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Background: Myeloproliferative neoplasms (MPN) are a diverse group of hematopoietic stem cell disorders. JAK2V617F gain-of-function is the most prevalent mutation, accounting for more than 60% of MPNs. PRMT5 was initially identified as a JAK-binding protein. Its enzymatic function catalyses the symmetric di-methylation of arginine on a variety of substrates, including histones and proteins of the splicing apparatus. It has been proposed that mutant JAK2 can phosphorylate PRMT5, leading to loss of methylation activity and promotion of erythropoiesis (Liu F. et al. Cancer Cell 2011). Based upon this study, it was proposed that enhancing PRMT5 activity may be a useful therapeutic measure (Skoda RC et al. Cancer Cell 2011). Aim: To determine the role of PRMT5 in JAK2V671F mutant hematopoiesis. Hypothesis: Inhibition of PRMT5 will exacerbate JAK2V617F hematopoiesis R esults: Using a conditional null allele, we deleted Prmt5 in embryonic development with the hematopoietic-specific VavCre transgene. This led to embryonic lethality at E9.5 due to absence of erythropoiesis but not other lineages. Similar embryonic lethality was observed using the erythroid specific EpoRCre transgene. Following a 350,000-compound library screen, we developed a potent and selective SAM-dependent inhibitor (CTx034) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. Consistent with the genetic evidence that PRMT5 is most important for erythropoiesis, CTx034 was a potent inhibitor of erythropoiesis in cultures derived from healthy human CD34+ cells. This suppression of erythropoiesis was associated with activation of p53. However, progenitor assays of bone marrow cells from patients with MPN showed that JAK2V617F erythropoiesis was more sensitive to CTx034 than normal erythropoiesis. We established JAK2V617F bone marrow chimeric mice to directly compare the in vivo effects of PRMT5 inhibition on mutant and wild-type erythropoiesis within the same animal. Remarkably, these studies showed normalization of spleen size and erythropoiesis, comparable to the current standard of care, Ruxolitinib (Figure 1A-B). Importantly, CTx034 was well tolerated in healthy animals with no suppression of hematopoiesis. One of the major therapeutic challenges for MPN is the eradication of the malignant clone, which is rarely achieved with Ruxolitinib. The addition of MDM2 inhibitors, which activate p53, are currently in trial. Importantly, CTx034 not only suppressed JAK2-mutant erythropoiesis but also activated p53 in JAK2-mutant progenitors, unlike Ruxolitinib (Figure 1C). This result strengthens the therapeutic rationale for PRMT5 inhibitors in MPN. To understand how CTx034 inhibits erythropoiesis, we initially considered direct methylation effects on JAK-STAT signalling and p53. Challenging previous reports, we could find no evidence that JAK alters PRMT5 activity, no evidence that PRMT5 inhibition perturbs JAK-STAT signalling and no evidence that PRMT5 methylates p53. To look more broadly, we performed RNA-seq analysis of CD34+ cells following 72 hours exposure to CTx034. Globally, this demonstrated a potent 'starvation' signal with suppression of protein synthesis despite activation of the upstream mTOR signalling pathway. This suppression of protein synthesis could be linked to three mechanisms. First, CTx034 inhibited methylation of the Sm core complex of the spliceosome, leading to alternate splicing (skipped exons and retained introns) affecting the elongation initiation factor 2 (EIF2) pathway. Second, PRMT5 directly interacts with the translation initiation complex (eIF4A, eIF4B, eIF4E and the poly(A)-binding protein 1, PABP1. Moreover, mass spectrometry identified PABP1 as a new target of PRMT5. Treatment with CTx034 did not alter protein abundance of any of these factors but decreased the RNA binding capacity of PABP1, thereby preventing the correct formation of the initiation of translation complex. Finally, CTx034 perturbed polysome formation with loss of methylation of RPS10. C onclusion: Challenging previous reports, we show that PRMT5 inhibitors are an attractive and novel therapeutic for JAK2V617F MPN by targeting initiation of translation, ribosome biogenesis and activation of p53. Disclosures Sonderegger: CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Toulmin:CRC Cancer Therapeutics: Research Funding. Lane:Novartis: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Stupple:CRC Cancer Therapeutics: Employment. Street:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Employment, Patents & Royalties. Jane:CRC Cancer Therapeutics: Patents & Royalties. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.

Background: Targeting leukemic stem cells without detrimental effects on hematopoietic stem cells is a major goal for improving cure rates for acute myeloid leukemia (AML). Strategies include targeting leukemia specific mutations or pathways where leukemic cells are more dependent, leading to so-called synthetic lethality. One potential target for the latter is PRMT5, an arginine methyltransferase that methylates arginine on histones and a large number of non-histone proteins including components of the spliceosome. PRMT5 is essential for the maintenance of normal hematopoietic stem cells, through p53-dependent and independent mechanisms. Aim: To determine the relative importance of PRMT5 in normal and leukemic stem cells using genetic and pharmacologic approaches. Hypothesis: Survival of leukemic stem cells will be more dependent on PRMT5 than normal hematopoietic stem cells. Results: Using a conditional-null allele, we deleted Prmt5 in two mouse models of AML; AML1-ETO and MLL-ENL. Deletion of Prmt5 in AML1-ETO leukemia dramatically improved survival, with relapse only occurring in the setting of Prmt5-expressing cells. In contrast, deletion of Prmt5 in MLL-ENL leukemia had little effect. After screening a 350,000-compound library, we developed a potent and selective SAM-dependent inhibitor (CTx293) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. We tested the in vivo activity of CTx293 in the AML1-ETO model generated on either a p53 wild-type or p53-/- background. Twice daily administration of CTx293 for 14 days demonstrated absolute p53 dependent activity, with prolongation of mean survival from 35 to 130 days (Figure 1A). To directly compare the effects of PRMT5 inhibition on leukemic and normal progenitors, we examined the numbers of cells within the same animal during treatment with CTx293. After 3 days, there was a two-fold reduction in both leukemic and normal progenitors. However, after 7 days treatment, leukemic progenitors had reduced more than 1000-fold whilst normal progenitors (in the same mouse) had recovered (Figure 1B). To understand this differential effect on normal and leukemic progenitors, we FACS-isolated cells after 3 days therapy. While, there was evidence of p53 activation in both normal and leukemic progenitors, the downstream effects were quite distinct, with leukemic progenitors showing activation of apoptosis. We tested the potency of CTx293 on primary human AML cells using a 14-day semi-solid agar growth assay. This demonstrated greater sensitivity of most AML samples (LD100 nM). Of note, TP53-mutant samples were more resistant. Finally, we demonstrate activity of single agent CTx293 in 4 patient-derived xenografts. Conclusion: We have used both genetic and pharmacologic approaches to show that PRMT5 is an attractive target for eliminating leukemic stem cells through a p53-dependent mechanism without toxicity to healthy stem cells. Disclosures Toulmin: CRC Cancer Therapeutics: Research Funding. Sonderegger:CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Street:CRC Cancer Therapeutics: Employment, Patents & Royalties; MERCK: Membership on an entity's Board of Directors or advisory committees. Stupple:CRC Cancer Therapeutics: Employment. Jane:CRC Cancer Therapeutics: Patents & Royalties. Wei:Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.

Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.