Dr. Ravi Majeti is an Associate Professor in the Department of Medicine, Division of Hematology, and Institute for Stem Cell Biology and Regenerative Medicine at Stanford University. 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 early human blood stem cell development and characterized acute myeloid leukemia (AML) stem cells. Dr. Majeti established his independent laboratory in 2009 with research focused on the molecular/genomic characterization and therapeutic targeting of leukemia stem cells in human hematologic malignancies, particularly acute myeloid leukemia (AML). Dr. Majeti is a recipient of the Burroughs Wellcome Fund Career Award for Medical Scientists and is a New York Stem Cell Foundation Robertson Investigator.
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 - Stanford Internal Medicine Residency (2013 - Present)
MSTP Admissions Committee, Stanford School of Medicine (2010 - Present)
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)
Residency:Brigham and Women's Hospital Harvard Medical School (2004) MA
Board Certification: Hematology, American Board of Internal Medicine (2007)
Fellowship:Stanford University Medical Center (2008) CA
Medical Education:University of California San Francisco (2002) 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.
Independent Studies (18)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Graduate and Fellowship Programs
A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells
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 Web of Science ID 000379366900021
View details for PubMedID 27213817
Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation.
Cell stem cell
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
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
Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia.
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
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 Web of Science ID 000350810200020
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
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 Web of Science ID 000331396500037
View details for PubMedID 24550281
Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia
SCIENCE TRANSLATIONAL MEDICINE
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
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 Web of Science ID 000285518000015
View details for PubMedID 21177505
Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma
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 Web of Science ID 000281523200014
View details for PubMedID 20813259
CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells
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 Web of Science ID 000268277000011
View details for PubMedID 19632179
Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood
CELL STEM CELL
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
Burning Fat Fuels Leukemic Stem Cell Heterogeneity.
Cell stem cell
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.
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
CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer
JOURNAL OF CLINICAL INVESTIGATION
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
Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo
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
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.
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
- An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis NATURE COMMUNICATIONS 2015; 6
- Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential PLOS ONE 2015; 10 (9)
- A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells MABS 2015; 7 (5): 946-956
Biology and Clinical Relevance of Acute Myeloid Leukemia Stem Cells
SEMINARS IN HEMATOLOGY
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 Web of Science ID 000357433800002
Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands
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 Web of Science ID 000351951800018
Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation
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 Web of Science ID 000350810200014
- Refractory warm IgM-mediated autoimmune hemolytic anemia associated with Churg-Strauss syndrome responsive to eculizumab and rituximab AMERICAN JOURNAL OF HEMATOLOGY 2015; 90 (1): 78-81
A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells.
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-leukemic evolution of hematopoietic stem cells: the importance of early mutations in leukemogenesis
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
- Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY 2014; 27 (3-4): 229-234
Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells
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 Web of Science ID 000342619600021
View details for PubMedID 24850755
- Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma NATURE COMMUNICATIONS 2014; 5
- Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms PLOS ONE 2014; 9 (3)
Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms.
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
Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma.
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
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
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 Web of Science ID 000325642600003
Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia
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
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
- The cancer stem cell model: B cell acute lymphoblastic leukaemia breaks the mould. EMBO molecular medicine 2013; 5 (1): 7-9
Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen
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
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
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 SIRP?, 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 Web of Science ID 000303249100065
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
2012; 109 (17): 6656-6661
Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-?, 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 Web of Science ID 000303249100064
View details for PubMedID 22451919
The CD47-SIRP alpha pathway in cancer immune evasion and potential therapeutic implications
CURRENT OPINION IN IMMUNOLOGY
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-? 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
Programmed cell removal: a new obstacle in the road to developing cancer
NATURE REVIEWS CANCER
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
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
Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy
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 SIRP? 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
Surprise! HSC Are Aberrant in Chronic Lymphocytic Leukemia
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
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
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
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/IL2R?-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
Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells
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
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
Human Acute Myelogenous Leukemia Stem Cells Revisited: There's More Than Meets the Eye
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
Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47
SCIENCE TRANSLATIONAL MEDICINE
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
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 2010; 6 (6): 502-503
Macrophages as mediators of tumor immunosurveillance
TRENDS IN IMMUNOLOGY
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/j.it.2010.04.001
View details for Web of Science ID 000279427000002
View details for PubMedID 20452821
Immunophenotypic features of acute myeloid leukemia with inv(3)(q21q26.2)/t(3;3)(q21;q26.2)
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.
AMER SOC HEMATOLOGY. 2009: 420-421
View details for Web of Science ID 000272725801195
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).
AMER SOC HEMATOLOGY. 2009: 646-647
View details for Web of Science ID 000272725801797
CD47 Is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis
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
Dysregulated gene expression networks in human acute myelogenous leukemia stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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 Web of Science ID 000263844100075
View details for PubMedID 19218430
The Adhesion Molecule Esam1 Is a Novel Hematopoietic Stem Cell Marker
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 Web of Science ID 000264706900016
View details for PubMedID 19074415
In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood
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