Abstract: Dysregulation of the protein arginine methyltransferase, PRMT5, occurs in a variety of malignancies and is associated with increased cancer cell proliferation. We investigated the role of PRMT5 in MPN pathogenesis through use of C220, a potent and selective small molecule PRMT5 inhibitor, and aimed to elucidate key PRMT5 targets contributing to the maintenance of MPN cells. We found that PRMT5 is overexpressed in MPN, most highly in PV. C220 dose-dependently reduced the proliferation of MPN cell lines and BaF3 cells expressing JAK2V617F in vitro. Human CD34+ cells from PV/PMF patients showed a high degree of sensitivity to C220 ex vivo. Consistent with the in vitrostudies, C220 treatment of SET2 xenografts in vivoled to a 71% reduction of median tumor volume, greater than that observed with JAK inhibition (59%). We assessed the efficacy of C220 in Jak2V617F- and MPLW515L-driven murine MPN models.In the conditional Jak2V617Fmodel of PV, mice treated with C220 displayed significant lower hematocrits (median 50.1% vs. 77.4 %, p & lt;0.0001), WBC (median 4.5 K/µl vs. 16.9 K/µl, p & lt;0.0001), reticulocytes (median 73 K/µl vs. 1522 K/µl, p & lt;0.0001) and splenomegaly (median weight 68 mg vs. 270 mg, p & lt;0.0001) compared to vehicle treatment. CD105+CD150- progenitors, committed erythroid progenitors (Pre-CFU-E), MEP, CD71-Ter119+ pro-erythroblasts and CD71+/Ter119+ erythroblasts were significantly decreased with C220 treatment [15.7% (p & lt;0.0001), 36% (p=0.0004), 58% (p=0.01), 10% (p & lt;0.0001) and 11% (p & lt;0.0001), respectively, relative to vehicle]. In the MPLW515Ladoptive transfer model of ET/MF, C220 attenuated leukocytosis (WBC 11.1 K/µl vs. 155.3 K/µl, p=0.023), splenomegaly (136 mg vs. 423 mg, p=0.003), hepatomegaly (1118 mg vs. 1677 mg, p=0.004), and BM fibrosis (reticulin score 0.5 vs. 2+). C220 significantly reduced serum cytokines in the Jak2V617F(Eotaxin, GM-CSF, IL-12p40, LIX, KC, MCP1, MIP1a, MIP-2, M-CSF, RANTES, IP-10, IL-1b) and MPLW515L(MIP1b, TNF- alpha, RANTES, IP-10 and IL-1b) model consistent with inhibition of systemic inflammation characteristic of MPN. We next assessed the efficacy of combined PRMT5 and JAK inhibition. Dual targeting of JAK and PRMT5 displayed additive effects on SET2 cell proliferation in vitro and was superior to monotherapy with either agent in vivo. In the Jak2V617Fmodel, dual JAK/PRMT5 inhibition further reduced key features such as splenomegaly (median weight 39 mg vs. 68 mg or 127 mg, p=0.0004 and p & lt;0.0001), elevated hematocrit (median 41.1% vs. 50.1% or 85.1%, p=0.0089 and p=0.0009), WBC (median 2.1 K/µl vs. 4.5 K/µl or 7.4 K/µl, p=0.0015 and p & lt;0.0001) and led to a significant decrease in erythroid progenitor populations compared to PRMT5 or JAK inhibitor monotherapy. Dual JAK/PRMT5 inhibition showed increased efficacy in the MPLW515L model with more significant suppression of leukocytosis (median WBC 3.1 K/µl vs. 176.7 K/µl, p=0.0436), splenomegaly (median weight 117 mg vs. 438 mg, p=0.0062) and hepatomegaly (median weight 1105 mg vs. 1573 mg, p=0.0188). To further understand the mechanism by which C220 leads to therapeutic efficacy in MPN, we performed RNA-seq in sorted MEP from the Jak2V617FBMT model and from SET2 xenografts after vehicle or C220 in vivotherapy. GSEA revealed E2F targets as one of the major gene sets down regulated upon treatment with C220. Consistent with the potent suppression of E2F1 target gene expression in vivo, we found that E2F1 and PRMT5 physically interact in JAK2-mutant MPN cells. Moreover, PRMT5 inhibition altered the methylation status of E2F1, leading to reduced expression of E2F1 downstream targets, including genes involved in cell cycle and DNA damage repair. Functional investigation of key downstream targets is ongoing. Our data provide evidence that PRMT5 is a credentialed therapeutic target in MPN, alone and in combination with JAK inhibition. Moreover, the therapeutic effects of PRMT5 inhibition in MPN include attenuated expression of E2F targets, which our data suggests is mediated by PRMT5 methylation of E2F1. These data demonstrate a novel link between PRMT5, E2F1 gene regulatory function, and the survival of MPN cells and provide a strong mechanism-based rationale for therapeutic studies of PRMT5 inhibitors in MPN. Based on these studies and others, PRT543, a novel and selective PRMT5 inhibitor, is currently being evaluated in a Phase I clinical trial in advanced cancers, including myelofibrosis (NCT03886831). Disclosures Bhagwat: Prelude Therapeutics: Employment. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Grego:Prelude Therapeutics: Employment. Mehta:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Levine:Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Celgene: Consultancy, Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Lilly: Honoraria; Amgen: Honoraria.

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 JAK2V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPLW515L 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 JAK2V617F+ MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2V617F/MPLW515L 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