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PRMT5 Inhibition Modulates E2F1 Methylation and Gene-Regulatory Networks Leading to Therapeutic Efficacy in JAK2^V617F-Mutant MPN.

ABSTRACT: We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation ex vivo. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2^V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPL^W515L model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis in vivo. PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. SIGNIFICANCE: Expression of PRMT5 and E2F targets is increased in JAK2^V617F+ MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2^V617F/MPL^W515L MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated.This article is highlighted in the In This Issue feature, p. 1611.

SUBMITTER: Pastore F

PROVIDER: S-EPMC7642059 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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PRMT5 Inhibition Modulates E2F1 Methylation and Gene-Regulatory Networks Leading to Therapeutic Efficacy in JAK2V617F-Mutant MPN.

Pastore Friederike F Bhagwat Neha N Pastore Alessandro A Radzisheuskaya Aliaksandra A Karzai Abdul A Krishnan Aishwarya A Li Bing B Bowman Robert L RL Xiao Wenbin W Viny Aaron D AD Zouak Anouar A Park Young C YC Cordner Keith B KB Braunstein Stephanie S Maag Jesper L JL Grego Alexander A Mehta Jaanvi J Wang Min M Lin Hong H Durham Benjamin H BH Koche Richard P RP Rampal Raajit K RK Helin Kristian K Scherle Peggy P Vaddi Kris K Levine Ross L RL

Cancer discovery 20200715 11

We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation ex vivo. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis ...[more]

PMID: 32669286

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Project description:BackgroundEvasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo.MethodsTo investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2V617F-driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2V617F-driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis.ResultsWe found that JAK2V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2V617F-driven MPN.ConclusionLCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN.

Project description:RationaleThe mechanisms driving atherothrombotic risk in individuals with JAK2 V617F ( Jak2 VF) positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood.ObjectiveThe goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2 VF expression.Methods and resultsIrradiated low-density lipoprotein receptor knockout ( Ldlr-/-) mice were transplanted with bone marrow from wild-type or Jak2 VF mice and fed a high-fat high-cholesterol Western diet. Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of Western diet, Jak2 VF mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of Western diet, Jak2 VF lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition, and costaining of erythrocytes and macrophages, suggesting erythrophagocytosis. Jak2 VF erythrocytes were more susceptible to phagocytosis by wild-type macrophages and showed decreased surface expression of CD47, a "don't-eat-me" signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2 VF macrophages displayed increased expression and production of proinflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAPK (mitogen-activated protein kinase) signaling, and reduced levels of MerTK (c-Mer tyrosine kinase), a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis.ConclusionsHematopoietic Jak2 VF expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2 VF caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated proinflammatory responses, likely contribute to plaque instability.

Project description:IntroductionSomatic mutations in myeloid growth factor pathway genes, such as JAK2, and genes involved in epigenetic regulation, such as TET2, in hematopoietic stem cells (HSCs) leads to clonal hematopoiesis of indeterminate potential (CHIP) which presents a risk factor for hematologic malignancy and cardiovascular disease. Smoking behavior has been repeatedly associated with the occurrence of CHIP but whether smoking is an environmental inflammatory stressor in promoting clonal expansion has not been investigated.MethodsWe performed in vivo smoke exposures in both wildtype (WT) mice and transplanted mice carrying Jak2V617F mutant and Tet2 knockout (Tet-/-) cells to determine the impact of cigarette smoke (CS) in the HSC compartment as well as favoring mutant cell expansion.ResultsWT mice exposed to smoke displayed increased oxidative stress in long-term HSCs and suppression of the hematopoietic stem and progenitor compartment but smoke exposure did not translate to impaired hematopoietic reconstitution in primary bone marrow transplants. Gene expression analysis of hematopoietic cells in the bone marrow identified an imbalance between Th17 and Treg immune cells suggesting a local inflammatory environment. We also observed enhanced survival of Jak2V617F cells exposed to CS in vivo and cigarette smoke extract (CSE) in vitro. WT bone marrow hematopoietic cells from WT/Jak2V617F chimeric mice exposed to CS demonstrated an increase in neutrophil abundance and distinct overexpression of bone marrow stromal antigen 2 (Bst2) and retinoic acid early transcript 1 (Raet1) targets. Bst2 and Raet1 are indicative of increased interferon signaling and cellular stress including oxidative stress and DNA damage, respectively. In chimeric mice containing both WT and Tet2-/- cells, we observed an increased percentage of circulating mutant cells in peripheral blood post-cigarette smoke exposure when compared to pre-exposure levels while this difference was absent in air-exposed controls.ConclusionAltogether, these findings demonstrate that CS results in an inflamed bone marrow environment that provides a selection pressure for existing CHIP mutations such as Jak2V617F and Tet2 loss-of-function.

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PRMT5 Inhibition Modulates E2F1 Methylation and Gene-Regulatory Networks Leading to Therapeutic Efficacy in JAK2^V617F-Mutant MPN.

Publications

PRMT5 Inhibition Modulates E2F1 Methylation and Gene-Regulatory Networks Leading to Therapeutic Efficacy in JAK2<sup>V617F</sup>-Mutant MPN.

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