Small-Molecule Inhibition of PRMT5 Induces Translational Stress and p53 in JAK2V617F Mutant Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPN) are a diverse group of hematopoietic stem cell disorders. JAK2^V617F 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 JAK2^V671F mutant hematopoiesis.

Hypothesis: Inhibition of PRMT5 will exacerbate JAK2^V617F hematopoiesis

Results: 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 JAK2^V617F erythropoiesis was more sensitive to CTx034 than normal erythropoiesis. We established JAK2^V617F 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.

Conclusion: Challenging previous reports, we show that PRMT5 inhibitors are an attractive and novel therapeutic for JAK2^V617F MPN by targeting initiation of translation, ribosome biogenesis and activation of p53.

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