Ann Mullally

All Publications

• Antibody targeting of mutant calreticulin in myeloproliferative neoplasms. Journal of cellular and molecular medicine Kramer, F., Mullally, A. 2023; 28 (5): e17896

  Abstract

  Mutations in calreticulin are one of the key disease-initiating mutations in myeloproliferative neoplasms (MPN). In MPN, mutant calreticulin translates with a novel C-terminus that leads to aberrant binding to the extracellular domain of the thrombopoietin receptor, MPL. This cell surface neoantigen has become an attractive target for immunological intervention. Here, we summarize recent advances in the development of mutant calreticulin targeting antibodies as a novel therapeutic approach in MPN.

  View details for DOI 10.1111/jcmm.17896

  View details for PubMedID 37551061

  View details for PubMedCentralID PMC10902560

• Biology and therapeutic targeting of molecular mechanisms in MPNs. Blood How, J., Garcia, J. S., Mullally, A. 2023; 141 (16): 1922-1933

  Abstract

  Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription signaling. As a result, JAK inhibitors have been the standard therapy for treatment of patients with myelofibrosis (MF). Although currently approved JAK inhibitors successfully ameliorate MPN-related symptoms, they are not known to substantially alter the MF disease course. Similarly, in essential thrombocythemia and polycythemia vera, treatments are primarily aimed at reducing the risk of cardiovascular and thromboembolic complications, with a watchful waiting approach often used in patients who are considered to be at a lower risk for thrombosis. However, better understanding of MPN biology has led to the development of rationally designed therapies, with the goal of not only addressing disease complications but also potentially modifying disease course. We review the most recent data elucidating mechanisms of disease pathogenesis and highlight emerging therapies that target MPN on several biologic levels, including JAK2-mutant MPN stem cells, JAK and non-JAK signaling pathways, mutant calreticulin, and the inflammatory bone marrow microenvironment.

  View details for DOI 10.1182/blood.2022017416

  View details for PubMedID 36534936

  View details for PubMedCentralID PMC10163317

• CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response. Leukemia Jutzi, J. S., Marneth, A. E., Jiménez-Santos, M. J., Hem, J., Guerra-Moreno, A., Rolles, B., Bhatt, S., Myers, S. A., Carr, S. A., Hong, Y., Pozdnyakova, O., van Galen, P., Al-Shahrour, F., Nam, A. S., Mullally, A. 2023; 37 (2): 359-369

  Abstract

  Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

  View details for DOI 10.1038/s41375-022-01781-0

  View details for PubMedID 36473980

• Two to tango! IL-13 and TGF-β drive myelofibrosis. Blood Jutzi, J. S., Mullally, A. 2022; 140 (26): 2767-2768

  View details for DOI 10.1182/blood.2022018859

  View details for PubMedID 36580345

  View details for PubMedCentralID PMC9837429

• Molecular Pathogenesis of Myeloproliferative Neoplasms. Current hematologic malignancy reports Rolles, B., Mullally, A. 2022; 17 (6): 319-329

  Abstract

  Myeloproliferative neoplasms (MPNs) are chronic hematological malignancies characterized by increased proliferation of MPN stem and myeloid progenitor cells with or without bone marrow fibrosis that typically lead to increased peripheral blood cell counts. The genetic and cytogenetic alterations that initiate and drive the development of MPNs have largely been defined, and we summarize these here.In recent years, advances in understanding the pathogenesis of MPNs have defined a long-preclinical phase in JAK2-mutant MPN, identified genetic loci associated with MPN predisposition and uncovered mechanistic insights in CALR-mutant MPN. The integration of molecular genetics into prognostic risk models is well-established in myelofibrosis and ongoing studies are interrogating the prognostic implications of concomitant mutations in ET and PV. Despite all these advances, the field is deficient in clonally selective therapies to effectively target the MPN clone at any stage of disease, from pre-clinical to advanced. Although the biological understanding of the pathogenesis of MPNs has progressed quickly, substantial knowledge gaps remain, including in the molecular mechanisms underlying MPN progression and myelofibrotic transformation. An ongoing goal for the MPN field is to translate advances in biological understanding to improved treatments for patients.

  View details for DOI 10.1007/s11899-022-00685-1

  View details for PubMedID 36336766

  View details for PubMedCentralID 2909585

• Mechanical checkpoint regulates monocyte differentiation in fibrotic niches. Nature materials Vining, K. H., Marneth, A. E., Adu-Berchie, K., Grolman, J. M., Tringides, C. M., Liu, Y., Wong, W. J., Pozdnyakova, O., Severgnini, M., Stafford, A., Duda, G. N., Hodi, F. S., Mullally, A., Wucherpfennig, K. W., Mooney, D. J. 2022; 21 (8): 939-950

  Abstract

  Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

  View details for DOI 10.1038/s41563-022-01293-3

  View details for PubMedID 35817965

  View details for PubMedCentralID PMC10197159

• Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPN. Blood Jutzi, J. S., Marneth, A. E., Ciboddo, M., Guerra-Moreno, A., Jiménez-Santos, M. J., Kosmidou, A., Dressman, J. W., Liang, H., Hamel, R. S., Lozano, P. R., Rumi, E., Doench, J., Gotlib, J. R., Krishnan, A., Elf, S., Al-Shahrour, F., Mullally, A. 2022

  Abstract

  Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and normalization of key MPN disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow, as compared to bone marrow-derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPN.

  View details for DOI 10.1182/blood.2022015629

  View details for PubMedID 35763665

• Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine. Science translational medicine Gigoux, M., Holmström, M. O., Zappasodi, R., Park, J. J., Pourpe, S., Bozkus, C. C., Mangarin, L. M., Redmond, D., Verma, S., Schad, S., George, M. M., Venkatesh, D., Ghosh, A., Hoyos, D., Molvi, Z., Kamaz, B., Marneth, A. E., Duke, W., Leventhal, M. J., Jan, M., Ho, V. T., Hobbs, G. S., Knudsen, T. A., Skov, V., Kjær, L., Larsen, T. S., Hansen, D. L., Lindsley, R. C., Hasselbalch, H., Grauslund, J. H., Lisle, T. L., Met, Ö., Wilkinson, P., Greenbaum, B., Sepulveda, M. A., Chan, T., Rampal, R., Andersen, M. H., Abdel-Wahab, O., Bhardwaj, N., Wolchok, J. D., Mullally, A., Merghoub, T. 2022; 14 (649): eaba4380

  Abstract

  The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

  View details for DOI 10.1126/scitranslmed.aba4380

  View details for PubMedID 35704596

  View details for PubMedCentralID PMC11182673

• Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses. Blood advances Knudsen, T. A., Skov, V., Stevenson, K., Werner, L., Duke, W., Laurore, C., Gibson, C. J., Nag, A., Thorner, A. R., Wollison, B., Hansen, D. L., Ellervik, C., El Fassi, D., de Stricker, K., Ocias, L. F., Brabrand, M., Bjerrum, O. W., Overgaard, U. M., Frederiksen, M., Kristensen, T. K., Kruse, T. A., Thomassen, M., Mourits-Andersen, T., Severinsen, M. T., Stentoft, J., Starklint, J., Neuberg, D. S., Kjaer, L., Larsen, T. S., Hasselbalch, H. C., Lindsley, R. C., Mullally, A. 2022; 6 (7): 2107-2119

  Abstract

  Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα-treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

  View details for DOI 10.1182/bloodadvances.2021004856

  View details for PubMedID 34507355

  View details for PubMedCentralID PMC9006286

• Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms. Experimental hematology Van Egeren, D., Kamaz, B., Liu, S., Nguyen, M., Reilly, C. R., Kalyva, M., DeAngelo, D. J., Galinsky, I., Wadleigh, M., Winer, E. S., Luskin, M. R., Stone, R. M., Garcia, J. S., Hobbs, G. S., Michor, F., Cortes-Ciriano, I., Mullally, A., Hormoz, S. 2022; 107: 14-19

  Abstract

  The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

  View details for DOI 10.1016/j.exphem.2021.12.364

  View details for PubMedID 34921959

  View details for PubMedCentralID PMC9332124

• Suppression of multiple anti-apoptotic BCL2 family proteins recapitulates the effects of JAK2 inhibitors in JAK2V617F driven myeloproliferative neoplasms. Cancer science Takei, H., Coelho-Silva, J. L., Tavares Leal, C., Queiroz Arantes Rocha, A., Mantello Bianco, T., Welner, R. S., Mishima, Y., Kobayashi, I. S., Mullally, A., Lima, K., Machado-Neto, J. A., Kobayashi, S. S., Lobo de Figueiredo-Pontes, L. 2022; 113 (2): 597-608

  Abstract

  Several lines of research suggest that Bcl-xL-mediated anti-apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock-in JAK2V617F mouse model and confirmed that Bcl-xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)-induced phenotype in the peripheral blood by conditional knock-in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. Mx1-Cre Jak2V617W/VF /Bcl2l1f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro-apoptotic stimuli in terminally differentiated erythroid progenitors. The pan-BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav-Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid-erythroid precursors. Therefore, disrupting the BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.

  View details for DOI 10.1111/cas.15210

  View details for PubMedID 34808021

  View details for PubMedCentralID PMC8819353

• Hydroxycarbamide effects on DNA methylation and gene expression in myeloproliferative neoplasms. Genome research Contreras Castillo, S., Montibus, B., Rocha, A., Duke, W., von Meyenn, F., McLornan, D., Harrison, C., Mullally, A., Schulz, R., Oakey, R. J. 2021; 31 (8): 1381-1394

  Abstract

  Hydroxycarbamide (HC, hydroxyurea) is a cytoreductive drug inducing cell cycle blockade. However, emerging evidence suggests that HC plays a role in the modulation of transcription through the activity of transcription factors and DNA methylation. Examining the global mechanism of action of HC in the context of myeloproliferative neoplasms (MPNs), for which HC is the first-line treatment, will provide a better understanding of its molecular effects. To explore the effects of HC genome-wide, transcriptomic analyses were performed on two clinically relevant cell types at different stages of differentiation treated with HC in a murine MPN model. This study was replicated in MPN patients by profiling genome-wide gene expression and DNA methylation using patient blood samples collected longitudinally, before and following HC exposure. The effects of HC on the transcriptome were not only associated with cell cycle interruption but also with hematopoietic functions. Moreover, a group of genes were restored to normal expression levels in murine hematopoietic stem cells (HSCs) following drug treatment, including the master regulator of hematopoiesis, RUNX1 In humans, HC significantly modifies DNA methylation levels in HSCs at several distal regulatory regions, which we show to be associated with SPI1 binding sites and at the SPI1 locus itself. We have identified novel targets of HC that include pivotal transcription factors involved in hematopoiesis, and for the first time we report abnormal methylation patterns in MPN patients at the master regulator gene SPI1 and its distal binding sites, which HC is able to restore to normal levels.

  View details for DOI 10.1101/gr.270066.120

  View details for PubMedID 34244229

  View details for PubMedCentralID PMC8327916

• Zinc-dependent multimerization of mutant calreticulin is required for MPL binding and MPN pathogenesis. Blood advances Rivera, J. F., Baral, A. J., Nadat, F., Boyd, G., Smyth, R., Patel, H., Burman, E. L., Alameer, G., Boxall, S. A., Jackson, B. R., Baxter, E. J., Laslo, P., Green, A. R., Kent, D. G., Mullally, A., Chen, E. 2021; 5 (7): 1922-1932

  Abstract

  Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.

  View details for DOI 10.1182/bloodadvances.2020002402

  View details for PubMedID 33821991

  View details for PubMedCentralID PMC8045488

• Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Cancer Cells in Myeloproliferative Neoplasms. Cell stem cell Van Egeren, D., Escabi, J., Nguyen, M., Liu, S., Reilly, C. R., Patel, S., Kamaz, B., Kalyva, M., DeAngelo, D. J., Galinsky, I., Wadleigh, M., Winer, E. S., Luskin, M. R., Stone, R. M., Garcia, J. S., Hobbs, G. S., Camargo, F. D., Michor, F., Mullally, A., Cortes-Ciriano, I., Hormoz, S. 2021; 28 (3): 514-523.e9

  Abstract

  Some cancers originate from a single mutation event in a single cell. Blood cancers known as myeloproliferative neoplasms (MPNs) are thought to originate when a driver mutation is acquired by a hematopoietic stem cell (HSC). However, when the mutation first occurs in individuals and how it affects the behavior of HSCs in their native context is not known. Here we quantified the effect of the JAK2-V617F mutation on the self-renewal and differentiation dynamics of HSCs in treatment-naive individuals with MPNs and reconstructed lineage histories of individual HSCs using somatic mutation patterns. We found that JAK2-V617F mutations occurred in a single HSC several decades before MPN diagnosis-at age 9 ± 2 years in a 34-year-old individual and at age 19 ± 3 years in a 63-year-old individual-and found that mutant HSCs have a selective advantage in both individuals. These results highlight the potential of harnessing somatic mutations to reconstruct cancer lineages.

  View details for DOI 10.1016/j.stem.2021.02.001

  View details for PubMedID 33621486

  View details for PubMedCentralID PMC7939520

• COVID-19 and myeloproliferative neoplasms: some considerations. Leukemia Kamaz, B., Mullally, A. 2021; 35 (1): 279-281

  View details for DOI 10.1038/s41375-020-01070-8

  View details for PubMedID 33106624

  View details for PubMedCentralID PMC7586362

• Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms. Nature Jayavelu, A. K., Schnöder, T. M., Perner, F., Herzog, C., Meiler, A., Krishnamoorthy, G., Huber, N., Mohr, J., Edelmann-Stephan, B., Austin, R., Brandt, S., Palandri, F., Schröder, N., Isermann, B., Edlich, F., Sinha, A. U., Ungelenk, M., Hübner, C. A., Zeiser, R., Rahmig, S., Waskow, C., Coldham, I., Ernst, T., Hochhaus, A., Jilg, S., Jost, P. J., Mullally, A., Bullinger, L., Mertens, P. R., Lane, S. W., Mann, M., Heidel, F. H. 2020; 588 (7836): 157-163

  Abstract

  Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.

  View details for DOI 10.1038/s41586-020-2968-3

  View details for PubMedID 33239784

  View details for PubMedCentralID 4290021

• Pregnancy outcomes, risk factors, and cell count trends in pregnant women with essential thrombocythemia. Leukemia research How, J., Leiva, O., Bogue, T., Fell, G. G., Bustoros, M. W., Connell, N. T., Connors, J. M., Ghobrial, I. M., Kuter, D. J., Mullally, A., Neuberg, D., Zwicker, J. I., Fogerty, A. E., Hobbs, G. S. 2020; 98: 106459

  Abstract

  Pregnancy in essential thrombocythemia (ET) is associated with increased risk of obstetric complications. We retrospectively evaluated risk factors in 121 pregnancies in 52 ET women seen at 3 affiliate hospitals. Univariable and multivariable analyses were performed at the α = 0.10 level. Cell counts were characterized throughout pregnancy and correlated with outcomes using logistic modeling. The overall live birth rate was 69 %. 48.7 % of all women experienced a pregnancy complication, the most common being spontaneous abortion, which occurred in 26 % of all pregnancies. Maternal thrombosis and hemorrhage rates were 2.5 % and 5.8 %. On multivariable analysis, aspirin use (OR 0.29, p = 0.014, 90 % CI 0.118-0.658) and history of prior pregnancy loss (OR 3.86, p = 0.011, CI 1.49-9.15) were associated with decreased and increased pregnancy complications, respectively. A Markov model was used to analyze the probability of a future pregnancy complication based on initial pregnancy outcome. An ET woman who suffers a pregnancy complication has a 0.594 probability of a subsequent pregnancy complication, compared to a 0.367 probability if she didn't suffer a complication. However, despite this elevated risk, overall prognosis is good, with a >50 % probability of a successful pregnancy by the third attempt. Platelet counts decreased by 43 % in ET during pregnancy, with nadir at delivery and prompt recovery in the postpartum period. Women with larger declines in gestational platelet counts were less likely to suffer complications (p = 0.083). Our study provides important guidance to physicians treating ET women during pregnancy, including counseling information regarding risk assessment and expected trajectory of platelet levels.

  View details for DOI 10.1016/j.leukres.2020.106459

  View details for PubMedID 33022566

• Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis. Blood advances Ng, J., Guo, F., Marneth, A. E., Ghanta, S., Kwon, M. Y., Keegan, J., Liu, X., Wright, K. T., Kamaz, B., Cahill, L. A., Mullally, A., Perrella, M. A., Lederer, J. A. 2020; 4 (19): 4965-4979

  Abstract

  Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin-Sca1+C-kit+CD150-CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

  View details for DOI 10.1182/bloodadvances.2020002556

  View details for PubMedID 33049055

  View details for PubMedCentralID PMC7556132

• Remodeling the Bone Marrow Microenvironment - A Proposal for Targeting Pro-inflammatory Contributors in MPN. Frontiers in immunology Jutzi, J. S., Mullally, A. 2020; 11: 2093

  Abstract

  Philadelphia-negative myeloproliferative neoplasms (MPN) are malignant bone marrow (BM) disorders, typically arising from a single somatically mutated hematopoietic stem cell. The most commonly mutated genes, JAK2, CALR, and MPL lead to constitutively active JAK-STAT signaling. Common clinical features include myeloproliferation, splenomegaly and constitutional symptoms. This review covers the contributions of cellular components of MPN pathology (e.g., monocytes, megakaryocytes, and mesenchymal stromal cells) as well as cytokines and soluble mediators to the development of myelofibrosis (MF) and highlights recent therapeutic advances. These findings outline the importance of malignant and non-malignant BM constituents to the pathogenesis and treatment of MF.

  View details for DOI 10.3389/fimmu.2020.02093

  View details for PubMedID 32983162

  View details for PubMedCentralID PMC7489333

• Murine Models of Myelofibrosis. Cancers Jacquelin, S., Kramer, F., Mullally, A., Lane, S. W. 2020; 12 (9)

  Abstract

  Myelofibrosis (MF) is subtype of myeloproliferative neoplasm (MPN) characterized by a relatively poor prognosis in patients. Understanding the factors that drive MF pathogenesis is crucial to identifying novel therapeutic approaches with the potential to improve patient care. Driver mutations in three main genes (janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL)) are recurrently mutated in MPN and are sufficient to engender MPN using animal models. Interestingly, animal studies have shown that the underlying molecular mutation and the acquisition of additional genetic lesions is associated with MF outcome and transition from early stage MPN such as essential thrombocythemia (ET) and polycythemia vera (PV) to secondary MF. In this issue, we review murine models that have contributed to a better characterization of MF pathobiology and identification of new therapeutic opportunities in MPN.

  View details for DOI 10.3390/cancers12092381

  View details for PubMedID 32842500

  View details for PubMedCentralID PMC7563264

• Busy signal: platelet-derived growth factor activation in myelofibrosis. Haematologica Marneth, A. E., Mullally, A. 2020; 105 (8): 1988-1990

  View details for DOI 10.3324/haematol.2020.253708

  View details for PubMedID 32739885

  View details for PubMedCentralID PMC7395275

• Fedratinib in myelofibrosis. Blood advances Mullally, A., Hood, J., Harrison, C., Mesa, R. 2020; 4 (8): 1792-1800

  Abstract

  Following the discovery of the JAK2V617F mutation in myeloproliferative neoplasms in 2005, fedratinib was developed as a small molecular inhibitor of JAK2. It was optimized to yield low-nanomolar activity against JAK2 (50% inhibitory concentration = 3 nM) and was identified to be selective for JAK2 relative to other JAK family members (eg, JAK1, JAK3, and TYK2). It quickly moved into clinical development with a phase 1 clinical trial opening in 2008, where a favorable impact on spleen and myelofibrosis (MF) symptom responses was reported. A phase 3 trial in JAK2 inhibitor treatment-naive MF patients followed in 2011 (JAKARTA); a phase 2 trial in MF patients resistant or intolerant to ruxolitinib followed in 2012 (JAKARTA-2). Clinical development suffered a major setback between 2013 and 2017 when the US Food and Drug Administration (FDA) placed fedratinib on clinical hold due to the development of symptoms concerning for Wernicke encephalopathy (WE) in 8 of 608 subjects (1.3%) who had received the drug. It was ultimately concluded that there was no evidence that fedratinib directly induces WE, but clear risk factors (eg, poor nutrition, uncontrolled gastrointestinal toxicity) were identified. In August 2019, the FDA approved fedratinib for the treatment of adults with intermediate-2 or high-risk MF. Notably, approval includes a "black box warning" on the risk of serious and fatal encephalopathy, including WE. FDA approval was granted on the basis of the JAKARTA studies in which the primary end points (ie, spleen and MF symptom responses) were met in ∼35% to 40% of patients (JAKARTA) and 25% to 30% of patients (JAKARTA-2), respectively.

  View details for DOI 10.1182/bloodadvances.2019000954

  View details for PubMedID 32343799

  View details for PubMedCentralID PMC7189288

• Distinct effects of ruxolitinib and interferon-alpha on murine JAK2V617F myeloproliferative neoplasm hematopoietic stem cell populations. Leukemia Austin, R. J., Straube, J., Bruedigam, C., Pali, G., Jacquelin, S., Vu, T., Green, J., Gräsel, J., Lansink, L., Cooper, L., Lee, S. J., Chen, N. T., Lee, C. W., Haque, A., Heidel, F. H., D'Andrea, R., Hill, G. R., Mullally, A., Milsom, M. D., Bywater, M., Lane, S. W. 2020; 34 (4): 1075-1089

  Abstract

  JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population. Whereas ruxolitinib inhibits Stat5 activation in erythroid progenitor populations, it fails to inhibit this same pathway in HSCs. In contrast, IFN-α has direct effects on HSCs. Furthermore, STAT1 phosphorylation and pathway activation is greater after IFN-α stimulation in Jak2V617F murine HSCs with increased induction of reactive oxygen species, DNA damage and reduction in quiescence after chronic IFN-α treatment. Interestingly, ruxolitinib does not block IFN-α induced reactive oxygen species and DNA damage in Jak2V617F murine HSCs in vivo. This work provides a mechanistic rationale informing how pegylated IFN-α reduces JAK2V617F allelic burden in the clinical setting and may inform future clinical efforts to combine ruxolitinib with pegylated IFN-α in patients with MPN.

  View details for DOI 10.1038/s41375-019-0638-y

  View details for PubMedID 31732720

  View details for PubMedCentralID PMC9221569

• The Molecular Genetics of Myeloproliferative Neoplasms. Cold Spring Harbor perspectives in medicine Marneth, A. E., Mullally, A. 2020; 10 (2)

  Abstract

  Activated JAK-STAT signaling is central to the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) and occurs as a result of MPN phenotypic driver mutations in JAK2, CALR, or MPL The spectrum of concomitant somatic mutations in other genes has now largely been defined in MPNs. With the integration of targeted next-generation sequencing (NGS) panels into clinical practice, the clinical significance of concomitant mutations in MPNs has become clearer. In this review, we describe the consequences of concomitant mutations in the most frequently mutated classes of genes in MPNs: (1) DNA methylation pathways, (2) chromatin modification, (3) RNA splicing, (4) signaling pathways, (5) transcription factors, and (6) DNA damage response/stress signaling. The increased use of molecular genetics for early risk stratification of patients brings the possibility of earlier intervention to prevent disease progression in MPNs. However, additional studies are required to decipher underlying molecular mechanisms and effectively target them.

  View details for DOI 10.1101/cshperspect.a034876

  View details for PubMedID 31548225

  View details for PubMedCentralID PMC6996444

• Both sides now: losses and gains of mutant CALR. Blood Mullally, A. 2020; 135 (2): 82-83

  Abstract

  In this issue of Blood, Di Buduo et al report on the consequences of loss of the interaction between mutant calreticulin (CALR) and endoplasmic reticulum (ER) resident protein 57 (ERp57) as it pertains to altered calcium signaling in myeloproliferative neoplasms (MPNs).

  View details for DOI 10.1182/blood.2019003820

  View details for PubMedID 31917838

  View details for PubMedCentralID PMC6952827

• Mutant calreticulin in myeloproliferative neoplasms. Blood How, J., Hobbs, G. S., Mullally, A. 2019; 134 (25): 2242-2248

  Abstract

  Recurrent mutations in calreticulin are present in ∼20% of patients with myeloproliferative neoplasms (MPNs). Since its discovery in 2013, we now have a more precise understanding of how mutant CALR, an endoplasmic reticulum chaperone protein, activates the JAK/STAT signaling pathway via a pathogenic binding interaction with the thrombopoietin receptor MPL to induce MPNs. In this Spotlight article, we review the current understanding of the biology underpinning mutant CALR-driven MPNs, discuss clinical implications, and highlight future therapeutic approaches.

  View details for DOI 10.1182/blood.2019000622

  View details for PubMedID 31562135

  View details for PubMedCentralID PMC6923668

• The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy. Blood advances Liu, S., Marneth, A. E., Alexe, G., Walker, S. R., Gandler, H. I., Ye, D. Q., Labella, K., Mathur, R., Toniolo, P. A., Tillgren, M., Gokhale, P. C., Barbie, D., Mullally, A., Stegmaier, K., Frank, D. A. 2018; 2 (23): 3428-3442

  Abstract

  To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

  View details for DOI 10.1182/bloodadvances.2018016733

  View details for PubMedID 30504235

  View details for PubMedCentralID PMC6290107

• JAK2 (and other genes) be nimble with MPN diagnosis, prognosis, and therapy. Hematology. American Society of Hematology. Education Program Ciboddo, M., Mullally, A. 2018; 2018 (1): 110-117

  Abstract

  Now that the spectrum of somatic mutations that initiate, propagate, and drive the progression of myeloproliferative neoplasms (MPNs) has largely been defined, recent efforts have focused on integrating this information into clinical decision making. In this regard, the greatest progress has been made in myelofibrosis, in which high-molecular-risk mutations have been identified and incorporated into prognostic models to help guide treatment decisions. In this chapter, we focus on advances in 4 main areas: (1) What are the MPN phenotypic driver mutations? (2) What constitutes high molecular risk in MPN (focusing on ASXL1)? (3) How do we risk-stratify patients with MPN? And (4) What is the significance of molecular genetics for MPN treatment? Although substantial progress has been made, we still have an incomplete understanding of the molecular basis for phenotypic diversity in MPN, and few rationally designed therapeutic approaches to target high-risk mutations are available. Ongoing research efforts in these areas are critical to understanding the biological consequences of genetic heterogeneity in MPN and to improving outcomes for patients.

  View details for DOI 10.1182/asheducation-2018.1.110

  View details for PubMedID 30504299

  View details for PubMedCentralID PMC6246021

• Gli1+ Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target. Cell stem cell Schneider, R. K., Mullally, A., Dugourd, A., Peisker, F., Hoogenboezem, R., Van Strien, P. M., Bindels, E. M., Heckl, D., Büsche, G., Fleck, D., Müller-Newen, G., Wongboonsin, J., Ventura Ferreira, M., Puelles, V. G., Saez-Rodriguez, J., Ebert, B. L., Humphreys, B. D., Kramann, R. 2018; 23 (2): 308-309

  View details for DOI 10.1016/j.stem.2018.07.006

  View details for PubMedID 30075132

• Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms. Science translational medicine Wolach, O., Sellar, R. S., Martinod, K., Cherpokova, D., McConkey, M., Chappell, R. J., Silver, A. J., Adams, D., Castellano, C. A., Schneider, R. K., Padera, R. F., DeAngelo, D. J., Wadleigh, M., Steensma, D. P., Galinsky, I., Stone, R. M., Genovese, G., McCarroll, S. A., Iliadou, B., Hultman, C., Neuberg, D., Mullally, A., Wagner, D. D., Ebert, B. L. 2018; 10 (436)

  Abstract

  Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2V617F, the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2V617F-expressing neutrophils and that PAD4 is required for Jak2V617F-driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2V617F-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

  View details for DOI 10.1126/scitranslmed.aan8292

  View details for PubMedID 29643232

  View details for PubMedCentralID PMC6442466

• Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood Elf, S., Abdelfattah, N. S., Baral, A. J., Beeson, D., Rivera, J. F., Ko, A., Florescu, N., Birrane, G., Chen, E., Mullally, A. 2018; 131 (7): 782-786

  Abstract

  Mutations in calreticulin (CALR) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms.

  View details for DOI 10.1182/blood-2017-08-800896

  View details for PubMedID 29288169

  View details for PubMedCentralID PMC5814933

• JAK2, CALR, MPL and ASXL1 mutational status correlates with distinct histological features in Philadelphia chromosome-negative myeloproliferative neoplasms. Haematologica Wong, W. J., Hasserjian, R. P., Pinkus, G. S., Breyfogle, L. J., Mullally, A., Pozdnyakova, O. 2018; 103 (2): e63-e68

  View details for DOI 10.3324/haematol.2017.178988

  View details for PubMedID 29146710

  View details for PubMedCentralID PMC5792288

• Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells. Journal of visualized experiments : JoVE Abdelfattah, N. S., Mullally, A. 2018

  Abstract

  Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

  View details for DOI 10.3791/56726

  View details for PubMedID 29364275

  View details for PubMedCentralID PMC5908454

• Kinase Inhibitors in the Treatment of Myeloid Malignancies. Hematology/oncology clinics of North America Mullally, A. 2017; 31 (4): ix-x

  View details for DOI 10.1016/j.hoc.2017.05.001

  View details for PubMedID 28673398

• The Development and Use of Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms. Hematology/oncology clinics of North America Hobbs, G. S., Rozelle, S., Mullally, A. 2017; 31 (4): 613-626

  Abstract

  Following the discovery of the JAK2V617F mutation, Janus kinase (JAK) 2 inhibitors were developed as rationally designed therapy in myeloproliferative neoplasms (MPNs). Although JAK2 inhibitors have clinical efficacy in MPN, they are not clonally selective for the JAK2V617F-mutant cells. Because activated JAK-signal transducer and activator of transcription (STAT) signaling is a common feature of MPN, JAK2 inhibitors are efficacious regardless of the specific MPN phenotypic driver mutation. The Food and Drug Administration approved the JAK1/JAK2 inhibitor, ruxolitinib, for the treatment of myelofibrosis and polycythemia vera. Additional JAK2 inhibitors are currently in advanced phased clinical trials.

  View details for DOI 10.1016/j.hoc.2017.04.002

  View details for PubMedID 28673391

• Gli1+ Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target. Cell stem cell Schneider, R. K., Mullally, A., Dugourd, A., Peisker, F., Hoogenboezem, R., Van Strien, P. M., Bindels, E. M., Heckl, D., Büsche, G., Fleck, D., Müller-Newen, G., Wongboonsin, J., Ventura Ferreira, M., Puelles, V. G., Saez-Rodriguez, J., Ebert, B. L., Humphreys, B. D., Kramann, R. 2017; 20 (6): 785-800.e8

  Abstract

  Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1+ mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1+ cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1+ cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.

  View details for DOI 10.1016/j.stem.2017.03.008

  View details for PubMedID 28457748

  View details for PubMedCentralID PMC6485654

• Gain of function in Jak2V617F-positive T-cells. Leukemia Nishanth, G., Wolleschak, D., Fahldieck, C., Fischer, T., Mullally, A., Perner, F., Schnöder, T. M., Just, S., Heidel, F. H., Schlüter, D. 2017; 31 (4): 1000-1003

  View details for DOI 10.1038/leu.2017.6

  View details for PubMedID 28074070

• Myeloproliferative neoplasm stem cells. Blood Mead, A. J., Mullally, A. 2017; 129 (12): 1607-1616

  Abstract

  Myeloproliferative neoplasms (MPNs) arise in the hematopoietic stem cell (HSC) compartment as a result of the acquisition of somatic mutations in a single HSC that provides a selective advantage to mutant HSC over normal HSC and promotes myeloid differentiation to engender a myeloproliferative phenotype. This population of somatically mutated HSC, which initiates and sustains MPNs, is termed MPN stem cells. In >95% of cases, mutations that drive the development of an MPN phenotype occur in a mutually exclusive manner in 1 of 3 genes: JAK2, CALR, or MPL The thrombopoietin receptor, MPL, is the key cytokine receptor in MPN development, and these mutations all activate MPL-JAK-STAT signaling in MPN stem cells. Despite common biological features, MPNs display diverse disease phenotypes as a result of both constitutional and acquired factors that influence MPN stem cells, and likely also as a result of heterogeneity in the HSC in which MPN-initiating mutations arise. As the MPN clone expands, it exerts cell-extrinsic effects on components of the bone marrow niche that can favor the survival and expansion of MPN stem cells over normal HSC, further sustaining and driving malignant hematopoiesis. Although developed as targeted therapies for MPNs, current JAK2 inhibitors do not preferentially target MPN stem cells, and as a result, rarely induce molecular remissions in MPN patients. As the understanding of the molecular mechanisms underlying the clonal dominance of MPN stem cells advances, this will help facilitate the development of therapies that preferentially target MPN stem cells over normal HSC.

  View details for DOI 10.1182/blood-2016-10-696005

  View details for PubMedID 28159736

  View details for PubMedCentralID PMC5413298

• Underlying mechanisms of the JAK2V617F mutation in the pathogenesis of myeloproliferative neoplasms. Der Pathologe Mullally, A. 2016; 37 (Suppl 2): 175-179

  Abstract

  Chronic myeloproliferative neoplasms (MPN) comprise a spectrum of clonal neoplastic disorders characterized by overproduction of terminally differentiated cells of the myeloid lineage. A common genetic basis for the BCR-ABL-negative MPN disorders was elucidated in 2005 with the identification of the JAK2V617F mutation in the majority of MPN patients. The discovery of JAK2V617F had a dramatic impact on the diagnosis and treatment of MPN. Testing for JAK2 mutations is now included in the World Health Organization (WHO) criteria for the diagnosis of MPN, and in 2011 the oral JAK2 kinase inhibitor ruxolitinib became the first Food and Drug Administration (FDA)-approved drug for the treatment of myelofibrosis. The drug is now also approved in Europe and Canada.

  View details for DOI 10.1007/s00292-016-0240-2

  View details for PubMedID 27796499

• Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation. Cancer cell Obeng, E. A., Chappell, R. J., Seiler, M., Chen, M. C., Campagna, D. R., Schmidt, P. J., Schneider, R. K., Lord, A. M., Wang, L., Gambe, R. G., McConkey, M. E., Ali, A. M., Raza, A., Yu, L., Buonamici, S., Smith, P. G., Mullally, A., Wu, C. J., Fleming, M. D., Ebert, B. L. 2016; 30 (3): 404-417

  Abstract

  More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

  View details for DOI 10.1016/j.ccell.2016.08.006

  View details for PubMedID 27622333

  View details for PubMedCentralID PMC5023069

• Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer discovery Elf, S., Abdelfattah, N. S., Chen, E., Perales-Patón, J., Rosen, E. A., Ko, A., Peisker, F., Florescu, N., Giannini, S., Wolach, O., Morgan, E. A., Tothova, Z., Losman, J. A., Schneider, R. K., Al-Shahrour, F., Mullally, A. 2016; 6 (4): 368-81

  Abstract

  Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN.The mechanism by which CALR mutations induce MPN remains unknown. In this report, we show that the positive charge of the CALR mutant C-terminus is necessary to transform hematopoietic cells by enabling binding between mutant CALR and the thrombopoietin receptor MPL.

  View details for DOI 10.1158/2159-8290.CD-15-1434

  View details for PubMedID 26951227

  View details for PubMedCentralID PMC4851866

• Haemophagocytic lymphohistiocytosis in adults: a multicentre case series over 7 years. British journal of haematology Schram, A. M., Comstock, P., Campo, M., Gorovets, D., Mullally, A., Bodio, K., Arnason, J., Berliner, N. 2016; 172 (3): 412-9

  Abstract

  Haemophagocytic lymphohistiocytosis (HLH) is a syndrome of uncontrolled immune activation that has gained increasing attention over the past decade. Although classically known as a familial disorder of children caused by mutations that affect cytotoxic T-cell function, an acquired form of HLH in adults is now widely recognized. This is often seen in the setting of malignancy, infection or rheumatological disorders. We performed a retrospective review across 3 tertiary care centres and identified 68 adults with HLH. The average age was 53 years (range 18-77 years) and 43 were male (63%). Underlying disorders included malignancy in 33 patients (49%), infection in 22 (33%), autoimmune disease in 19 (28%) and idiopathic HLH in 15 (22%). Patients were treated with disease-specific therapy and immunomodulatory agents. After a median follow-up of 32·2 months, 46 patients had died (69%). The median overall survival was 4 months (95% CI: 0·0-10·2 months). Patients with malignancy had a worse prognosis compared to those without (median survival 2·8 months versus 10·7 months, P = 0·007). HLH is a devastating disorder with a high mortality. Further research is needed to improve treatment and outcomes.

  View details for DOI 10.1111/bjh.13837

  View details for PubMedID 26537747

• RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability. Cell reports Chen, E., Ahn, J. S., Sykes, D. B., Breyfogle, L. J., Godfrey, A. L., Nangalia, J., Ko, A., DeAngelo, D. J., Green, A. R., Mullally, A. 2015; 13 (11): 2345-2352

  Abstract

  JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

  View details for DOI 10.1016/j.celrep.2015.11.037

  View details for PubMedID 26686625

  View details for PubMedCentralID PMC4691544

• Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition. Nature medicine Wen, Q. J., Yang, Q., Goldenson, B., Malinge, S., Lasho, T., Schneider, R. K., Breyfogle, L. J., Schultz, R., Gilles, L., Koppikar, P., Abdel-Wahab, O., Pardanani, A., Stein, B., Gurbuxani, S., Mullally, A., Levine, R. L., Tefferi, A., Crispino, J. D. 2015; 21 (12): 1473-80

  Abstract

  Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-β. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

  View details for DOI 10.1038/nm.3995

  View details for PubMedID 26569382

  View details for PubMedCentralID PMC4674320

• Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology. Haematologica Ishikawa, Y., Maeda, M., Pasham, M., Aguet, F., Tacheva-Grigorova, S. K., Masuda, T., Yi, H., Lee, S. U., Xu, J., Teruya-Feldstein, J., Ericsson, M., Mullally, A., Heuser, J., Kirchhausen, T., Maeda, T. 2015; 100 (4): 439-51

  Abstract

  Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.

  View details for DOI 10.3324/haematol.2014.119537

  View details for PubMedID 25552701

  View details for PubMedCentralID PMC4380716

• Marked hyperferritinemia does not predict for HLH in the adult population. Blood Schram, A. M., Campigotto, F., Mullally, A., Fogerty, A., Massarotti, E., Neuberg, D., Berliner, N. 2015; 125 (10): 1548-52

  Abstract

  Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of uncontrolled immune activation that has gained increasing attention during the last decade. The diagnosis of HLH is based on a constellation of clinical and laboratory abnormalities, including elevated serum ferritin levels. In the pediatric population, marked hyperferritinemia is specific for HLH. To determine what conditions are associated with profoundly elevated ferritin in the adult population, we performed a retrospective analysis in a large academic health care system. We identified 113 patients with serum ferritin levels higher than 50,000 µg/L. The most frequently observed conditions included renal failure, hepatocellular injury, infections, and hematologic malignancies. Our results suggest that marked hyperferritinemia can be seen in a variety of conditions and is not specific for HLH in adults.

  View details for DOI 10.1182/blood-2014-10-602607

  View details for PubMedID 25573993

• Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice. Blood Bender, M., Giannini, S., Grozovsky, R., Jönsson, T., Christensen, H., Pluthero, F. G., Ko, A., Mullally, A., Kahr, W. H., Hoffmeister, K. M., Falet, H. 2015; 125 (6): 1014-24

  Abstract

  Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2(fl/fl) Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2(fl/fl) Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver-derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

  View details for DOI 10.1182/blood-2014-07-587857

  View details for PubMedID 25468568

  View details for PubMedCentralID PMC4319232

• Distinct effects of concomitant Jak2V617F expression and Tet2 loss in mice promote disease progression in myeloproliferative neoplasms. Blood Chen, E., Schneider, R. K., Breyfogle, L. J., Rosen, E. A., Poveromo, L., Elf, S., Ko, A., Brumme, K., Levine, R., Ebert, B. L., Mullally, A. 2015; 125 (2): 327-35

  Abstract

  Signaling mutations (eg, JAK2V617F) and mutations in genes involved in epigenetic regulation (eg, TET2) are the most common cooccurring classes of mutations in myeloproliferative neoplasms (MPNs). Clinical correlative studies have demonstrated that TET2 mutations are enriched in more advanced phases of MPNs such as myelofibrosis and leukemic transformation, suggesting that they may cooperate with JAK2V617F to promote disease progression. To dissect the effects of concomitant Jak2V617F expression and Tet2 loss within distinct hematopoietic compartments in vivo, we generated Jak2V617F/Tet2 compound mutant genetic mice. We found that the combination of Jak2V617F expression and Tet2 loss resulted in a more florid MPN phenotype than that seen with either allele alone. Concordant with this, we found that Tet2 deletion conferred a strong functional competitive advantage to Jak2V617F-mutant hematopoietic stem cells (HSCs). Transcriptional profiling revealed that both Jak2V617F expression and Tet2 loss were associated with distinct and nonoverlapping gene expression signatures within the HSC compartment. In aggregate, our findings indicate that Tet2 loss drives clonal dominance in HSCs, and Jak2V617F expression causes expansion of downstream precursor cell populations, resulting in disease progression through combinatorial effects. This work provides insight into the functional consequences of JAK2V617F-TET2 comutation in MPNs, particularly as it pertains to HSCs.

  View details for DOI 10.1182/blood-2014-04-567024

  View details for PubMedID 25281607

  View details for PubMedCentralID PMC4287639

• How does JAK2V617F contribute to the pathogenesis of myeloproliferative neoplasms? Hematology. American Society of Hematology. Education Program Chen, E., Mullally, A. 2014; 2014 (1): 268-76

  Abstract

  A decade on from the discovery of the JAK2V617F mutation in the majority of patients with myeloproliferative neoplasms (MPNs), JAK2V617F is now firmly installed in the hematology curriculum of medical students and the diagnostic-testing algorithm of clinicians. Furthermore, the oral JAK1/JAK2 inhibitor ruxolitinib, rationally designed to target activated JAK2 signaling in MPN, has been approved by the Food and Drug Administration (FDA) of the United States for the past 3 years for the treatment of intermediate- and advanced-phase myelofibrosis. Notwithstanding this, JAK2V617F continues to stimulate the MPN research community and novel insights into understanding the mechanisms by which JAK2V617F contributes to the pathogenesis of MPN are continually emerging. In this chapter, we focus on recent advances in 4 main areas: (1) the molecular processes coopted by JAK2V617F to induce MPN, (2) the role that JAK2V617F plays in phenotypic diversity in MPN, (3) the functional impact of JAK2V617F on hematopoietic stem cells, and (4) therapeutic strategies to target JAK2V617F. Although great strides have been made, significant deficits still exist in our understanding of the precise mechanisms by which JAK2V617F-mutant hematopoietic stem cells emerge and persist to engender clonal hematopoiesis in MPN and in developing strategies to preferentially target the JAK2V617F-mutant clone therapeutically. Critically, although myelofibrosis remains arguably the greatest clinical challenge in JAK2V617F-mediated MPN, the current understanding of myelofibrosis-specific disease biology remains quite rudimentary. Therefore, many important biological questions pertaining to JAK2V617F will continue to engage and challenge the MPN research community in the coming decade.

  View details for DOI 10.1182/asheducation-2014.1.268

  View details for PubMedID 25696866

• Hit the spleen, JAK! Blood Lane, S. W., Mullally, A. 2014; 124 (19): 2898-900

  Abstract

  In this issue of Blood, Wang et al report on the response of splenic-derived hematopoietic stem and progenitor cells from patients with myelofibrosis (MF) to the Janus kinase (JAK) inhibitor, AZD1480.

  View details for DOI 10.1182/blood-2014-09-600635

  View details for PubMedID 25377558

• JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response. Proceedings of the National Academy of Sciences of the United States of America Chen, E., Ahn, J. S., Massie, C. E., Clynes, D., Godfrey, A. L., Li, J., Park, H. J., Nangalia, J., Silber, Y., Mullally, A., Gibbons, R. J., Green, A. R. 2014; 111 (42): 15190-5

  Abstract

  Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

  View details for DOI 10.1073/pnas.1401873111

  View details for PubMedID 25288776

  View details for PubMedCentralID PMC4210350

• Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS. Cancer cell Schneider, R. K., Ademà, V., Heckl, D., Järås, M., Mallo, M., Lord, A. M., Chu, L. P., McConkey, M. E., Kramann, R., Mullally, A., Bejar, R., Solé, F., Ebert, B. L. 2014; 26 (4): 509-20

  Abstract

  The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

  View details for DOI 10.1016/j.ccr.2014.08.001

  View details for PubMedID 25242043

  View details for PubMedCentralID PMC4199102

• Loss of function of TET2 cooperates with constitutively active KIT in murine and human models of mastocytosis. PloS one De Vita, S., Schneider, R. K., Garcia, M., Wood, J., Gavillet, M., Ebert, B. L., Gerbaulet, A., Roers, A., Levine, R. L., Mullally, A., Williams, D. A. 2014; 9 (5): e96209

  Abstract

  Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM.

  View details for DOI 10.1371/journal.pone.0096209

  View details for PubMedID 24788138

  View details for PubMedCentralID PMC4008566

• Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia. The Journal of experimental medicine Järås, M., Miller, P. G., Chu, L. P., Puram, R. V., Fink, E. C., Schneider, R. K., Al-Shahrour, F., Peña, P., Breyfogle, L. J., Hartwell, K. A., McConkey, M. E., Cowley, G. S., Root, D. E., Kharas, M. G., Mullally, A., Ebert, B. L. 2014; 211 (4): 605-12

  Abstract

  Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.

  View details for DOI 10.1084/jem.20131033

  View details for PubMedID 24616378

  View details for PubMedCentralID PMC3978274

• Sinister symbiosis: pathological hematopoietic-stromal interactions in CML. Cell stem cell Mullally, A., Ebert, B. L. 2013; 13 (3): 257-8

  Abstract

  The impact of myeloid malignancies on the nonhematopoietic components of the bone marrow remains poorly understood. In this issue of Cell Stem Cell, Schepers et al. (2013) describe how malignant myeloid cells alter the endosteal hematopoietic stem cell (HSC) niche, resulting in the expansion of osteoblastic lineage cells that preferentially support malignant HSCs.

  View details for DOI 10.1016/j.stem.2013.08.009

  View details for PubMedID 24012363

  View details for PubMedCentralID PMC3842099

• Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-α in a murine model of polycythemia vera. Blood Mullally, A., Bruedigam, C., Poveromo, L., Heidel, F. H., Purdon, A., Vu, T., Austin, R., Heckl, D., Breyfogle, L. J., Kuhn, C. P., Kalaitzidis, D., Armstrong, S. A., Williams, D. A., Hill, G. R., Ebert, B. L., Lane, S. W. 2013; 121 (18): 3692-702

  Abstract

  Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNα treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNα treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.

  View details for DOI 10.1182/blood-2012-05-432989

  View details for PubMedID 23487027

  View details for PubMedCentralID PMC3643767

• miR-433 is aberrantly expressed in myeloproliferative neoplasms and suppresses hematopoietic cell growth and differentiation. Leukemia Lin, X., Rice, K. L., Buzzai, M., Hexner, E., Costa, F. F., Kilpivaara, O., Mullally, A., Soares, M. B., Ebert, B. L., Levine, R., Licht, J. D. 2013; 27 (2): 344-52

  Abstract

  BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.

  View details for DOI 10.1038/leu.2012.224

  View details for PubMedID 22864358

• Myeloproliferative neoplasm animal models. Hematology/oncology clinics of North America Mullally, A., Lane, S. W., Brumme, K., Ebert, B. L. 2012; 26 (5): 1065-81

  Abstract

  Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow into irradiated syngeneic mice established the field of MPN animal modeling. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN-associated genetic abnormalities on hematopoietic stem and progenitor cells (HSPCs). Xenograft models have allowed the study of primary human MPN-propagating cells in vivo. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models.

  View details for DOI 10.1016/j.hoc.2012.07.007

  View details for PubMedID 23009938

  View details for PubMedCentralID PMC3459181

• Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy NATURE Koppikar, P., Bhagwat, N., Kilpivaara, O., Manshouri, T., Adli, M., Hricik, T., Liu, F., Saunders, L. M., Mullally, A., Abdel-Wahab, O., Leung, L., Weinstein, A., Marubayashi, S., Goel, A., Goenen, M., Estrov, Z., Ebert, B. L., Chiosis, G., Nimer, S. D., Bernstein, B. E., Verstovsek, S., Levine, R. L. 2012; 489 (7414): 155-U222

  Abstract

  The identification of somatic activating mutations in JAK2 (refs 1–4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

  View details for DOI 10.1038/nature11303

  View details for Web of Science ID 000308347000054

  View details for PubMedID 22820254

• Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera. Blood Mullally, A., Poveromo, L., Schneider, R. K., Al-Shahrour, F., Lane, S. W., Ebert, B. L. 2012; 120 (1): 166-72

  Abstract

  In the current model of the pathogenesis of polycythemia vera (PV), the JAK2V617F mutation arises in hematopoietic stem cells (HSCs) that maintain the disease, while erythroid precursor populations expand, resulting in excessive red blood cell production. We examined the role of these specific cell populations using a conditional Jak2V617F knockin murine model. We demonstrate that the most immature long-term (LT) HSCs are solely responsible for initiating and maintaining the disease in vivo and that Jak2V617F mutant LT-HSCs dominate hematopoiesis over time. When we induced Jak2V617F expression in erythropoietin receptor expressing precursor cells, the mice developed elevated hematocrit, expanded erythroid precursors, and suppressed erythropoietin levels. However, the disease phenotype was significantly attenuated compared with mice expressing Jak2V617F in LT-HSCs. In addition to developing a PV phenotype, all mice transplanted with Jak2V617F LT-HSCs underwent myelofibrotic transformation over time. These findings recapitulate the development of post-PV myelofibrosis in human myeloproliferative neoplasms. In aggregate, these results demonstrate the distinct roles of LT-HSCs and erythroid precursors in the pathogenesis of PV.

  View details for DOI 10.1182/blood-2012-01-402396

  View details for PubMedID 22627765

  View details for PubMedCentralID PMC3390954

• EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L LEUKEMIA Wernig, G., Kharas, M. G., Mullally, A., Leeman, D. S., Okabe, R., George, T., Clary, D. O., Gilliland, D. G. 2012; 26 (4): 720-727

  Abstract

  About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

  View details for DOI 10.1038/leu.2011.261

  View details for PubMedID 22005786

• Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q(-) syndrome BLOOD Kumar, M. S., Narla, A., Nonami, A., Mullally, A., Dimitrova, N., Ball, B., McAuley, J. R., Poveromo, L., Kutok, J. L., Galili, N., Raza, A., Attar, E., Gilliland, D. G., Jacks, T., Ebert, B. L. 2011; 118 (17): 4666-4673

  Abstract

  Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.

  View details for DOI 10.1182/blood-2010-12-324715

  View details for Web of Science ID 000296368700029

  View details for PubMedID 21873545

  View details for PubMedCentralID PMC3208282

• Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells BLOOD Dutt, S., Narla, A., Lin, K., Mullally, A., Abayasekara, N., Megerdichian, C., Wilson, F. H., Currie, T., Khanna-Gupta, A., Berliner, N., Kutok, J. L., Ebert, B. L. 2011; 117 (9): 2567-2576

  Abstract

  Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q-syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q-syndrome, DBA, and perhaps other bone marrow failure syndromes.

  View details for DOI 10.1182/blood-2010-07-295238

  View details for Web of Science ID 000288207400008

  View details for PubMedID 21068437

  View details for PubMedCentralID PMC3062351

• Mutations with epigenetic effects in myeloproliferative neoplasms and recent progress in treatment: Proceedings from the 5th International Post-ASH Symposium BLOOD CANCER JOURNAL Tefferi, A., Abdel-Wahab, O., Cervantes, F., Crispino, J. D., Finazzi, G., Girodon, F., Gisslinger, H., Gotlib, J., Kiladjian, J., Levine, R. L., Licht, J. D., Mullally, A., Odenike, O., Pardanani, A., Silver, R. T., Solary, E., Mughal, T. 2011; 1

  Abstract

  Immediately following the 2010 annual American Society of Hematology (ASH) meeting, the 5th International Post-ASH Symposium on Chronic Myelogenous Leukemia and BCR-ABL1-Negative Myeloproliferative Neoplasms (MPNs) took place on 7-8 December 2010 in Orlando, Florida, USA. During this meeting, the most recent advances in laboratory research and clinical practice, including those that were presented at the 2010 ASH meeting, were discussed among recognized authorities in the field. The current paper summarizes the proceedings of this meeting in BCR-ABL1-negative MPN. We provide a detailed overview of new mutations with putative epigenetic effects (TET oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), isocitrate dehydrogenase (IDH) and enhancer of zeste homolog 2 (EZH2)) and an update on treatment with Janus kinase (JAK) inhibitors, pomalidomide, everolimus, interferon-α, midostaurin and cladribine. In addition, the new 'Dynamic International Prognostic Scoring System (DIPSS)-plus' prognostic model for primary myelofibrosis (PMF) and the clinical relevance of distinguishing essential thrombocythemia from prefibrotic PMF are discussed.

  View details for DOI 10.1038/bcj.2011.4

  View details for Web of Science ID 000298815400001

  View details for PubMedID 23471017

  View details for PubMedCentralID PMC3255279

• STATistical power of clonal analysis: differential STAT1 pathway activation downstream of the JAK2V617F mutation. Cancer cell Mullally, A., Ebert, B. L. 2010; 18 (5): 405-6

  Abstract

  The biological basis of the phenotypic diversity observed in JAK2V617F-positive myeloproliferative neoplasms is poorly understood. In this issue of Cancer Cell, Chen et al. describe that interferon and STAT1 signaling are activated in essential thrombocythemia but not in polycythemia vera. STAT1 promotes megakaryopoiesis and thus contributes to an essential thrombocythemia phenotype.

  View details for DOI 10.1016/j.ccr.2010.10.037

  View details for PubMedID 21075304

  View details for PubMedCentralID PMC3001244

• CNS relapse in acute promyeloctyic leukemia. Journal of clinical oncology : official journal of the American Society of Clinical Oncology Housman, E., Chang, P., Lane, S. W., Blinder, R., Galinsky, I., Kesari, S., Ho, V. T., Stone, R. M., Mullally, A. 2010; 28 (24): e409-11

  View details for DOI 10.1200/JCO.2009.27.1577

  View details for PubMedID 20530270

• NF1 inactivation revs up Ras in adult acute myelogenous leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research Mullally, A., Ebert, B. L. 2010; 16 (16): 4074-6

  Abstract

  Mutations in the Ras pathway are common in myeloid malignancies. NF1, a tumor suppressor and negative regulator of Ras, is inactivated in a subset of adult acute myelogenous leukemia (AML) cases. Loss of NF1 function sensitizes cells to inhibition of mammalian target of rapamycin (mTOR), a downstream effector of Ras activation, highlighting a potential therapeutic opportunity for some AML patients.

  View details for DOI 10.1158/1078-0432.CCR-10-1438

  View details for PubMedID 20587590

• Physiological Jak2V617F Expression Causes a Lethal Myeloproliferative Neoplasm with Differential Effects on Hematopoietic Stem and Progenitor Cells CANCER CELL Mullally, A., Lane, S. W., Ball, B., Megerdichian, C., Okabe, R., Al-Shahrour, F., Paktinat, M., Haydu, J. E., Housman, E., Lord, A. M., Wernig, G., Kharas, M. G., Mercher, T., Kutok, J. L., Gilliland, D. G., Ebert, B. L. 2010; 17 (6): 584-596

  Abstract

  We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

  View details for DOI 10.1016/j.ccr.2010.05.015

  View details for Web of Science ID 000278952300010

  View details for PubMedID 20541703

  View details for PubMedCentralID PMC2909585

• Frequent TET2 mutations in systemic mastocytosis: clinical, KITD816V and FIP1L1-PDGFRA correlates. Leukemia Tefferi, A., Levine, R. L., Lim, K. H., Abdel-Wahab, O., Lasho, T. L., Patel, J., Finke, C. M., Mullally, A., Li, C. Y., Pardanani, A., Gilliland, D. G. 2009; 23 (5): 900-4

  Abstract

  TET2 (TET oncogene family member 2) is a candidate tumor suppressor gene located at chromosome 4q24, and was recently reported to be mutated in approximately 14% of patients with JAK2V617F-positive myeloproliferative neoplasms. We used high-throughput DNA sequence analysis to screen for TET2 mutations in bone marrow-derived DNA from 48 patients with systemic mastocytosis (SM), including 42 who met the 2008 WHO (World Health Organization) diagnostic criteria for SM and 6 with FIP1L1-PDGFRA. Twelve (29%) SM, but no FIP1L1-PDGFRA patients, had TET2 mutations. A total of 17 mutations (13 frameshift, 2 nonsense and 2 missense) were documented in 2 (15%) of 13 indolent SM patients, 2 (40%) of 5 aggressive SM, and 8 (35%) of 23 SM associated with a clonal non-mast cell-lineage hematopoietic disease (P=0.52). KITD816V was detected by PCR sequencing in 50 or 20% of patients with or without TET2 mutation (P=0.05), respectively. Multivariable analysis showed a significant association between the presence of TET2 mutation and monocytosis (P=0.0003) or female sex (P=0.05). The association with monocytosis was also observed in non-indolent SM (n=29), in which the presence of mutant TET2 did not affect survival (P=0.98). We conclude that TET2 mutations are frequent in SM, segregate with KITD816V and influence phenotype without necessarily altering prognosis.

  View details for DOI 10.1038/leu.2009.37

  View details for PubMedID 19262599

  View details for PubMedCentralID PMC4654631

• TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis. Leukemia Tefferi, A., Pardanani, A., Lim, K. H., Abdel-Wahab, O., Lasho, T. L., Patel, J., Gangat, N., Finke, C. M., Schwager, S., Mullally, A., Li, C. Y., Hanson, C. A., Mesa, R., Bernard, O., Delhommeau, F., Vainchenker, W., Gilliland, D. G., Levine, R. L. 2009; 23 (5): 905-11

  Abstract

  High-throughput DNA sequence analysis was used to screen for TET2 mutations in bone marrow-derived DNA from 239 patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). Thirty-two mutations (19 frameshift, 10 nonsense, 3 missense; mostly involving exons 4 and 12) were identified for an overall mutational frequency of approximately 13%. Specific diagnoses included polycythemia vera (PV; n=89), essential thrombocythemia (ET; n=57), primary myelofibrosis (PMF; n=60), post-PV MF (n=14), post-ET MF (n=7) and blast phase PV/ET/MF (n=12); the corresponding mutational frequencies were approximately 16, 5, 17, 14, 14 and 17% (P=0.50). Mutant TET2 was detected in approximately 17 and approximately 7% of JAK2V617F-positive and -negative cases, respectively (P=0.04). However, this apparent clustering of the two mutations was accounted for by an independent association between mutant TET2 and advanced age; mutational frequency was approximately 23% in patients > or =60 years old versus approximately 4% in younger patients (P<0.0001). The presence of mutant TET2 did not affect survival, leukemic transformation or thrombosis in either PV or PMF; a correlation with hemoglobin <10 g per 100 ml in PMF was noted (P=0.05). We conclude that TET2 mutations occur in both JAK2V617F-positive and -negative MPN, are more prevalent in older patients, display similar frequencies across MPN subcategories and disease stages, and hold limited prognostic relevance.

  View details for DOI 10.1038/leu.2009.47

  View details for PubMedID 19262601

  View details for PubMedCentralID PMC4654629

• A germline JAK2 SNP is associated with predisposition to the development of JAK2(V617F)-positive myeloproliferative neoplasms. Nature genetics Kilpivaara, O., Mukherjee, S., Schram, A. M., Wadleigh, M., Mullally, A., Ebert, B. L., Bass, A., Marubayashi, S., Heguy, A., Garcia-Manero, G., Kantarjian, H., Offit, K., Stone, R. M., Gilliland, D. G., Klein, R. J., Levine, R. L. 2009; 41 (4): 455-9

  Abstract

  Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2(V617F)) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2(V617F)-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2(V617F) is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.

  View details for DOI 10.1038/ng.342

  View details for PubMedID 19287384

  View details for PubMedCentralID PMC3676425

• Beyond HLA: the significance of genomic variation for allogeneic hematopoietic stem cell transplantation. Blood Mullally, A., Ritz, J. 2007; 109 (4): 1355-62

  Abstract

  The last 2 years have seen much excitement in the field of genetics with the identification of a formerly unappreciated level of "structural variation" within the normal human genome. Genetic structural variants include deletions, duplications, and inversions in addition to the recently discovered, copy number variants. Single nucleotide polymorphisms are the most extensively evaluated variant within the genome to date. Combining our knowledge from these studies with our rapidly accumulating understanding of structural variants, it is apparent that the extent of genetic dissimilarity between any 2 individuals is considerable and much greater than that which was previously recognized. Clearly, this more diverse view of the genome has significant implications for allogeneic hematopoietic stem cell transplantation, not least in the generation of transplant antigens but also in terms of individual susceptibility to transplant-related toxicities. With advances in DNA sequencing technology we now have the capacity to perform genome-wide analysis in a high throughput fashion, permitting a detailed genetic analysis of patient and donor prior to transplantation. Understanding the significance of this additional genetic information and applying it in a clinically meaningful way will be one of the challenges faced by transplant clinicians in the future.

  View details for DOI 10.1182/blood-2006-06-030858

  View details for PubMedID 17008540

• Wasted sheep and premature infants: the role of trace metals in hematopoiesis. Blood reviews Mullally, A. M., Vogelsang, G. B., Moliterno, A. R. 2004; 18 (4): 227-34

  Abstract

  Trace element deficiencies and toxicities are not commonly encountered in clinical practice, particularly in regions where there is access to adequate nutrition and occupational exposures are regulated. However, specific clinical scenarios associated with trace metal deficiency and toxicity states do exist. Often, clues to the presence of these states may lie in the development of blood count abnormalities. The consultant haematologist is frequently involved in the care of these patients, and it is with this audience that this review is intended. This review will focus on the trace metals required for normal hematopoiesis including their function, metabolism, deficiency and toxicity states, and the clinical situations underlying these. As much of the information regarding trace metal disease states has arisen from veterinary medicine and from human case reports, these will be summarized and highlighted in this review.

  View details for DOI 10.1016/S0268-960X(03)00067-5

  View details for PubMedID 15501551