JAK2V617F Myeloproliferative Neoplasms Research Articles

Endothelial Damage in JAK2V617F Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis.

JAK2V617F-mutated myeloproliferative neoplasms (MPN) exhibit abnormal proliferation of bone marrow progenitors and increased risk of thrombosis, specifically in splanchnic veins (SVT). The contribution of the endothelium to the development of the prothrombotic phenotype was explored. Plasma and serum samples from JAK2V617F MPN patients with (n=26) or without (n=7) thrombotic debut and different treatments, were obtained (n=33). Cultured endothelial cells (ECs) were exposed to serum samples from these patients and from healthy donors as controls. Changes in markers of inflammation (VCAM-1, ICAM-1), cell permeability (VE-cadherin), production of VWF, extracellular matrix (ECM) reactivity, and activation of intracellular signaling pathways related to stress, proliferation, inflammation (Akt, p44/42, IkBa), and JAK2/STAT3 pathway, were assessed by immunofluorescence, flow adhesion, SDS-PAGE and immunoblot. Additionally, circulating markers of endothelial activation and damage (VWF, sVCAM-1, sTNFRI, thrombomodulin, angiopoietin-2, a2-antiplasmin activity, PAI-1) were evaluated in Patients' plasma. The in vitro studies showed that EC exposure to MPN thrombotic patients' sera resulted in increased VCAM-1 and ICAM-1, and reduced VE-cadherin expression (p<0.05) at the cell surface. Production and release of VWF to the ECM were higher (p<0.05), with increased platelet adhesion after perfusing whole blood, being more noticeable in response to sera from non-treated patients. Furthermore, intracellular activation of Akt, p44/42, IkBa and JAK2/STAT3 was observed. Moreover, plasma levels of VWF, TNF-R1, VCAM-1, thrombomodulin, and angiopoietin-2 were higher in JAK2V617F+ MPN patients with thrombosis. The present findings suggest that circulating factors in MPNs with SVT debut induce endothelial proinflammatory and prothrombotic phenotypes, which are modulated in vitro with MPN treatment.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5592.

Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53−/− mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53−/−) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53−/−-reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.

Exploiting Synthetic Lethality between Germline BRCA1 Haploinsufficiency and PARP Inhibition in JAK2V617F-Positive Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/- Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/- cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/- cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/- cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.

Single Cell Transcriptomics Reveal That HMGA1 Chromatin Regulators Drive Transcriptional Networks Involved in Megakaryocyte Expansion, Platelet Activation, and Interferon Signaling in JAK2 V617F Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling, thromboses, abnormal bleeding, and increased risk of transformation to myelofibrosis (MF) or acute myeloid leukemia (AML). Importantly, clinical outcomes are poor after transformation and actionable mechanisms that drive progression remain elusive. We discovered that High Mobility Group A1 ( HMGA1) chromatin regulators are required for transformation to AML in preclinical models of MPN by up-regulating transcriptional networks involved in GATA2 and proliferation (Li et al, Blood, 2022). Unexpectedly, loss of just a single Hmga1 allele within HSC prevents progression to MF in JAK2 V617F mice. Intriguingly, Hmga1 also drives fibrosis and tumor progression in pancreatic tumor models (Chia et al, JCI 2023). We therefore sought to elucidate mechanisms underlying HMGA1 early in progression that could be targeted in therapy to prevent MF. Methods : To elucidate Hmga1 function early in MPN, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells from JAK2 V617F mice at 16 weeks old before the development of MF with both Hmga1 alleles intact ( Hmga1+/+) compared to those with haploinsufficiency ( Hmga1+/-). We used gene set enrichment analysis (GSEA) to identify Hmga1-dependent transcriptional networks, CellChat to define cell interactions, and trajectory analysis to assess differentiation. To identify relevant networks in patients, we assessed HMGA1 pathways in transcriptomes from platelets, peripheral blood mononuclear cells, or CD34 + stem and progenitor cells from MPN patients and healthy, age-matched controls via bulk or scRNAseq. Results: We discovered that loss of just a single Hmga1 allele in HSPC is sufficient to dampen expansion of long-term, quiescent HSC (qHSC), megakaryocyte-biased HSC (Mk-HSC), and megakaryocyte-erythroid-biased HSC (MEP-HSC) while expanding HSC with a lymphoid bias (Ly-HSC) in JAK2 V617Fmice by scRNAseq. Pathway analysis in the most quiescent HSC clusters reveal that Hmga1 induces pathways involved in interferon α (IFNα) and interferon γ (IFNγ) signaling. Trajectory analysis show that JAK2 V617F qHSC with intact Hmga1 have diminished differentiation capacity, leading to decreased Ly-HSC. Because Hmga1 haploinsufficiency dramatically decreased megakaryocyte hyperplasia and thrombocytosis in JAK2 V617Fmice, we focused on HMGA1 transcriptional networks in Mk-HSC and MEP-HSC. Similar to qHSC, Hmga1 up-regulates networks involved in IFNα and IFNγ signaling in Mk- and MEP-biased HSC. In contrast to the qHSC, Hmga1 activates transcriptional networks involved in cell cycle progression in HSC poised to differentiate into Mk and MEP, including: 1) MYC Target pathways, 2) E2F Targets, and, 3) Mitotic Spindle genes. In addition, gene networks involved in oxidative phosphorylation are activated, which associate with proliferative states. Unexpectedly, we also discovered that Hmga1 up-regulates genes involved in coagulation and platelet activation, including genes encoding Platelet factor 4, von Willebrand factor, glycoprotein 2b, glycoprotein 3a, and P-selectin. Because megakaryocytes are associated with inflammatory signals in MPN, we queried inflammatory gene networks, which revealed that Hmga1 induces genes encoding IL6, IL1-β, and gene networks involved in signaling through TNFα and NF-κB. CellChat shows that Hmga1 increases MIF receptor-ligand signaling. Moreover, HMGA1 and similar networks are up-regulated in transcriptomes of platelets, PBMC, and CD34 + cells from patients with JAK2 V617F MPN. Conclusions: We uncovered a previously unknown epigenetic program in JAK2 V617FMPN whereby Hmga1 induces transcriptional networks that drive expansion in mutant HSC with greatest impact on HSC biased to differentiate into megakaryocytes (Mk-HSC, MEP-HSC). Mechanistically, Hmga1 up-regulates genes involved in cell cycle progression, platelet activation, and inflammatory signaling in Mk-biased HSC. HMGA1 also induces genes involved in IFNα and IFNγ signaling. Together, our studies reveal a new paradigm whereby HMGA1 drives progression early in MPN by activating transcriptional networks required for megakaryocyte expansion, inflammatory signaling, and IFN networks, the latter of which may sensitize JAK2 mutant HSC to IFN therapy.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Combined Myeloid and Lymphoid Malignancies in 13 Elderly Patients

Disclosures Myeloid malignancies such as myelodysplasia (MDS) often evolve from age related clonal hematopoiesis (CH) with progression of variant allele frequencies. The Philadelphia negative myeloproliferative neoplasms (MPN) polythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are associated with mutations in JAK2, CALR, or MPL. Multiple myeloma (MM) and monoclonal gammopathies (MGUS) are felt to be age related, and the malignant cell is of lymphoid origin. Burkitt's Lymphoma is associated with EBV exposure and dysregulation of c-myc. However the etiology of most lymphoproliferative disorders (LPD) is not defined, but immune dysregulation may be causative. The combination of myeloid and lymphoid malignancies are not recognized to be a common occurrence; however within a three year period 13 patients with combined myeloid and lymphoid malignancies were detected in a single community practice. DATA Five patients had combined myeloid and lymphoid malignancies diagnosed concurrently. Three had a lymphoid disorder and then developed a myeloid disorder. Five had a myeloid disorder followed by a lymphoid disorder. Three patients had multiple myeloma (MM) and two had MGUS. Initially all patients responded to standard therapies, One patient at 87 years died from end stage renal disease likely related to her myeloma. Another patient with CLL and PV died at 92 years from an epithelial malignancy. Concurrent diagnoses Age at diagnosis (years) CLL ET 76MGUS MDS 77MM MDS 78MM MDS (RSBA) 85 5) DLBCL MF. 60 LPD/MM followed by a Age at initial diagnosis (years) and time to second diagnosis myeloid disorder. 1) MZL MF 58 15 months 2) MM ET 83 7 months 3) CLL PV 86 4 years Myeloid disorder followed by a LPD/MGUS MDS DLBCL 76 3 monthsMDS MGUS 78 30 monthsTP CLL 15 60 yearsET MyF 77 65 monthsET CLL 60 15 years (CLL chronic lymphocytic leukemia, DLBCL diffuse large B cell lymphoma, MF myelofibrosis, MyF mycosis fungoides, MZL mantle zone lymphoma, RSBA ringed sideroblastic anemia, TP inherited thrombocytopenia) JAK2 mutations were found in three of the four ET patients and in the one PV patient. One MF patient and one ET patient were negative for the JAK2 and CALR mutations. The mutation status of the other MF patient was not known. At diagnosis the patient with inherited thrombocytopenia was suspected to have a myeloid disorder. Combined myeloid and lymphoid malignancies is not well defined and is a seemingly uncommon event. Yet an Italian study demonstrated that MPN patients with JAK2 mutations have a 2.8-3.4 fold increased risk of developing a LPD compared with an age matched population (1). The linkage is unclear. Immune senescence may be important for the development of LPD observed in the elderly. Also long term latency of many years between the JAK2V617F mutation acquisition and MPN presentation has been documented (2). The occurrence of both myeloid disorders and LPD is not well recognized. Future studies may provide insights into the mechanisms underlying these disorders. Increasing genetic complexity and immune senescence associated with aging likely plays a major role. The limitations of this study are its small size, single community investigator, retrospective clinical nature, and the lack of gene sequencing and other molecular studies in most if not all of the patients. This observation was noted over three years in a single community practice which strongly implies that combined occurrence may be more common than recognized and suggests that further investigation is warranted. Boxer none, Denes none

JAK2 V617F Impairs Lymphoid Differentiation in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2 V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2 V617F clone does not contribute to mature lymphocytes. We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2 V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2 V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2 V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2 V617F alleles were largely absent in T and B cells relative to HSCs. Lymphopoiesis declines with age, potentially explaining the absence of JAK2 V617F in lymphocytes from older patients with MPN even if JAK2 V617F has no effect on lymphopoiesis. This “JAK2 V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2mutated lymphocytes. However, we found that JAK2 V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2 V617F neutral” hypothesis are not supported by patient data. The alternative explanation is that JAK2 V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2 V617F adverse” hypothesis by tracking JAK2 V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2 V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2 V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2 V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2 V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2 V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation. To further define the extent to which JAK2 V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2 V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2 V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2 V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis. To determine the effect of JAK2 V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2 V617F or wild type JAK2 (JAK2 WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2 V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2 V617F HSCs displayed a competitive disadvantage relative to their JAK2 WT counterparts during lymphopoiesis in vivo. Myeloid proliferation in MPN driven by JAK2 V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2 V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2 V617F interferes with T cell differentiation.

Trp53-Loss in Hematopoietic Stem Cells Drives the Evolutionary Process of Leukemic Transformation in a Jak2V617F-Driven Myeloproliferative Neoplasms Mouse Model

Myeloproliferative neoplasms (MPNs) are clonal disorders resulting from genetic lesions in hematopoietic stem cells (HSCs). The development of post-MPN AML is treatment resistant and associated with very poor outcomes. This leukemic transformation is due to TP53 inactivation in approximately 50% of cases. Existing mouse models with double mutant Jak2V617F and Trp53 have demonstrated that Trp53-lossalone is sufficient to induce leukemic transformation in Jak2V617F-driven MPN. These models were generated either by crossing Jak2V617F transgenic mice with Trp53R172H knock-in or Trp53 knock-out mice, or by retroviral expression of Jak2V617F in Trp53 knock-out bone morrow (BM) cells. These germline mutated models do not accurately model the sequential somatic Trp53 mutations that arise in Jak2V617F MPN, or track their clonal expansion in vivo. Here, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice to generate a novel mouse model of high molecular risk MPN that transforms to AML. We isolated lineage -/low Sca1 +c-Kit + (LSK) cells from BM of donor mice expressing a conditional allele of mutant Jak2 from its endogenous locus ( Jak2fl-V617F) in combination with an inducible Cas9-IRES-GFP allele and CreER-fusion allele, both from the Rosa26 locus ( Rosa26CreER/lsl-Cas9-IRES-GFP). LSKs were transduced with lentivirus expressing a sgRNA targeting exon 4 of Trp53 (sgTrp53), located in the DNA-binding domain of Trp53. These transfected sgTrp53 LSKs, along with control groups transduced with either empty vector (EV) or a sgRNA targeting Catsper1 (sgCatsper1, off target cutting control), were transplanted into lethally irradiated recipients. Successful engraftment and CreER activity, induced by a 2-week period of Tamoxifen chow, were confirmed by flow cytometry. Validation of Trp53-editing was performed using amplicon sequencing, revealing the most common mutations were frameshift mutations. A subset of these frameshift mutations exhibited an extremely high frequency, conferring a selective advantage within the cell populations in sgTrp53 mice. We further examined the levels of p53 induced by nutlin-3a (an MDM2 inhibitor) in edited BM cells from three groups of mice and found that Trp53 editing leads to a reduction in p53 levels and prevents the loss of viability in response to MDM2 inhibition. This indicates that CRISPR-Cas9 mediated Trp53 mutations result in a loss of function. SgTrp53 mice recapitulated the evolution of leukemic transformation of MPN, showing a PV phenotype for 8 weeks post CreER activation, characterized by elevated hematocrit but normal leukocytes and platelet counts, consistent with the phenotype exhibited by the EV and sgCatsper1 recipients. Subsequently, after 12 weeks, this MPN phenotype transformed to acute leukemia. This disease progression was associated with significantly shortened survival of sgTrp53 recipients, compared to the sgCatsper1 or EV control groups. Histopathologic analysis further supported these findings, showing leukocytosis and the presence of immature blast cells in the PB, spleen and BM of sgTrp53 mice, whereas such cells were absent in the control groups. Notably, the frequency of blast cells was correlated with the frequency of Trp53 frameshift mutations, consistent with what has been reported in MPN patients. Additionally, CRISPR-Cas9 mediated Trp53-loss primarily affected the erythroid lineage, driving expansion of erythroblasts, megakaryocyte-erythroid progenitors (MEP), and Lineage -/low Sca1 -c-Kit + in sgTrp53 mice. The erythroblasts showed aberrant co-expression of CD41 and CD71. Through secondary transplants with unfractionated spleen cells, whole BM cells, or sorted MEP cell populations, we confirmed that the leukemia driven by CRISPR-cas9 mediated Trp53-loss in the Jak2V617F-driven PV is transplantable, and that the MEP compartment in leukemic sgTrp53 mice contains the leukemia initiation population. In total, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice, faithfully replicating the process of clonal expansion and evolution observed in the leukemic transformation of MPN. These mouse models of secondary AML are valuable for testing new therapies targeting disease initiating stem cells. Moreover, our models represent the distinct stages of MPN progression, offering a platform to evaluate preventive and therapeutic interventions at various stages of this disease.

HMGA1 Chromatin Regulators Drive Immune Evasion in MPN Progression through Epigenetic Rewiring to Repress Antigen Presentation

Introduction: Myeloproliferative Neoplasms (MPN) are clonal blood diseases caused by hyperactive JAK/STAT signaling and overproduction of myeloid lineages. MPN patients are at increased risk for transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), both resulting in poor clinical outcomes. Importantly, targetable mechanisms to prevent progression remain elusive. We discovered that HMGA1 chromatin regulators are required for leukemogenesis and progression to MF in JAK2 V617Fmice (Li et al, Blood 2022). Emerging evidence suggests that immune escape is a fundamental pathway required for progression in diverse tumors. We therefore sought to: 1) determine whether HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be modulated in therapy. Methods: To identify pathways regulated by HMGA1 in JAK2 V617F MPN, we performed multi-omics sequencing (seq), including RNAseq, chromatin immunoprecipitation (ChIPseq), and assays of chromatin accessibility (ATACseq) in JAK2 V617Fmutant AML cell lines (DAMI, SET-2, UKE-1) + HMGA1 depletion. HMGA1 was inactivated via CRISPR or short hairpin RNA (shRNA). We used gene set enrichment analysis (GSEA) to dissect molecular mechanisms underlying HMGA1. Transcriptional networks were validated (mRNA, protein) via quantitative(q)-PCR, immunoblots, and flow cytometry. ChIP-PCR was used to assess chromatin binding of HMGA1, repressive histones, and the CCCTC-binding factor (CTCF). Human T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) for co-culture assays. To identify pathways relevant in patients, we assessed gene expression in MPN cohorts. Results: To elucidate transcriptional networks governed by HMGA1 in MPN, we integrated multiomics approaches (RNA/ChIP/ATACseq) in JAK2 V617F mutant AML cells, which revealed that HMGA1 represses genes involved in interferon γ (IFNγ) signaling and antigen presentation, including genes encoding major histocompatibility complex (MHC) class I and II proteins. HMGA1 also represses the CD74 gene which encodes a chaperone protein that assembles MHC Class II at the cell surface. Silencing HMGA1 via CRISPR or shRNA results in up-regulation of CD74 and MHC genes, with greatest induction of MHC Class II genes ( HLA-DRA, DRB1, DPA1, DPB1). Similarly, HMGA1 depletion increases protein levels of MHC Class II and CD74 by flow cytometry and/or immunoblots. To determine how HMGA1 represses MHC Class II genes, we examined genome architecture modulated by HMGA1 at the MHC Class II loci. By ChIP-PCR, HMGA1 recruits repressive histone marks to promoter regions for MHC Class II genes. Strikingly, HMGA1 depletion enhances chromatin accessibility at critical enhancers (super enhancers) involved in MHC Class II gene expression which overlap with binding sites for CTCF, a chromatin regulator involved in genome organization. Further analyses revealed that HMGA1 chromatin occupancy precludes binding by CTCF, whereas HMGA1 silencing allows CTCF binding to sites within the MHC Class II loci required for MHC Class II gene expression. Because the histone deacetylase inhibitor, entinostat, induces MHC Class II genes in solid tumors, we assessed MHC Class II gene expression with entinostat and HMGA1 depletion. HMGA1 silencing enhances up-regulation of MHC Class II genes by entinostat. Finally, to test the functional significance of this pathway in immune evasion, we performed co-culture experiments with T cells and JAK2 V617F AML cells which showed that HMGA1 silencing in AML cells results in T cell activation and cytotoxic T cell killing of JAK2 V617F AML cells. To examine the relevance of this pathway in patients, we interrogated RNAseq from PBMCs and found that HMGA1 is up-regulated and associated with repression in MHC Class II genes in 30% of MPN patients after progression to AML. Conclusions: We discovered a novel epigenetic program whereby HMGA1 drives immune evasion during MPN progression by repressing MHC Class II genes. We also define a new mechanism of gene repression by HMGA1 through chromatin compaction and repressive histone marks that preclude CTCF binding to the MHC Class II loci. Both HMGA1 depletion and entinostat up-regulate MHC Class II genes, thus unveiling HMGA1 as a therapeutic target to stimulate an immune attack and prevent MPN progression.

Platelets and neutrophils cooperate to induce increased neutrophil extracellular trap formation in JAK2V617F myeloproliferative neoplasms

BackgroundNeutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. ObjectivesThis study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. MethodsNETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. ResultsIn PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. ConclusionOur study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.

Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2V617F myeloproliferative neoplasms

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are chronic malignancies associated with high-risk complications and suboptimal responses to JAK inhibitors such as ruxolitinib. A better understanding of cellular changes induced by ruxolitinib is required to develop new combinatory therapies to improve treatment efficacy. Here, we demonstrate that ruxolitinib induced autophagy in JAK2V617F cell lines and primary MPN patient cells through the activation of protein phosphatase 2A (PP2A). Inhibition of autophagy or PP2A activity along with ruxolitinib treatment reduced proliferation and increased the death of JAK2V617F cells. Accordingly, proliferation and clonogenic potential of JAK2V617F-driven primary MPN patient cells, but not of normal hematopoietic cells, were markedly impaired by ruxolitinib treatment with autophagy or PP2A inhibitor. Finally, preventing ruxolitinib-induced autophagy with a novel potent autophagy inhibitor Lys05 improved leukemia burden reduction and significantly prolonged the mice’s overall survival compared with ruxolitinib alone. This study demonstrates that PP2A-dependent autophagy mediated by JAK2 activity inhibition contributes to resistance to ruxolitinib. Altogether, our data support that targeting autophagy or its identified regulator PP2A could enhance sensitivity to ruxolitinib of JAK2V617F MPN cells and improve MPN patient care.

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions

BackgroundVascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. ObjectivesWe sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. MethodsWe assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. ResultsWe found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. ConclusionsBy using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte–endothelial adhesion.

Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2V617F mutation

BackgroundJAK2V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis. ObjectivesOur main objective was to explore and compare platelet function depending on the MPN molecular marker. MethodsWe analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels. ResultsWe observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN. ConclusionsThese results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR.

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression.

Myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed single-cell RNA sequencing on serial MPN and sAML patient stem and progenitor cells, identifying aberrantly increased expression of DUSP6 underlying disease transformation. Pharmacologic dual-specificity phosphatase (DUSP)6 targeting led to inhibition of S6 and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling while also reducing inflammatory cytokine production. DUSP6 perturbation further inhibited ribosomal S6 kinase (RSK)1, which we identified as a second indispensable candidate associated with poor clinical outcome. Ectopic expression of DUSP6 mediated JAK2-inhibitor resistance and exacerbated disease severity in patient-derived xenograft (PDX) models. Contrastingly, DUSP6 inhibition potently suppressed disease development across Jak2V617F and MPLW515L MPN mouse models and sAML PDXs without inducing toxicity in healthy controls. These findings underscore DUSP6 in driving disease transformation and highlight the DUSP6-RSK1 axis as a vulnerable, druggable pathway in myeloid malignancies.

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations activating JAK2 in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. MPN patients are at increased risk for transformation to bone marrow fibrosis (myelofibrosis) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. Importantly, targetable mechanisms to prevent progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in hematopoietic stem cells and aberrantly overexpressed in MPN with highest levels after transformation to myelofibrosis and acute leukemia (Li & Kim et al Blood 2022, Xian et al Nature Commun 2017). We recently discovered that HMGA1 is required for progression to leukemia in murine models of JAK2V617F MPN leukemia. Further, loss of just a single Hmga1 allele prevents progression to myelofibrosis in JAK2V617F transgenic mice. Emerging evidence suggests that immune escape represents a fundamental pathway involved in progression in diverse tumors. We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy. Methods: To identify gene networks regulated by HMGA1 in JAK2V617F MPN, we performed multi-omics sequencing analyses, including RNAseq, chromatin immunoprecipitation (ChIPseq), and assays of chromatin accessibility (ATACseq) from JAK2V617Fmutant AML cell lines (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNAs (shRNA) targeting 2 different sequences. We used gene set enrichment analysis to dissect molecular mechanisms underlying HMGA1. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays. To validate these pathways in human MPN, we assessed gene expression in independent MPN patient cohorts. Results: To elucidate transcriptional networks governed by HMGA1 during MPN progression to leukemia, we integrated RNAseq, ChIPseq, and ATACseq in JAK2V617F mutant AML cells (DAMI, SET-2) which revealed that HMGA1 represses genes involved in antigen presentation (Interferon γ response networks), including genes encoding major histocompatibility complex (MHC) class I and II antigens. Silencing HMGA1 via CRISPR or shRNA results in up-regulation of MHC class I and II antigen genes, with greatest induction of MHC class II genes HLA-DRA/DRB1. Similarly, HMGA1 depletion increases cell surface expression of HLA-DR antigens by flow cytometry. Further, inhibiting HMGA1 in JAK2V617Fmutant AML cells results in up-regulating the class II major histocompatibility complex transactivator (CIITA) and invariant chain (CD74), which function in inducing MHC class II gene expression and chaperoning structure assembly, respectively. To identify underlying mechanisms, we intersected ChIPseq with ATACseq, which revealed HMGA1 occupancy downstream of the HLA-DRB1 locus, including regions at +6.27kb, +6.7kb, +14kb, and +15kb from the transcription start site. Further, preliminary results suggest that HMGA1 recruits repressive histone marks to repress HLA-DRB1 at these sites. To target HMGA1 networks, we queried CMAP, which identified the histone deacytelyase inhibitor (HDACi), entinostat. Strikingly, entinostat not only up-regulates MHC class II genes and antigens, but is also cytotoxic in JAK2V617F mutant AML cells. Finally, HMGA1 depletion enhances sensitivity to entinostat, which synergizes with JAK inhibitor, Ruxolitinib, to further induce MHC gene expression. Conclusions: We discovered a novel epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and modulating transcriptional networks to repress MHC antigen presentation. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN after progression to AML. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks with entinostat could activate an immune attack and prevent MPN progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Escape from Type II JAK2 Inhibition in MPN Depends on AXL/MAPK Activation and Is Targetable

Myeloproliferative neoplasms (MPN) are characterized by dysregulated JAK2 signaling. Clinical type I JAK2 inhibitors as ruxolitinib are hampered by limited disease-modifying effects and occurrence of resistance. Based on pre-clinical data, novel type II JAK2 inhibitors as CHZ868 effectively stabilize inactive JAK2, reduce MPN clones and overcome resistance to ruxolitinib. We study whether MPN cells could escape from type ll JAK2 inhibition, and characterize therapeutic approaches. To assess potential escape from type II JAK inhibition, JAK2V617F MPN cells incl. SET2, UKE-1 and Ba/F3 EPOR Jak2V617F were exposed long-term to CHZ868. Outgrowing cells showed significantly increased IC50 to CHZ868 and reduced susceptibility for apoptosis induction. No second-site mutations in JAK2 were detected. Phosphoproteomic analysis identified >2500 differentially phosphorylated sites with most prominent activation of the MAPK pathway in JAK inhibitor resistant cells. Type II JAK inhibition persistently suppressed JAK2 - STAT3/5 signaling, while MAPK target activation incl. MEK and ERK was prominent also by phospho-immunoblot suggesting MAPK pathway signaling as a driver of escape from type II JAK inhibition. To characterize the molecular basis, we profiled differential gene expression and chromatin accessibility by RNA-sequencing and ATAC-sequencing in MPN cells escaping JAK inhibition. Paired analysis of RNA-seq/ATAC-seq revealed enrichment of H3K27me3 and PRC2 signatures with overall more compacted chromatin. Expression of AXL tyrosine kinase known to signal through the MAPK pathway, was significantly increased along with enriched RAS signaling target gene patterns by GSEA. Motif analysis suggested differential transcriptional programs with loss of ETS and increased accessibility of GATA transcription factor family binding sites. Enhanced AXL expression was confirmed by immunoblotting and depletion of AXL by AXL-specific shRNA knock-down resensitized resistant cells to CHZ868 with decreased IC50. Since these findings pointed towards a role of AXL activation for escape of MPN cells from type II JAK inhibition, we targeted AXL activation using respective inhibitors. AXL inhibitor bemcentinib and AXL/FLT3 inhibitor gilteritinib reduced proliferation of resistant cells with up to 5.6-fold and 3.4-fold decreased IC50, respectively. Given that type II JAK inhibition with CHZ868 maintained suppression of JAK2-STAT3/5 activation in resistant cells, we combined JAK2 and AXL inhibition. Notably, dual JAK2 / AXL inhibition with CHZ868 / bemcentinib or CHZ868 / gilteritinib showed most pronounced inhibitory effects with IC50 similar to values seen in JAK inhibitor sensitive cells and effective induction of apoptosis. Resistant MPN cells engrafted subcutaneously into immunocompromised NSG mice and resulted in robust tumor growth despite JAK2 inhibitor treatment. However, AXL inhibition with bemcentinib reduced progression of type II JAK inhibitor resistant tumors. Combined JAK2 and AXL inhibition showed most pronounced suppression of subcutaneous tumor growth with minimal progression. Given that inhibitors of MAPK signaling targeting e.g. MEK-ERK activation, are in clinical use for other malignancies with favorable tolerability profiles, we then characterized combined JAK2 / MAPK inhibition to overcome AXL-MAPK driven escape of MPN cells from type II JAK inhibitors. Combination of CHZ868 and MEK inhibitor trametinib significantly reduced subcutaneous tumor growth in NSG mice similarly to JAK2 / AXL inhibition. Expression of MAPK pathway effectors as DUSP6 was substantially reduced. Furthermore, intravenous engraftment of JAK inhibitor resistant cells into NSG mice led to substantial bone marrow (BM) infiltration shown by luciferase imaging and hCD45 immunohistochemistry despite treatment with CHZ868. Combined JAK2 / MAPK inhibition with CHZ868 and trametinib significantly reduced BM infiltration by luciferase imaging and histology supporting that escape from type II JAK inhibition is dependent on AXL-MAPK activation. In summary, we report on a novel mechanism of AXL-MAPK driven escape from type II JAK2 inhibition which is targetable in vitro and in vivo. Our findings highlight that rationally designed combination therapy approaches can restore sensitivity to JAK inhibitor therapy in MPN, which may have potential implications for other malignant settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

An inherited gain-of-function risk allele in EPOR predisposes to familial JAK2V617F myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are mainly sporadic but inherited variants have been associated with higher risk development. Here, we identified an EPOR variant (EPORP488S ) in a large family diagnosed with JAK2V617F -positive polycythaemia vera (PV) or essential thrombocytosis (ET). We investigated its functional impact on JAK2V617F clonal amplification in patients and found that the variant allele fraction (VAF) was low in PV progenitors but increase strongly in mature cells. Moreover, we observed that EPORP488S alone induced a constitutive phosphorylation of STAT5 in cell lines or primary cells. Overall, this study points for searching inherited-risk alleles affecting the JAK2/STAT pathway in MPN.

HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy.Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP).Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617F murine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression.Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. [Display omitted] DisclosuresRampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.