAD+-dependent signaling and respiration, and impaired phagocytosis and resolution of inflammation. Innate immune challenge triggered upstream KP activation but paradoxically suppressed cell-autonomous NAD+ synthesis by limiting the conversion of downstream quinolinate to NAD+, a profile recapitulated in aging macrophages. Increasing de novo NAD+ generation in immune-challenged or aged macrophages restored oxidative phosphorylation and homeostatic immune responses. Thus, KP-derived NAD+ operates as a metabolic switch to specify macrophage effector responses. Breakdown of de novo NAD+ synthesis may underlie declining NAD+ levels and rising innate immune dysfunction in aging and age-associated diseases.

    View details for PubMedID 30478397

  • Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis. Science advances Tvorogov, D., Thomas, D., Liau, N. P., Dottore, M., Barry, E. F., Lathi, M., Kan, W. L., Hercus, T. R., Stomski, F., Hughes, T. P., Tergaonkar, V., Parker, M. W., Ross, D. M., Majeti, R., Babon, J. J., Lopez, A. F. 2018; 4 (11): eaat3834


    Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2V617F mutant CD34+ progenitors after drug washout. Type I inhibitor-induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients.

    View details for PubMedID 30498775

  • Identification of the Human Skeletal Stem Cell. Cell Chan, C. K., Gulati, G. S., Sinha, R., Tompkins, J. V., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E. Y., Leavitt, T., Lu, W., Nguyen, A., Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43


    Stem cell regulation and hierarchical organization ofhuman skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

    View details for PubMedID 30241615

  • Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation CELL Buenrostro, J. D., Corces, M., Lareau, C. A., Wu, B., Schep, A. N., Aryee, M. J., Majeti, R., Chang, H. Y., Greenleaf, W. J. 2018; 173 (6): 1535-+


    Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

    View details for PubMedID 29706549

  • Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells NATURE IMMUNOLOGY Karamitros, D., Stoilova, B., Aboukhalil, Z., Hamey, F., Reinisch, A., Samitsch, M., Quek, L., Otto, G., Repapi, E., Doondeea, J., Usukhbayar, B., Calvo, J., Taylor, S., Goardon, N., Six, E., Pflumio, F., Porcher, C., Majeti, R., Gottgens, B., Vyas, P. 2018; 19 (1): 85-+
  • Engineering complex genotypes in primary haematopoietic cells using Cas9/sgRNA and AAV donor vectors Bak, R. O., Dever, D. P., Reinisch, A., Cruz, D., Majeti, R., Porteus, M. H. MARY ANN LIEBERT, INC. 2017: A17
  • Proposed Terminology and Classification of Pre-Malignant Neoplastic Conditions: A Consensus Proposal EBIOMEDICINE Valent, P., Akin, C., Arock, M., Bock, C., George, T. I., Galli, S. J., Gotlib, J., Haferlach, T., Hoermann, G., Hermine, O., Jaeger, U., Kenner, L., Kreipe, H., Majeti, R., Metcalfe, D. D., Orfao, A., Reiter, A., Sperr, W. R., Staber, P. B., Sotlar, K., Schiffer, C., Superti-Furga, G., Horny, H. 2017; 26: 17–24
  • Preleukemic Hematopoietic Stem Cells in Human Acute Myeloid Leukemia FRONTIERS IN ONCOLOGY Corces, M., Chang, H. Y., Majeti, R. 2017; 7: 263


    Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by an uncontrolled proliferation of undifferentiated myeloid lineage cells. Decades of research have demonstrated that AML evolves from the sequential acquisition of genetic alterations within a single lineage of hematopoietic cells. More recently, the advent of high-throughput sequencing has enabled the identification of a premalignant phase of AML termed preleukemia. Multiple studies have demonstrated that AML can arise from the accumulation of mutations within hematopoietic stem cells (HSCs). These HSCs have been termed "preleukemic HSCs" as they represent the evolutionary ancestors of the leukemia. Through examination of the biological and clinical characteristics of these preleukemic HSCs, this review aims to shed light on some of the unexplored questions in the field. We note that some of the material discussed is speculative in nature and is presented in order to motivate future work.

    View details for PubMedID 29164062

  • Systematic discovery of mutation-specific synthetic lethals by mining pan-cancer human primary tumor data. Nature communications Sinha, S., Thomas, D., Chan, S., Gao, Y., Brunen, D., Torabi, D., Reinisch, A., Hernandez, D., Chan, A., Rankin, E. B., Bernards, R., Majeti, R., Dill, D. L. 2017; 8: 15580-?


    Two genes are synthetically lethal (SL) when defects in both are lethal to a cell but a single defect is non-lethal. SL partners of cancer mutations are of great interest as pharmacological targets; however, identifying them by cell line-based methods is challenging. Here we develop MiSL (Mining Synthetic Lethals), an algorithm that mines pan-cancer human primary tumour data to identify mutation-specific SL partners for specific cancers. We apply MiSL to 12 different cancers and predict 145,891 SL partners for 3,120 mutations, including known mutation-specific SL partners. Comparisons with functional screens show that MiSL predictions are enriched for SLs in multiple cancers. We extensively validate a SL interaction identified by MiSL between the IDH1 mutation and ACACA in leukaemia using gene targeting and patient-derived xenografts. Furthermore, we apply MiSL to pinpoint genetic biomarkers for drug sensitivity. These results demonstrate that MiSL can accelerate precision oncology by identifying mutation-specific targets and biomarkers.

    View details for DOI 10.1038/ncomms15580

    View details for PubMedID 28561042

  • Optimizing Next-Generation AML Therapy: Activity of Mutant IDH2 Inhibitor AG-221 in Preclinical Models CANCER DISCOVERY Thomas, D., Majeti, R. 2017; 7 (5): 459–61
  • Disrupting the CD47-SIRP alpha anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors SCIENCE TRANSLATIONAL MEDICINE Gholamin, S., Mitra, S. S., Feroze, A. H., Liu, J., Kahn, S. A., Zhang, M., Esparza, R., Richard, C., Ramaswamy, V., Remke, M., Volkmer, A. K., Willingham, S., Ponnuswami, A., McCarty, A., Lovelace, P., Storm, T. A., Schubert, S., Hutter, G., Narayanan, C., Chu, P., Raabe, E. H., Harsh, G., Taylor, M. D., Monje, M., Cho, Y., Majeti, R., Volkmer, J. P., Fisher, P. G., Grant, G., Steinberg, G. K., Vogel, H., Edwards, M., Weissman, I. L., Cheshier, S. H. 2017; 9 (381)


    Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

    View details for DOI 10.1126/scitranslmed.aaf2968

    View details for PubMedID 28298418

  • The role of mutations in the cohesin complex in acute myeloid leukemia INTERNATIONAL JOURNAL OF HEMATOLOGY Mazumdar, C., Majeti, R. 2017; 105 (1): 31-36
  • Proposed Terminology and Classification of Pre-Malignant Neoplastic Conditions: A Consensus Proposal. EBioMedicine Valent, P. n., Akin, C. n., Arock, M. n., Bock, C. n., George, T. I., Galli, S. J., Gotlib, J. n., Haferlach, T. n., Hoermann, G. n., Hermine, O. n., Jäger, U. n., Kenner, L. n., Kreipe, H. n., Majeti, R. n., Metcalfe, D. D., Orfao, A. n., Reiter, A. n., Sperr, W. R., Staber, P. B., Sotlar, K. n., Schiffer, C. n., Superti-Furga, G. n., Horny, H. P. 2017; 26: 17–24


    Cancer evolution is a step-wise non-linear process that may start early in life or later in adulthood, and includes pre-malignant (indolent) and malignant phases. Early somatic changes may not be detectable or are found by chance in apparently healthy individuals. The same lesions may be detected in pre-malignant clonal conditions. In some patients, these lesions may never become relevant clinically whereas in others, they act together with additional pro-oncogenic hits and thereby contribute to the formation of an overt malignancy. Although some pre-malignant stages of a malignancy have been characterized, no global system to define and to classify these conditions is available. To discuss open issues related to pre-malignant phases of neoplastic disorders, a working conference was organized in Vienna in August 2015. The outcomes of this conference are summarized herein and include a basic proposal for a nomenclature and classification of pre-malignant conditions. This proposal should assist in the communication among patients, physicians and scientists, which is critical as genome-sequencing will soon be offered widely for early cancer-detection.

    View details for PubMedID 29203377

  • Optimizing Next-Generation AML Therapy: Activity of Mutant IDH2 Inhibitor AG-221 in Preclinical Models. Cancer discovery Thomas, D. n., Majeti, R. n. 2017; 7 (5): 459–61


    AG-221 or enasidenib is a first-in-class selective inhibitor of mutated isocitrate dehydrogenase 2 (IDH2) with early demonstrated clinical efficacy in acute myeloid leukemia as a single agent, yet with persistence of mutant IDH2 clones. Two articles in this issue of Cancer Discovery provide further insight into the biological activity of AG-221 in promoting differentiation of IDH2-mutant cells and reversing aberrant DNA methylation over time, and demonstrating preclinical activity in combination with a targeted FLT3 kinase inhibitor to eliminate IDH2-mutant clones. Cancer Discov; 7(5); 459-61. ©2017 AACR.See related article by Yen et al., p. 478See related article by Shih et al., p. 494.

    View details for PubMedID 28461409

  • Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice. Nature protocols Reinisch, A. n., Hernandez, D. C., Schallmoser, K. n., Majeti, R. n. 2017; 12 (10): 2169–88


    Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function. The ossicles, therefore, allow for accelerated and superior engraftment of primary patient-derived acute myeloid leukemia (AML) and other hematologic malignancies such as myelofibrosis (MF) in mice. The humanized ossicles are formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). Human hematopoietic cells can subsequently be transplanted directly into the ossicle marrow space or by intravenous injection. Using this method, a humanized engraftable BM microenvironment can be formed within 6-10 weeks. Engraftment of human hematopoietic cells can be evaluated by flow cytometry 8-16 weeks after transplantation. This protocol describes a robust and reproducible in vivo methodology for the study of normal and malignant human hematopoiesis in a more physiologic setting.

    View details for PubMedID 28933777

  • The CD47 Macrophage Checkpoint as a New Immunotherapy Target Sikic, B. I., Padda, S. K., Shah, S. A., Colevas, D., Narayanan, S., Fisher, G. A., Supan, D., Wakelee, H. A., Aoki, R., Pegram, M. D., Villalobos, V. M., Liu, J., Takimoto, C., Chao, M. P., Volkmer, J. P., Majeti, R., Weissman, I. L. ELSEVIER SCIENCE INC. 2017: S108–S109
  • Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes ONCOTARGET Matre, P., Velez, J., Jacamo, R., Qi, Y., Su, X., Cai, T., Chan, S. M., Lodi, A., Sweeney, S. R., Ma, H., Davis, R. E., Baran, N., Haferlach, T., Su, X., Flores, E. R., Gonzalez, D., Konoplev, S., Samudio, I., Dinardo, C., Majeti, R., Schimmer, A. D., Li, W., Wang, T., Tiziani, S., Konopleva, M. 2016; 7 (48): 79708-79721
  • Sticking It to the Niche: CD98 Mediates Critical Adhesive Signals in AML CANCER CELL Reinisch, A., Majeti, R. 2016; 30 (5): 662–64


    In this issue of Cancer Cell, Bajaj and colleagues report that CD98, a heterodimeric protein highly expressed in acute myeloid leukemia (AML) and largely dispensable for basal hematopoiesis, plays an important role in facilitating leukemia stem cell adhesion to bone marrow vasculature and is a potential therapeutic target in AML.

    View details for PubMedID 27846387

  • CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells. Nature Dever, D. P., Bak, R. O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C. E., Pavel-Dinu, M., Saxena, N., Wilkens, A. B., Mantri, S., Uchida, N., Hendel, A., Narla, A., Majeti, R., Weinberg, K. I., Porteus, M. H. 2016


    The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

    View details for DOI 10.1038/nature20134

    View details for PubMedID 27820943

  • The role of mutations in the cohesin complex in acute myeloid leukemia. International journal of hematology Mazumdar, C., Majeti, R. 2016: -?


    Mutations in the members of the cohesin complex have recently been identified as early events in acute myeloid leukemia (AML) pathogenesis. Studies conducted by our lab and others have shown that cohesin mutations or knockdown of cohesin subunits impair hematopoietic differentiation and enforce stem cell programs in both human and mouse hematopoiesis. Furthermore, studies in both models demonstrated global changes in chromatin accessibility and structure, in particular increased accessibility at binding sites for hematopoietic stem and progenitor cell (HSPC) transcription factors. These results suggest that mutations in the cohesin complex may contribute to leukemogenesis through modulation of HSPC chromatin accessibility. Future studies will be necessary to determine the detailed mechanisms mediating these phenotypes.

    View details for PubMedID 27796738

  • SIRP alpha-Antibody Fusion Proteins Selectively Bind and Eliminate Dual Antigen-Expressing Tumor Cells CLINICAL CANCER RESEARCH Piccione, E. C., Juarez, S., Tseng, S., Liu, J., Stafford, M., Narayanan, C., Wang, L., Weiskopf, K., Majeti, R. 2016; 22 (20): 5109-5119


    CD47 is highly expressed on a variety of tumor cells. The interaction of CD47 with signal regulatory protein alpha (SIRPα), a protein on phagocytic cells, transmits a "don't eat me" signal that negatively regulates phagocytosis. CD47-SIRPα antagonists enable phagocytosis by disrupting the inhibitory signal and can synergize with Fc-mediated pro-phagocytic signals for potent elimination of tumor cells. A potential limitation of therapeutic CD47-SIRPα antagonists is that expression of CD47 on normal cells may create sites of toxicity or an "antigen sink." To overcome these limitations and address selective tumor targeting, we developed SIRPabodies to improve the therapeutic benefits of CD47-SIRPα blockade specifically toward tumor.SIRPabodies were generated by grafting the wild-type SIRPα either to the N-terminus or to the C-terminus of the heavy chain of rituximab. Selective tumor binding was tested using CFSE-labeled human primary CLL cells in the presence of 20-fold excess of human RBCs. NSG mice were transplanted with Raji-luciferase cells and were assigned to controls versus SIRPabody treatment. Cynomolgus nonhuman primates were administered a single intravenous infusion of SIRPabody at 3, 10, or 30 mg/kg.SIRPabodies selectively bound to dual antigen-expressing tumor cells in the presence of a large antigen sink. SIRPabody reduced tumor burden and extended survival in mouse xenograft lymphoma models. SIRPabody caused no significant toxicity in nonhuman primates.These findings establish SIRPabodies as a promising approach to deliver the therapeutic benefit of CD47-SIRPα blockade specifically toward tumor cells. SIRPabodies may be applied to additional cancer types by grafting SIRPα onto other tumor-specific therapeutic antibodies. Clin Cancer Res; 22(20); 5109-19. ©2016 AACR.

    View details for DOI 10.1158/1078-0432.CCR-15-2503

    View details for Web of Science ID 000385632700019

    View details for PubMedID 27126995

  • Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells. Oncotarget Huang, M., Garcia, J. S., Thomas, D., Zhu, L., Nguyen, L. X., Chan, S. M., Majeti, R., Medeiros, B. C., Mitchell, B. S. 2016


    The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.

    View details for DOI 10.18632/oncotarget.12493

    View details for PubMedID 27732946

  • Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics Corces, M. R., Buenrostro, J. D., Wu, B., Greenside, P. G., Chan, S. M., Koenig, J. L., Snyder, M. P., Pritchard, J. K., Kundaje, A., Greenleaf, W. J., Majeti, R., Chang, H. Y. 2016; 48 (10): 1193-1203


    We define the chromatin accessibility and transcriptional landscapes in 13 human primary blood cell types that span the hematopoietic hierarchy. Exploiting the finding that the enhancer landscape better reflects cell identity than mRNA levels, we enable 'enhancer cytometry' for enumeration of pure cell types from complex populations. We identify regulators governing hematopoietic differentiation and further show the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia (AML), chromatin accessibility uncovers unique regulatory evolution in cancer cells with a progressively increasing mutation burden. Single AML cells exhibit distinctive mixed regulome profiles corresponding to disparate developmental stages. A method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of preleukemic hematopoietic stem cell characteristics. Thus, regulome dynamics can provide diverse insights into hematopoietic development and disease.

    View details for DOI 10.1038/ng.3646

    View details for PubMedID 27526324

  • Burning Fat Fuels Leukemic Stem Cell Heterogeneity. Cell stem cell Thomas, D., Majeti, R. 2016; 19 (1): 1-2


    Obese leukemia patients exhibit reduced survival after chemotherapy, suggesting an important role of adipose tissue in disease progression. In this issue of Cell Stem Cell, Ye et al. (2016) reveal metabolic heterogeneity in leukemic stem cell (LSC) subpopulations and show that chemotherapy-resistant CD36+ LSCs co-opt gonadal adipose tissue to support their metabolism and survival.

    View details for DOI 10.1016/j.stem.2016.06.014

    View details for PubMedID 27392217

  • ASH1L Links Histone H3 Lysine 36 Dimethylation to MLL Leukemia. Cancer discovery Zhu, L., Li, Q., Wong, S. H., Huang, M., Klein, B. J., Shen, J., Ikenouye, L., Onishi, M., Schneidawind, D., Buechele, C., Hansen, L., Duque-Afonso, J., Zhu, F., Martin, G. M., Gozani, O., Majeti, R., Kutateladze, T. G., Cleary, M. L. 2016; 6 (7): 770-783


    Numerous studies in multiple systems support that histone H3 lysine 36 dimethylation (H3K36me2) is associated with transcriptional activation; however, the underlying mechanisms are not well defined. Here, we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, a mixed-lineage leukemia (MLL)-associated protein that colocalizes with MLL, ASH1L, and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild-type MLL proteins to chromatin at key leukemia target genes and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely, KDM2A, an H3K36me2 demethylase and Polycomb group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation, and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention.Epigenetic regulators play vital roles in cancer pathogenesis and represent a new frontier in therapeutic targeting. Our studies provide basic mechanistic insight into the role of H3K36me2 in transcription activation and MLL leukemia pathogenesis and implicate ASH1L histone methyltransferase as a promising target for novel molecular therapy. Cancer Discov; 6(7); 770-83. ©2016 AACR.See related commentary by Balbach and Orkin, p. 700This article is highlighted in the In This Issue feature, p. 681.

    View details for DOI 10.1158/2159-8290.CD-16-0058

    View details for PubMedID 27154821

    View details for PubMedCentralID PMC4930721

  • CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer JOURNAL OF CLINICAL INVESTIGATION Weiskopf, K., Jahchan, N. S., Schnorr, P. J., Cristea, S., Ring, A. M., Maute, R. L., Volkmer, A. K., Volkmer, J., Liu, J., Lim, J. S., Yang, D., Seitz, G., Thuyen Nguyen, T., Wu, D., Jude, K., Guerston, H., Barkal, A., Trapani, F., George, J., Poirier, J. T., Gardner, E. E., Miles, L. A., de Stanchina, E., Lofgren, S. M., Vogel, H., Winslow, M. M., Dive, C., Thomas, R. K., Rudin, C. M., van de Rijn, M., Majeti, R., Garcia, K. C., Weissman, I. L., Sage, J. 2016; 126 (7): 2610-2620


    Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

    View details for DOI 10.1172/JCI81603

    View details for Web of Science ID 000379094800024

    View details for PubMedID 27294525

    View details for PubMedCentralID PMC4922696

  • SUPER-ENHANCER ANALYSIS DEFINES NOVEL AML AND MDS SUB-TYPES SENSITIVE TO SY-1425, A POTENT AND SELECTIVE RARA AGONIST Mckeown, M., Lee, E., Fiore, C., Eaton, M., Lopez, J., Corces-Zimmerman, R., Majeti, R., Stephens, K., Fritz, C., Olson, E. FERRATA STORTI FOUNDATION. 2016: 325
  • FACS-Based Enrichment of a Highly Purified HBB-Targeted Hematopoietic Stem and Progenitor Cell Population Using rAAV6 and CRISPR/Cas9 Dever, D. P., Bak, R. O., Camarena, J., Saxena, N., Reinisch, A., Nicolas, C. E., Wilkens, A. B., Hendel, A., Uchida, N., Majeti, R., Weinberg, K. I., Porteus, M. H. NATURE PUBLISHING GROUP. 2016: S17
  • CRISPR/Cas9 and rAAV6-Mediated Targeted Integration at the CCR5 Locus in Hematopoietic Stem and Progenitor Cells Bak, R. O., Dever, D. P., Saxena, N., Camarena, J., Reinisch, A., Wilkens, A. B., Nicolas, C. E., Hendel, A., Majeti, R., Weinberg, K. I., Porteus, M. H. NATURE PUBLISHING GROUP. 2016: S19
  • Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo PLOS ONE Zhang, M., Hutter, G., Kahn, S. A., Azad, T. D., Gholamin, S., Xu, C. Y., Liu, J., Achrol, A. S., Richard, C., Sommerkamp, P., Schoen, M. K., McCracken, M. N., Majeti, R., Weissman, I., Mitra, S. S., Cheshier, S. H. 2016; 11 (4)


    Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

    View details for DOI 10.1371/journal.pone.0153550

    View details for Web of Science ID 000374541200027

    View details for PubMedID 27092773

    View details for PubMedCentralID PMC4836698

  • Alkylator-Induced and Patient-Derived Xenograft Mouse Models of Therapy-Related Myeloid Neoplasms Model Clinical Disease and Suggest the Presence of Multiple Cell Subpopulations with Leukemia Stem Cell Activity. PloS one Jonas, B. A., Johnson, C., Gratzinger, D., Majeti, R. 2016; 11 (7)


    Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their de novo counterparts. Here, we report the development of mouse models of t-AML/MDS. First, we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU), resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia, myelodysplasia, and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second, we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall, we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics.

    View details for DOI 10.1371/journal.pone.0159189

    View details for PubMedID 27428079

    View details for PubMedCentralID PMC4948781

  • Clonal evolution of preleukemic hematopoietic stem cells in acute myeloid leukemia EXPERIMENTAL HEMATOLOGY Sykes, S. M., Kokkaliaris, K. D., Milsom, M. D., Levine, R. L., Majeti, R. 2015; 43 (12): 989–92


    Acute myeloid leukemia (AML) is an aggressive blood cancer that results from an abnormal expansion of uncontrollably proliferating myeloid progenitors that have lost the capacity to differentiate. AML encompasses many genetically distinct subtypes that predominantly develop de novo. However, AML can also arise from premalignant myeloid conditions, such as myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPNs), or develop as the result of exposure to genotoxic agents used to treat unrelated malignancies. Although numerous distinct cytogenetic and molecular abnormalities associated with AML were discovered prior to the turn of the millennium, recent advances in whole genome sequencing and global genomic approaches have resulted in an explosion of newly identified molecular abnormalities. However, even with these advances, our understanding of how these mutations contribute to the etiology, pathogenesis, and therapeutic responses of AML remains largely unknown. Recently the International Society for Experimental Hematology (ISEH) hosted a webinar entitled "Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells (HSCs) in AML" in which two AML mavens, Ross Levine, MD, and Ravindra Majeti, MD, PhD, discussed some of their recent, groundbreaking studies that have shed light on how many of these newly identified mutations contribute to leukemogenesis and therapy resistance in AML. Here, we provide a brief overview of this webinar and discuss the basic scientific and clinical implications of the data presented.

    View details for PubMedID 26455528

    View details for PubMedCentralID PMC5492948

  • Deciphering the cancer methylome with Boolean implications to find novel drivers of aberrant DNA methylation and actionable drug targets Sinha, S., Thomas, D., Majeti, R., Dill, D. L. AMER ASSOC CANCER RESEARCH. 2015
  • An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis NATURE COMMUNICATIONS Jung, N., Dai, B., Gentles, A. J., Majeti, R., Feinberg, A. P. 2015; 6


    Acute myeloid leukaemia (AML) is characterized by subpopulations of leukaemia stem cells (LSCs) that are defined by their ability to engraft in immunodeficient mice. Here we show an LSC DNA methylation signature, derived from xenografts and integration with gene expression that is comprised of 71 genes and identifies a key role for the HOXA cluster. Most of the genes are epigenetically regulated independently of underlying mutations, although several are downstream targets of epigenetic modifier genes mutated in AML. The LSC epigenetic signature is associated with poor prognosis independent of known risk factors such as age and cytogenetics. Analysis of early haematopoietic progenitors from normal individuals reveals two distinct clusters of AML LSC resembling either lymphoid-primed multipotent progenitors or granulocyte/macrophage progenitors. These results provide evidence for DNA methylation variation between AML LSCs and their blast progeny, and identify epigenetically distinct subgroups of AML likely reflecting the cell of origin.

    View details for DOI 10.1038/ncomms9489

    View details for Web of Science ID 000364941300001

    View details for PubMedID 26444494

    View details for PubMedCentralID PMC4633733

  • A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells MABS Piccione, E. C., Juarez, S., Liu, J., Tseng, S., Ryan, C. E., Narayanan, C., Wang, L., Weiskopf, K., Majeti, R. 2015; 7 (5): 946-956


    Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an 'antigen sink' that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.

    View details for DOI 10.1080/19420862.2015.1062192

    View details for Web of Science ID 000360218900015

  • Biology and Clinical Relevance of Acute Myeloid Leukemia Stem Cells SEMINARS IN HEMATOLOGY Reinisch, A., Chan, S. M., Thomas, D., Majeti, R. 2015; 52 (3): 150-164


    Evidence for the cancer stem cell model was first demonstrated in xenotransplanted blood and bone marrow samples from patients with acute myeloid leukemia (AML) almost two decades ago, supporting the concept that a rare clonal and mutated leukemic stem cell (LSC) population is sufficient to drive leukemic growth. The inability to eliminate LSCs with conventional therapies is thought to be the primary cause of disease relapse in AML patients, and as such, novel therapies with the ability to target this population are required to improve patient outcomes. An important step towards this goal is the identification of common immunophenotypic surface markers and biological properties that distinguish LSCs from normal hematopoietic stem and progenitor cells (HSPCs) across AML patients. This work has resulted in the development of a large number of potential LSC-selective therapies that target cell surface molecules, intracellular signaling pathways, and the bone marrow microenvironment. Here, we will review the basic biology, immunophenotypic detection, and clinical relevance of LSCs, as well as emerging biological and small-molecule strategies that either directly target LSCs or indirectly target these cells through modulation of their microenvironment.

    View details for DOI 10.1053/j.seminhematol.2015.03.008

    View details for PubMedID 26111462

  • Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands CELL Moraga, I., Wernig, G., Wilmes, S., Gryshkova, V., Richter, C. P., Hong, W., Sinha, R., Guo, F., Fabionar, H., Wehrman, T. S., Krutzik, P., Demharter, S., Plo, I., Weissman, I. L., Minary, P., Majeti, R., Constantinescu, S. N., Piehler, J., Garcia, K. C. 2015; 160 (6): 1196-1208


    Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

    View details for DOI 10.1016/j.cell.2015.02.011

    View details for PubMedID 25728669

  • Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood Reinisch, A., Etchart, N., Thomas, D., Hofmann, N. A., Fruehwirth, M., Sinha, S., Chan, C. K., Senarath-Yapa, K., Seo, E., Wearda, T., Hartwig, U. F., Beham-Schmid, C., Trajanoski, S., Lin, Q., Wagner, W., Dullin, C., Alves, F., Andreeff, M., Weissman, I. L., Longaker, M. T., Schallmoser, K., Majeti, R., Strunk, D. 2015; 125 (2): 249-260


    In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a bone marrow cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13 and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs) as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

    View details for DOI 10.1182/blood-2014-04-572255

    View details for PubMedID 25406351

  • Refractory warm IgM-mediated autoimmune hemolytic anemia associated with Churg-Strauss syndrome responsive to eculizumab and rituximab AMERICAN JOURNAL OF HEMATOLOGY Chao, M. P., Hong, J., Kunder, C., Lester, L., Schrier, S. L., Majeti, R. 2015; 90 (1): 78-81

    View details for DOI 10.1002/ajh.23791

    View details for Web of Science ID 000346771400022

    View details for PubMedID 24942207

  • A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells. mAbs Piccione, E. C., Juarez, S., Liu, J., Tseng, S., Ryan, C. E., Narayanan, C., Wang, L., Weiskopf, K., Majeti, R. 2015; 7 (5): 946-956


    Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an 'antigen sink' that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.

    View details for DOI 10.1080/19420862.2015.1062192

    View details for PubMedID 26083076

  • Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential. PloS one Liu, J., Wang, L., Zhao, F., Tseng, S., Narayanan, C., Shura, L., Willingham, S., Howard, M., Prohaska, S., Volkmer, J., Chao, M., Weissman, I. L., Majeti, R. 2015; 10 (9)

    View details for DOI 10.1371/journal.pone.0137345

    View details for PubMedID 26390038

  • Mutation in Wilms' Tumor 1 Induces DNA Hypermethylation of PRC2 Targets, Blocks Myelomonocytic Differentiation, and Defines a Novel Subtype of AML Responsive to EZH2 Inhibition Thomas, D., Sinha, S., Yu, L., Jung, N., Dai, B., Gentles, A. J., Feinberg, A. P., Dill, D., Majeti, R. AMER SOC HEMATOLOGY. 2014
  • Pre-leukemic evolution of hematopoietic stem cells: the importance of early mutations in leukemogenesis LEUKEMIA Corces-Zimmerman, M. R., Majeti, R. 2014; 28 (12): 2276-2282


    Cancer has been shown to result from the sequential acquisition of genetic alterations in a single lineage of cells. In leukemia, increasing evidence has supported the idea that this accumulation of mutations occurs in self-renewing hematopoietic stem cells (HSCs). These HSCs containing some, but not all, leukemia-specific mutations have been termed as pre-leukemic. Multiple recent studies have sought to understand these pre-leukemic HSCs and determine to what extent they contribute to leukemogenesis. These studies have elucidated patterns in mutation acquisition in leukemia, demonstrated resistance of pre-leukemic cells to standard induction chemotherapy and identified these pre-leukemic cells as a putative reservoir for the generation of relapsed disease. When combined with decades of research on clonal evolution in leukemia, mouse models of leukemogenesis, and recent massively parallel sequencing-based studies of primary patient leukemia, studies of pre-leukemic HSCs begin to piece together the evolutionary puzzle of leukemogenesis. These results have broad implications for leukemia treatment, targeted therapies, minimal residual disease monitoring and early detection screening.

    View details for DOI 10.1038/leu.2014.211

    View details for Web of Science ID 000346177500002

    View details for PubMedID 25005245

    View details for PubMedCentralID PMC4262622

  • Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY Majeti, R. 2014; 27 (3-4): 229-234
  • Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells. Blood Nguyen, L. X., Chan, S. M., Ngo, T. D., Raval, A., Kim, K. K., Majeti, R., Mitchell, B. S. 2014; 124 (4): 579-589


    The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.

    View details for DOI 10.1182/blood-2013-12-544726

    View details for PubMedID 24850755

  • Proffered Paper: Overcoming immune evasion in ovarian and breast cancer with anti-CD47 antibody blockade: A novel class of immune therapy Volkmer, A. K., Willingham, S. B., Tseng, S. R., Ho, P. Y., Volkmer, J. P., Sikic, B. I., Majeti, R., Weissman, I. L. ELSEVIER SCI LTD. 2014: S13
  • Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma NATURE COMMUNICATIONS Green, M. R., Vicente-Duenas, C., Romero-Camarero, I., Liu, C. L., Dai, B., Gonzalez-Herrero, I., Garcia-Ramirez, I., Alonso-Escudero, E., Iqbal, J., Chan, W. C., Campos-Sanchez, E., Orfao, A., Pintado, B., Flores, T., Blanco, O., Jimenez, R., Angel Martinez-Climent, J., Garcia Criado, F. J., Garcia Cenador, M. B., Zhao, S., Natkunam, Y., Lossos, I. S., Majeti, R., Melnick, A., Cobaleda, C., Alizadeh, A. A., Sanchez-Garcia, I. 2014; 5


    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by 'hit-and-run' oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a 'hit-and-run' role in lymphomagenesis.

    View details for DOI 10.1038/ncomms4904

    View details for Web of Science ID 000338831000001

  • OVERCOMING IMMUNE EVASION IN PEDIATRIC BRAIN TUMORS: A PRE-CLINICAL DEVELOPMENT STUDY USING A HUMANIZED ANTI-CD47 ANTIBODY Gholamin, S., Mitra, S., Feroze, A., Zhang, M., Esparza, R., Kahn, S., Richard, C., Achrol, A., Volkmer, A., Liu, J., Volkmer, J., Majeti, R., Weissman, I., Cheshier, S. OXFORD UNIV PRESS INC. 2014: 138
  • Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms. PloS one Lee, J. Y., Hong, W., Majeti, R., Stearns, T. 2014; 9 (3)


    Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.

    View details for DOI 10.1371/journal.pone.0092641

    View details for PubMedID 24658090

  • Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia. Best practice & research. Clinical haematology Majeti, R. n. 2014; 27 (3-4): 229–34


    Massively parallel DNA sequencing has uncovered recurrent mutations in many human cancers. In acute myeloid leukemia (AML), cancer genome/exome resequencing has identified numerous recurrently mutated genes with an average of 5 mutations in each case of de novo AML. In order for these multiple mutations to accumulate in a single lineage of cells, they are serially acquired in clones of self-renewing hematopoietic stem cells (HSC), termed pre-leukemic HSC. Isolation and characterization of pre-leukemic HSC have shown that their mutations are enriched in genes involved in regulating DNA methylation, chromatin modifications, and the cohesin complex. On the other hand, genes involved in regulating activated signaling are generally absent. Pre-leukemic HSC have been found to persist in clinical remission and may ultimately give rise to relapsed disease through the acquisition of novel mutations. Thus, pre-leukemic HSC may constitute a key cellular reservoir that must be eradicated for long-term cures.

    View details for PubMedID 25455271

  • Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms. PloS one Lee, J. Y., Hong, W., Majeti, R., Stearns, T. 2014; 9 (3)

    View details for DOI 10.1371/journal.pone.0092641

    View details for PubMedID 24658090

  • Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma. Nature communications Green, M. R., Vicente-Dueñas, C., Romero-Camarero, I., Long Liu, C., Dai, B., González-Herrero, I., García-Ramírez, I., Alonso-Escudero, E., Iqbal, J., Chan, W. C., Campos-Sanchez, E., Orfao, A., Pintado, B., Flores, T., Blanco, O., Jiménez, R., Martínez-Climent, J. A., Criado, F. J., Cenador, M. B., Zhao, S., Natkunam, Y., Lossos, I. S., Majeti, R., Melnick, A., Cobaleda, C., Alizadeh, A. A., Sánchez-García, I. 2014; 5: 3904-?


    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by 'hit-and-run' oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a 'hit-and-run' role in lymphomagenesis.

    View details for DOI 10.1038/ncomms4904

    View details for PubMedID 24887457

  • Role of DNMT3A, TET2, and IDH1/2 mutations in pre-leukemic stem cells in acute myeloid leukemia INTERNATIONAL JOURNAL OF HEMATOLOGY Chan, S. M., Majeti, R. 2013; 98 (6): 648-657


    Aberrant changes in the epigenome are now recognized to be important in driving the development of multiple human cancers including acute myeloid leukemia. Recent advances in sequencing technologies have led to the identification of recurrent mutations in genes that regulate DNA methylation including DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 2 (TET2), and isocitrate dehydrogenase 1 (IDH1) and IDH2. These mutations have been shown to promote self-renewal and block differentiation of hematopoietic stem/progenitor cells. Acquisition of these mutations in hematopoietic stem cells can lead to their clonal expansion resulting in a pre-leukemic stem cell (pre-LSC) population. Pre-LSCs retain the ability to differentiate into the full spectrum of mature daughter cells but can become fully transformed with the acquisition of additional driver mutations. Here, we review the effects of mutations in DNMT3A, TET2, and IDH1/2 on mouse and human hematopoiesis, the current understanding of their role in pre-LSCs, and therapeutic strategies to eliminate this population which may serve as a cellular reservoir for relapse.

    View details for DOI 10.1007/s12185-013-1407-8

    View details for Web of Science ID 000328481700005

    View details for PubMedID 23949914

  • Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib LEUKEMIA Huang, M., Thomas, D., Li, M. X., Feng, W., Chan, S. M., Majeti, R., Mitchell, B. S. 2013; 27 (10): 1970-1980


    Mutations in exon 12 of the NPM1 gene (NPMc+) define a distinct subset of acute myelogenous leukemias (AML) in which the NPMc+ protein localizes aberrantly to the leukemic cell cytoplasm. We have found that introduction of the most common NPMc+ variant into K562 and 32D cells sensitizes these cells to apoptosis induced by drugs such as bortezomib and arsenic trioxide that induce reactive oxygen species (ROS) formation and that cytotoxicity is prevented in the presence of N-acetyl-1-cysteine, a ROS scavenger. The substitution of tryptophan288 by cysteine occurs in the great majority of NPM1c+ mutations. Mutagenesis of C288 to alanine re-localizes NPMc+ from the cytoplasm to the nucleolus and attenuates the sensitivity of cells expressing this mutation to bortezomib and arsenic trioxide. Primary AML leukemic cells expressing NPMc+ are also significantly more sensitive than other AML cells to apoptosis induced by both drugs at pharmacologically achievable doses. We conclude that the presence of a cysteine moiety at position 288 results in the cytoplasmic localization of NPM1c+ and the increased sensitivity to bortezomib and arsenic trioxide. These data suggest that bortezomib and arsenic trioxide may have increased therapeutic efficacy in NPM1c+ leukemias.Leukemia accepted article preview online, 23 July 2013. doi:10.1038/leu.2013.222.

    View details for DOI 10.1038/leu.2013.222

    View details for PubMedID 23877794

  • Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia LEUKEMIA Craddock, C., Quek, L., Goardon, N., Freeman, S., Siddique, S., Raghavan, M., Aztberger, A., Schuh, A., Grimwade, D., IVEY, A., Virgo, P., Hills, R., McSkeane, T., Arrazi, J., Knapper, S., Brookes, C., Davies, B., Price, A., Wall, K., Griffiths, M., Cavenagh, J., Majeti, R., Weissman, I., Burnett, A., Vyas, P. 2013; 27 (5): 1028-1036


    Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5'-azacitidine and sodium valproate (VAL-AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL-AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

    View details for DOI 10.1038/leu.2012.312

    View details for Web of Science ID 000318698300005

    View details for PubMedID 23223186

  • Clonal evolution of acute leukemia genomes ONCOGENE Jan, M., Majeti, R. 2013; 32 (2): 135-140


    In large part, cancer results from the accumulation of multiple mutations in a single cell lineage that are sequentially acquired and subject to an evolutionary process where selection drives the expansion of more fit subclones. Owing to the technical challenge of distinguishing and isolating distinct cancer subclones, many aspects of this clonal evolution are poorly understood, including the diversity of different subclones in an individual cancer, the nature of the subclones contributing to relapse, and the identity of pre-cancerous mutations. These issues are not just important to our understanding of cancer biology, but are also clinically important given the need to understand the nature of subclones responsible for the refractory and relapsed disease that cause significant morbidity and mortality in patients. Recently, advanced genomic techniques have been used to investigate clonal diversity and evolution in acute leukemia. Studies of pediatric acute lymphoblastic leukemia (ALL) demonstrated that in individual patients there are multiple genetic subclones of leukemia-initiating cells, with a complex clonal architecture. Separate studies also investigating pediatric ALL determined that the clonal basis of relapse was variable and complex, with relapse often evolving from a clone ancestral to the predominant de novo leukemia clone. Additional studies in both ALL and acute myeloid leukemia have identified pre-leukemic mutations in some individual cases. This review will highlight these recent reports investigating the clonal evolution of acute leukemia genomes and discuss the implications for clinical therapy.

    View details for DOI 10.1038/onc.2012.48

    View details for Web of Science ID 000314075500001

    View details for PubMedID 22349821

  • The cancer stem cell model: B cell acute lymphoblastic leukaemia breaks the mould. EMBO molecular medicine McClellan, J. S., Majeti, R. 2013; 5 (1): 7-9

    View details for DOI 10.1002/emmm.201202207

    View details for PubMedID 23229878

    View details for PubMedCentralID PMC3569649

  • Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen BLOOD Ochsenreither, S., Majeti, R., Schmitt, T., Stirewalt, D., Keilholz, U., Loeb, K. R., Wood, B., Choi, Y. E., Bleakley, M., Warren, E. H., Hudecek, M., Akatsuka, Y., Weissman, I. L., Greenberg, P. D. 2012; 119 (23): 5492-5501


    Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1-specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell-based therapy approaches.

    View details for DOI 10.1182/blood-2011-07-365890

    View details for Web of Science ID 000307391400023

    View details for PubMedID 22529286

    View details for PubMedCentralID PMC3369684

  • The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Willingham, S. B., Volkmer, J., Gentles, A. J., Sahoo, D., Dalerba, P., Mitra, S. S., Wang, J., Contreras-Trujillo, H., Martin, R., Cohen, J. D., Lovelace, P., Scheeren, F. A., Chao, M. P., Weiskopf, K., Tang, C., Volkmer, A. K., Naik, T. J., Storm, T. A., Mosley, A. R., Edris, B., Schmid, S. M., Sun, C. K., Chua, M., Murillo, O., Rajendran, P., Cha, A. C., Chin, R. K., Kim, D., Adorno, M., Raveh, T., Tseng, D., Jaiswal, S., Enger, P. O., Steinberg, G. K., Li, G., So, S. K., Majeti, R., Harsh, G. R., van de Rijn, M., Teng, N. N., Sunwoo, J. B., Alizadeh, A. A., Clarke, M. F., Weissman, I. L. 2012; 109 (17): 6662-6667


    CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

    View details for DOI 10.1073/pnas.1121623109

    View details for PubMedID 22451913

  • Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Edris, B., Weiskopf, K., Volkmer, A. K., Volkmer, J., Willingham, S. B., Contreras-Trujillo, H., Liu, J., Majeti, R., West, R. B., Fletcher, J. A., Beck, A. H., Weissman, I. L., van de Rijn, M. 2012; 109 (17): 6656-6661


    Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

    View details for DOI 10.1073/pnas.1121629109

    View details for PubMedID 22451919

  • Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia Jan, M., Snyder, T. M., Corces-Zimmerman, M., Weissman, I. L., Quake, S. R., Majeti, R. AMER ASSOC CANCER RESEARCH. 2012
  • The CD47-SIRP alpha pathway in cancer immune evasion and potential therapeutic implications CURRENT OPINION IN IMMUNOLOGY Chao, M. P., Weissman, I. L., Majeti, R. 2012; 24 (2): 225-232


    Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47-signal-regulatory protein-α pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies.

    View details for DOI 10.1016/j.coi.2012.01.010

    View details for Web of Science ID 000303187600017

    View details for PubMedID 22310103

    View details for PubMedCentralID PMC3319521

  • Programmed cell removal: a new obstacle in the road to developing cancer. Nature reviews. Cancer Chao, M. P., Majeti, R., Weissman, I. L. 2012; 12 (1): 58-67


    The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

    View details for DOI 10.1038/nrc3171

    View details for PubMedID 22158022

  • Programmed cell removal: a new obstacle in the road to developing cancer NATURE REVIEWS CANCER Chao, M. P., Majeti, R., Weissman, I. L. 2012; 12 (1): 58-67


    The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

    View details for DOI 10.1038/nrc3171

    View details for Web of Science ID 000298369300014

  • Treatment advances have not improved the early death rate in acute promyelocytic leukemia HAEMATOLOGICA-THE HEMATOLOGY JOURNAL McClellan, J. S., Kohrt, H. E., Coutre, S., Gotlib, J. R., Majeti, R., Alizadeh, A. A., Medeiros, B. C. 2012; 97 (1): 133-136


    Early mortality in acute promyelocytic leukemia has been reported to occur in less than 10% of patients treated in clinical trials. This study reports the incidence and clinical features of acute promyelocytic leukemia patients treated at Stanford Hospital, CA, USA since March 1997, focusing on early mortality. We show that the risk of early death in acute promyelocytic leukemia patients is higher than previously reported. In a cohort of 70 patients who received induction therapy at Stanford Hospital, 19% and 26% died within seven and 30 days of admission, respectively. High early mortality was not limited to our institution as evaluation of the Surveillance, Epidemiology and End Results Database demonstrated that 30-day mortality for acute promyelocytic leukemia averaged 20% from 1977-2007 and did not improve significantly over this interval. Our findings show that early death is now the greatest contributor to treatment failure in this otherwise highly curable form of leukemia.

    View details for DOI 10.3324/haematol.2011.046490

    View details for Web of Science ID 000299870500022

    View details for PubMedID 21993679

    View details for PubMedCentralID PMC3248942

  • Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy BLOOD Chao, M. P., Tang, C., Pachynski, R. K., Chin, R., Majeti, R., Weissman, I. L. 2011; 118 (18): 4890-4901


    Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRPα and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

    View details for DOI 10.1182/blood-2011-02-338020

    View details for Web of Science ID 000296714500018

    View details for PubMedID 21828138

    View details for PubMedCentralID PMC3208297

  • Surprise! HSC Are Aberrant in Chronic Lymphocytic Leukemia CANCER CELL Alizadeh, A. A., Majeti, R. 2011; 20 (2): 135-136


    In this issue of Cancer Cell, Kikushige et al. report the surprising finding that, in human chronic lymphocytic leukemia (CLL), hematopoietic stem cells (HSC) aberrantly generate clonal B cells with CLL-like phenotypes, which implicate HSC in the pathogenesis of this mature lymphoid malignancy and has major implications for CLL therapies.

    View details for DOI 10.1016/j.ccr.2011.08.001

    View details for Web of Science ID 000294099700002

    View details for PubMedID 21840478

  • Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments BLOOD Gibbs, K. D., Gilbert, P. M., Sachs, K., Zhao, F., Blau, H. M., Weissman, I. L., Nolan, G. P., Majeti, R. 2011; 117 (16): 4226-4233


    The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

    View details for DOI 10.1182/blood-2010-07-298232

    View details for Web of Science ID 000289807600012

    View details for PubMedID 21357764

    View details for PubMedCentralID PMC3087474

  • Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jan, M., Chao, M. P., Cha, A. C., Alizadeh, A. A., Gentles, A. J., Weissman, I. L., Majeti, R. 2011; 108 (12): 5009-5014


    Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

    View details for DOI 10.1073/pnas.1100551108

    View details for Web of Science ID 000288712200061

    View details for PubMedID 21383193

    View details for PubMedCentralID PMC3064328

  • Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells ONCOGENE Majeti, R. 2011; 30 (9): 1009-1019


    Accumulating evidence indicates that many human cancers are organized as a cellular hierarchy initiated and maintained by self-renewing cancer stem cells. This cancer stem cell model has been most conclusively established for human acute myeloid leukemia (AML), although controversies still exist regarding the identity of human AML stem cells (leukemia stem cell (LSC)). A major implication of this model is that, in order to eradicate the cancer and cure the patient, the cancer stem cells must be eliminated. Monoclonal antibodies have emerged as effective targeted therapies for the treatment of a number of human malignancies and, given their target antigen specificity and generally minimal toxicity, are well positioned as cancer stem cell-targeting therapies. One strategy for the development of monoclonal antibodies targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, monoclonal antibodies targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.

    View details for DOI 10.1038/onc.2010.511

    View details for Web of Science ID 000287964100001

    View details for PubMedID 21076471

  • Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia CANCER RESEARCH Chao, M. P., Alizadeh, A. A., Tang, C., Jan, M., Weissman-Tsukamoto, R., Zhao, F., Park, C. Y., Weissman, I. L., Majeti, R. 2011; 71 (4): 1374-1384


    Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.

    View details for DOI 10.1158/0008-5472.CAN-10-2238

    View details for Web of Science ID 000287352600020

    View details for PubMedID 21177380

    View details for PubMedCentralID PMC3041855

  • Human Acute Myelogenous Leukemia Stem Cells Revisited: There's More Than Meets the Eye CANCER CELL Majeti, R., Weissman, I. L. 2011; 19 (1): 9-10


    In this issue of Cancer Cell, Goardon et al. revise earlier conclusions regarding acute myelogenous leukemia (AML) stem cells by demonstrating that in the majority of patients, they reside in two hierarchically related populations most similar to normal hematopoietic progenitors. These findings have implications for therapeutic targeting of these cells.

    View details for DOI 10.1016/j.ccr.2011.01.007

    View details for Web of Science ID 000287290300005

    View details for PubMedID 21251611

    View details for PubMedCentralID PMC3045274

  • Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47 SCIENCE TRANSLATIONAL MEDICINE Chao, M. P., Jaiswal, S., Weissman-Tsukamoto, R., Alizadeh, A. A., Gentles, A. J., Volkmer, J., Weiskopf, K., Willingham, S. B., Raveh, T., Park, C. Y., Majeti, R., Weissman, I. L. 2010; 2 (63)


    Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

    View details for DOI 10.1126/scitranslmed.3001375

    View details for Web of Science ID 000288444900003

    View details for PubMedID 21178137

  • Second-line mitoxantrone, etoposide, and cytarabine for acute myeloid leukemia: A single-center experience AMERICAN JOURNAL OF HEMATOLOGY Kohrt, H. E., Patel, S., Ho, M., Owen, T., Pollyea, D. A., Majeti, R., Gotlib, J., Coutre, S., Liedtke, M., Berube, C., Alizadeh, A. A., Medeiros, B. C. 2010; 85 (11): 877-881


    The majority of patients with acute myeloid leukemia (AML) will require second-line chemotherapy for either relapsed or refractory disease. Currently, only allogeneic hematopoietic cell transplantation (HCT) offers a curative option in this setting and no preferred regimen has been established. The reported efficacy of second-line regimens is widely disparate, thus limiting informed clinical decision making. A retrospective review of 77 patients receiving therapy between 2001 and 2008 with relapsed, 42, and refractory, 35, AML was performed to determine overall response rate and survival following mitoxantrone (8 mg/m(2)/day), etoposide (100 mg/m(2)/day), and cytarabine (1,000 mg/m(2)/day) chemotherapy administered over 5 days. Among 77 patients (median age of 54 years and 64% intermediate risk karyotype) with median follow-up of 153 days, 18% achieved a complete response and 8% a morphologic leukemia-free state. Fifty-seven (74%) experienced treatment failure, 10 of whom achieved a remission after additional therapy. Median overall survival (OS) was 6.8 months. Among patients achieving a response, 50% received consolidation with allogeneic HCT, autologous HCT (5%), or consolidation chemotherapy alone (45%). A nonsignificant trend in overall response (50%, 27%, and 23.8%) and median OS (8.3, 6.8, and 4.7 months) was observed by cytogenetic stratification into favorable, intermediate, and unfavorable risk. Patients with refractory versus relapsed disease had similar overall responses (20% and 31%, P = 0.41) and median OS (5.3 and 7.6 months, P = 0.36). Despite risk stratification by the European Prognostic Index, our series demonstrates inferior rates of response and survival, illustrating the limited activity of this regimen in our cohort.

    View details for DOI 10.1002/ajh.21857

    View details for Web of Science ID 000283568200010

    View details for PubMedID 20872554

  • Metastatic Cancer Stem Cells: An Opportunity for Improving Cancer Treatment? CELL STEM CELL Diehn, M., Majeti, R. 2010; 6 (6): 502-503

    View details for DOI 10.1016/j.stem.2010.05.001

    View details for Web of Science ID 000278840700006

    View details for PubMedID 20569685

  • Macrophages as mediators of tumor immunosurveillance TRENDS IN IMMUNOLOGY Jaiswal, S., Chao, M. P., Majeti, R., Weissman, I. L. 2010; 31 (6): 212-219


    Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

    View details for DOI 10.1016/

    View details for Web of Science ID 000279427000002

    View details for PubMedID 20452821

    View details for PubMedCentralID PMC3646798

  • Immunophenotypic features of acute myeloid leukemia with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) LEUKEMIA RESEARCH Medeiros, B. C., Kohrt, H. E., Arber, D. A., Bangs, C. D., Cherry, A. M., Majeti, R., Kogel, K. E., Azar, C. A., Patel, S., Alizadeh, A. A. 2010; 34 (5): 594-597


    Immunophenotypic identification of myeloid specific antigens is an important diagnostic tool in the management of patients with acute myeloid leukemia (AML). These antigens allow determination of cell of origin and degree of differentiation of leukemia blasts. AML with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is a relatively rare subtype of AML. The immunophenotypic characteristics of inv(3) AML patients are somewhat limited. We identified 14 new cases of hematological disorders with increased myeloid blasts carrying inv(3)(q21q26.2)/t(3;3)(q21;q26.2). Also, we identified another 13 cases previously published in the literature, where the immunophenotype of inv(3)(q21q26.2) was documented. As a group, patients with AML with inv(3)(q21q26.2) had high levels of early myeloid (CD13, CD33, CD117 and MPO) and uncommitted markers (CD34, HLA-DR and CD56) and a high rate of monosomy 7 in addition to the inv(3)(q21q26.2). Differential karyotype and expression of certain antigens were noted in patients with de novo AML with inv(3)(q21q26.2) vs. those with inv(3)(q21q26.2)-containing blasts.

    View details for DOI 10.1016/j.leukres.2009.08.029

    View details for Web of Science ID 000276945300009

    View details for PubMedID 19781775

  • Early Mortality in Acute Promyelocytic Leukemia May Be Higher Than Previously Reported. 51st Annual Meeting and Exposition of the American-Society-of-Hematology Alizadeh, A. A., McClellan, J. S., Gotlib, J. R., Coutre, S., Majeti, R., Kohrt, H. E., Medeiros, B. C. AMER SOC HEMATOLOGY. 2009: 420–21
  • Is Time of the Essence in Adult Acute Myeloid Leukemia (AML)? Time to Blast Clearance and Time to Induction Therapy Fail to Predict Overall Survival (OS). 51st Annual Meeting and Exposition of the American-Society-of-Hematology Kohrt, H. E., Patel, S., Ho, M., Owen, T., Majeti, R., Gotlib, J. R., Coutre, S., Medeiros, B. C., Alizadeh, A. A. AMER SOC HEMATOLOGY. 2009: 646–47
  • CD47 Is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis CELL Jaiswal, S., Jamieson, C. H., Pang, W. W., Park, C. Y., Chao, M. P., Majeti, R., Traver, D., van Rooijen, N., Weissman, I. L. 2009; 138 (2): 271-285


    Macrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPalpha, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

    View details for DOI 10.1016/j.cell.2009.05.046

    View details for Web of Science ID 000268277000010

    View details for PubMedID 19632178

    View details for PubMedCentralID PMC2775564

  • Dysregulated gene expression networks in human acute myelogenous leukemia stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Majetl, R., Becker, M. W., Tian, Q., Lee, T. M., Yan, X., Liu, R., Chiang, J., Hood, L., Clarke, M. F., Weissman, I. L. 2009; 106 (9): 3396-3401


    We performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, we identified 3,005 differentially expressed genes. Using 2 independent analyses, we identified multiple pathways that are aberrantly regulated in leukemic stem cells compared with normal HSC. Several pathways, including Wnt signaling, MAP Kinase signaling, and Adherens Junction, are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions, including Ribosome and T Cell Receptor Signaling pathway. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations, can yield an understanding of the critical pathways regulating cancer stem cells.

    View details for DOI 10.1073/pnas.0900089106

    View details for PubMedID 19218430

  • The Adhesion Molecule Esam1 Is a Novel Hematopoietic Stem Cell Marker STEM CELLS Ooi, A. G., Karsunky, H., Majeti, R., Butz, S., Vestweber, D., Ishida, T., Quertermous, T., Weissman, I. L., Forsberg, E. C. 2009; 27 (3): 653-661


    Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

    View details for DOI 10.1634/stemcells.2008-0824

    View details for PubMedID 19074415

  • In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood NATURE PROTOCOLS Park, C. Y., Majeti, R., Weissman, I. L. 2008; 3 (12): 1932-1940


    Establishment of robust xenograft models is critical to studying human hematopoiesis in a physiologic setting. Using a recently developed immunodeficient mouse strain, we have established long-term multilineage human grafts and demonstrated their serially transplantability using limited numbers of purified human hematopoietic stem cells (HSCs). Herein, we describe our protocol for the isolation of human HSC (Lin-CD34+CD38-CD90+) from umbilical cord blood (CB) as well as the xenotransplantation system that allows stable engraftment of human hematopoietic cells with as few as ten HSCs. Isolation of CB mononuclear cells requires 2-3 h, and cells may be cryopreserved before transplantation. Isolation of HSC requires approximately 2-3 h, and transplantation requires 1 h. Short-term and long-term engraftment is assessed 4-6 weeks and 10-12 weeks post-transplantation, respectively, with preparation and analysis time requiring 4-8 h at each time point.

    View details for DOI 10.1038/nprot.2008.194

    View details for Web of Science ID 000265781700012

    View details for PubMedID 19180077