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In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and that underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B-cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B-cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown (Kdm1a-KD) in Eμ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and pro-apoptotic pathways. Genetic KDM1A depletion also affected milieu components (T-, stromal, monocytic cells), resulting in significant reductions of their capacity to support CLL cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA-seq) and H3K4me3 marks (ChIP-seq) in Eµ-TCL1A vs. iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL by altering histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell-leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL, namely via tumor-cell intrinsic mechanisms and impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

XLSX file - 24K, PCR Primer sequences.

PDF file - 113K, Bcl2 protein expression in wild-type and Tyk2 knockout mice.

PDF file - 74K, Knockdown efficiency and BCL2 expression in JURKAT cells.

XLSX file - 525K, Summary of inducible shRNA screen on three T-ALL cell lines.

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PDF file - 32K, Expressions of IL-10 and IL-10 receptors, and effect of an anti-IL10 neutralizing antibody on growth of T-ALL cell lines.

XLSX file - 30K, Targeting sequences for shRNAs.

PDF file - 68K, Cell cycle distribution after TYK2 knockdown in T-ALL cells.

XLSX file - 14291K, TYK2 genotype in 17 T-ALL cell lines.

XLSX file - 35K, Sensitivities to TYK2 knockdown and JAK Inhibitor I treatment, and phosphorylation status of downstream signaling molecules in T-ALL cell lines and Ba/F3 clones.

PDF file - 61K, Validation of shRNA constructs.

XLSX file - 36K, Cell viability profile by RAPID assay.

PDF file - 273K, Confirmation of CD3 expression on OP9 DL1 outgrowth cells.

PDF file - 24K, Effect of JAK kinase inhibitors with varying TYK2-specificity on growth and viability of T-ALL cells.

XLSX file - 23K, Clinical, biologic and molecular characteristics of the T-ALL patient samples analyzed. See separate file.

PDF file - 105K, Signaling and cell cycle distribution after JAK inhibitor I treatment.

Supplementary Figure 1. Pro-inflammatory cytokines are elevated in MF mice and reversed with JAK inhibitor treatment. Supplementary Figure 2. Principal component analysis of MF cells. Supplementary Figure 3. Single cell analysis of immunophenotypically-defined subpopulations from MF and control mice. Supplementary Figure 4. Elevated IL8 expression in MF patients. Supplementary Figure 5. Complete removal of Il6 from the mouse system shows only minor effects on MPLW515L disease. Supplementary Figure 6. Peripheral blood and cytometric flow analysis of MPLW515L transplanted mice with and without Stat3 deletion. Supplementary Figure 7. Constitutive activation of STAT3 in the BM of MPLW515L mice and primary MF patients. Supplementary Figure 8. Schematic illustration of bone marrow transplantation experiments using Stat3-deficient mice or littermate control mice as donors. Supplementary Figure 9. Excision of Stat3 post engraftment prolongs survival and reduces leukocytosis. Supplementary Figure 10. Mutant-restricted deletion of Stat3 does not affect disease severity in vivo. Supplementary Figure 11. Single cell analysis of populations from MF and control mice. Supplementary Figure 12. Mutant and non-mutant hematopoietic cells feature a higher percentage of cytokine secreting cells. Supplementary Figure 13. Single cell analysis of populations from Jak2V617F MF and control mice. Supplementary Figure 14. Mutant and non-mutant hematopoietic cells feature a higher percentage of cytokine secreting cells. Supplementary Figure 15. JAK1/2 inhibition reduces cytokine expression from CD45.1-positive wild-type cells and CD45.2-positive mutant cell population. Supplementary Table 1. Primary myelofibrosis patient characteristics. Supplementary Table 2. Genetic alterations of patient PMF#11.

Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-kB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and the hMPLW515L murine model of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients.

Myeloid malignancies are characterized by the stepwise acquisition of different somatic mutations in hematopoietic stem and progenitor cells (HSPCs) that promote subsequent leukemic transformation. When these mutations, including in the epigenetic regulator TET2, are found in the blood cells of individuals without any signs of hematologic malignancy, this condition is termed clonal hematopoiesis (CH). The incidence of CH increases with age and has been recognized as a risk factor for the development of secondary heme malignancies and cardiovascular disease. Nonetheless, currently no therapies exist to alter the natural course of CH. Accumulating evidence indicates that inflammatory signals can enhance myeloproliferation of CH HSPCs suggesting a key role of inflammatory stressors in the promotion of the clonal advantage of Tet2-mutant stem cells. However, whether hijacking this inflammatory signaling will prevent clonal expansion and leukemogenesis is currently unknown. The members of the Janus family of nonreceptor tyrosine kinases (JAK) transmit a diversity of ligand-mediated signals and act as a signaling-hub for inflammation. Our central hypothesis is that CH and progression to acute myeloid leukemia (AML) occurs in the setting of inflammatory stress and that mutant clonal expansion is mediated by JAK/STAT inflammatory signaling, primarily through JAK1, a non-essential gene in adult hematopoiesis. We previously showed that Jak1 is critical for stress hematopoiesis in HSPCs. To assess whether Tet2-mediated clonal expansion requires Jak1, signaling, we established a conditional Scl driven Cre-inducible deletion model of Tet2-/- and Jak1-/-. We have adapted a bone marrow derived endothelial cell organoid system that allows to maintain and expand HSPCs during longer periods of time. In this setting Tet2-/- HSPCs show increased sensitivity to IL3, a Jak1-dependent cytokine that mediates exit of quiescence in HSPCs. The loss of competitive advantage of Tet2-/- Jak1-/- cells persists even in the context of co-culture with Jak1-/- Tet2-wildtype cells, and to a lesser extent in the presence of the Jak1 inhibitor Itacitinib, suggesting a denser requirement for Jak1 in CH mutant clones compared to wild-type HSPCs. Ex-vivo colony forming assays and in vivo competitive transplants demonstrate that the self-renewal abilities of Tet2-mutant HSPCs require Jak1 signaling. Furthermore, the extramedullary hematopoiesis observed in a primary model of Tet2-/- pre-leukemic myeloproliferation, was also dependent on Jak1. Moreover, studies in Tet2-/-/Flt3ITD and Mll-AF9 AML models showed that pharmacologic Jak1 inhibition abrogated ex vivo colony formation in both AMLs. This study is significant because targeting JAK/STAT mediated inflammatory signaling in CH-mutant HSPCs has the ability to translate into a precision interception strategy aimed at preventing clonal expansion and leukemic transformation. Citation Format: Pablo Sánchez Vela, Anthony Martinez Benitez, Aishwarya Krishnan, Maria Kleppe, Sheng F. Cai, Ross L. Levine. Targeting JAK1 signaling for molecular prevention in clonal hematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P073.

SUMMARYPro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

Chemotherapy and radiation therapy are the foundations of adjuvant therapy for early-stage breast cancer. As a complication of cytotoxic regimens, breast cancer patients are at risk for therapy-related myeloid neoplasms (t-MNs). These t-MNs are commonly refractory to antileukemic therapies and result in poor patient outcomes. We previously demonstrated that somatic mutations in leukemia-related genes are present in the tumor-infiltrating leukocytes (TILeuks) of a subset of early breast cancers. Here, we performed genomic analysis of microdissected breast cancer tumor cells and TILeuks from seven breast cancer patients who subsequently developed leukemia. In four patients, mutations present in the leukemia were detected in breast cancer TILeuks. This finding suggests that TILeuks in the primary breast cancer may harbor the ancestor of the future leukemogenic clone. Additional research is warranted to ascertain whether infiltrating mutant TILeuks could constitute a biomarker for the development of t-MN and to determine the functional consequences of mutant TILeuks.

Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of AML driven by the most common AF10 fusion proteins, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a comprehensive map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent anti-oncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.STATEMENT OF SIGNIFICANCEGene fusions of AF10/MLLT10 are recurrent in acute myeloid and lymphoid leukemia and are associated with extremely poor survival outcomes. We show that the JAK1 kinase is required for activation of the AF10 fusion oncotranscriptome and for leukemogenesis. Since a number of JAK/STAT pathways inhibitors are in clinical development or approved for use, our studies may help develop a therapeutic strategy for AF10-rearranged leukemias.

Summary Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo . Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo .

Supplemental Digital Content is available in the text, Despite recent advances, the myeloproliferative neoplasms (MPNs) are attended by considerable morbidity and mortality. Janus kinase (Jak) inhibitors such as ruxolitinib manage symptoms but do not substantially change the natural history of the disease. In this report, we show the effects of IMG-7289, an irreversible inhibitor of the epigenetically active lysine-specific demethylase 1 (LSD1) in mouse models of MPN. Once-daily treatment with IMG-7289 normalized or improved blood cell counts, reduced spleen volumes, restored normal splenic architecture, and reduced bone marrow fibrosis. Most importantly, LSD1 inhibition lowered mutant allele burden and improved survival. IMG-7289 selectively inhibited proliferation and induced apoptosis of JAK2 V617F cells by concomitantly increasing expression and methylation of p53, and, independently, the pro-apoptotic factor PUMA and by decreasing the levels of its antiapoptotic antagonist BCLXL. These data provide a molecular understanding of the disease-modifying activity of the LSD1 inhibitor IMG-7289 that is currently undergoing clinical evaluation in patients with high-risk myelofibrosis. Moreover, low doses of IMG-7289 and ruxolitinib synergize in normalizing the MPN phenotype in mice, offering a rationale for investigating combination therapy.

SummaryAlthough clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

Background: In the last few decades, theoretical models of cancer growth and progression have long focused on the aberrations of cancer cells alone, such as the abnormal mitotic and invasive characteristics of cancer cells. More recent research across multiple solid tumors suggests a critical interplay between solid tumors and immune regulating cells. Mounting evidence suggests that the immune system can tip the scales of cancer progression, eliciting either an anti-tumor or pro-tumor immune response depending upon varying stimulating and inhibitory factors. Here, we are the first to demonstrate novel mutations including leukemogenic mutations among tumor infiltrating lymphocytes in breast cancer patients. Methods: We obtained 17 primary breast cancer samples from patients who presented for either a lumpectomy or mastectomy as part of an IRB approved biospecimen protocol. Of the 17 patient samples, 13 had triple negative breast cancer, 2 had ER+, HER2+ disease, and 2 had ER+, HER2- disease. In the 17 samples, we used fluorescent activated cell sorting to separate CD45-positive hematopoietic cells from CD45-negative epithelial cells. We then performed exome sequencing of tumor-infiltrating hematopoietic cells to investigate for the presence of pathogenic mutations in tumor-associated leukocytes. In this first step, we identified candidate mutations in known cancer genes, including BCOR, NOTCH2, TET2, NF1, EZH2, and JAK1. As a validation step, we then performed capture-based sequencing of tumor-infiltrating leukocytes in 20 breast cancer samples matched to each patient’s germline DNA sample (buccal swab). In 10 of the 20 patients, we identified and validated somatic mutations. Of note, 6 of these patients harbored mutations known to be associated with leukemia, including DNTM3A, TET2, and BCOR. Most of these mutations were present in at least 5-20% of reads. This suggests that these mutations were present in enriched subclones and were not rare alleles occurring in a minority of hematopoietic stem cells. Lastly, we performed 454 deep sequencing analysis of microdissected tumor DNA samples and confirmed the absence of these mutations in breast cancer cells. Conclusion: Our data demonstrate somatic mutations in tumor infiltrating leukocytes in breast tumors which were not identified in matched germline or tumor DNA samples. Notably, some of these mutations have been implicated in the pathogenesis of lymphoid and myeloid malignancies. This observation suggests a unique relationship between cancer cells and mutant infiltrating leukocytes. We are now investigating the functional interaction between cancer cells and hematopoietic cells. Our findings reframe our understanding of carcinogenesis and offer novel opportunities for cancer detection and treatment. Citation Format: Elizabeth A Comen, Maria Kleppe, Hannah Wen, Britta Weigelt, Lennart Bastian, Brian Blum, Franck T Rapaport, Matt Keller, Nicolas Socci, Agnes Viale, Daoqi You, Robert Benezra, Edi Brogi, Jorge Reis-Filho, Michael Berger, Ross Levine, Larry Norton. Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-4.

The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. Significance: Our results demonstrate that JAK–STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs. Cancer Discov; 5(3); 316–31. ©2015 AACR. See related commentary by Belver and Ferrando, p. 234 This article is highlighted in the In This Issue feature, p. 213

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens., The complex microenvironmental signalling pathways that govern haematopoietic stem cell (HSC) activity remain poorly defined. Here, the authors identify endothelial NF-κB signalling as regulating regenerative HSC function, accelerating haematopoietic recovery following myelosuppressive injury in mice.

The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells. Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. Disclosures Levine: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Recent studies have implicated the innate immunity system in the pathogenesis of myelodysplastic syndromes (MDS). Toll-like receptor (TLR) genes encode key innate immunity signal initiators. We recently identified multiple genes, known to be regulated by TLRs, to be overexpressed in MDS bone marrow (BM) CD34+ cells, and hypothesized that TLR signaling is abnormally activated in MDS. We analyzed a large cohort of MDS cases and identified TLR1, TLR2 and TLR6 to be significantly overexpressed in MDS BM CD34+ cells. Deep sequencing followed by Sanger resequencing of TLR1, TLR2, TLR4 and TLR6 genes uncovered a recurrent genetic variant, TLR2-F217S, in 11% of 149 patients. Functionally, TLR2-F217S results in enhanced activation of downstream signaling including NF-κB activity after TLR2 agonist treatment. In cultured primary BM CD34+ cells of normal donors, TLR2 agonists induced histone demethylase JMJD3 and interleukin-8 gene expression. Inhibition of TLR2 in BM CD34+ cells from patients with lower-risk MDS using short hairpin RNA resulted in increased erythroid colony formation. Finally, RNA expression levels of TLR2 and TLR6, as well as presence of TLR2-F217S, are associated with distinct prognosis and clinical characteristics. These findings indicate that TLR2-centered signaling is deregulated in MDS, and that its targeting may have potential therapeutic benefit in MDS.

Over the last years, we have witnessed significant improvement in our ability to elucidate the genetic events, which contribute to the pathogenesis of acute and chronic leukemias, and also in patients with myeloproliferative neoplasms (MPN). However, despite significant insight into the role of specific mutations, including the JAK2V617F mutation, in MPN pathogenesis, the precise mechanisms by which specific disease alleles contribute to leukemic transformation in MPN remain elusive. Here we review recent studies aimed at understanding the role of downstream signaling pathways in MPN initiation and phenotype, and discuss how these studies have begun to lead to novel insights with biologic, clinical, and therapeutic relevance.

JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation invivo. Jak1-deficient HSCs exhibit decreased competitiveness invivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued byexpression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.

SummaryGenomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Certain biological features are inherent traits of cancer, yet some of them still hold mysteries for researchers and clinicians. The heterogeneity of a tumor mass is an old concept that has lately become both a puzzling factor and a feature that should be harnessed to better understand tumor vulnerabilities. Improved scientific approaches to further determine and uncover the meaning of these heterogeneous features are still needed to translate findings into ways to develop therapies, identify drug response biomarkers and stratify patients. In Bedside to Bench, Maria Kleppe and Ross L. Levine look at recent clinical cancer trials that have advanced the field and discuss main questions regarding the role of tumor heterogeneity in predicting therapeutic response and tumor progression. In addition, they raise awareness of the relevance of the interactions among different tumor entities and their contribution to the malignancy of the whole tumor. In Bench to Bedside, Kornelia Polyak peruses studies that uncover specific mutations conferring endocrine drug resistance in breast tumors and that add to our knowledge of the evolution and architecture of tumors, and she discusses how this can be used to implement drug regimens.