ASH 2019 | Protein arginine methyltransferase 5 (PRMT5) inhibition: a promising preclinical target for MPN

The majority of patients with myeloproliferative neoplasms (MPNs) carry the Janus kinase 2 (JAK2) mutation, V617F. This activating mutation has been shown to trigger cell proliferation and inhibit apoptosis.^1-3 The protein arginine methyltransferase 5 (PRMT5) is physiologically involved in epigenetic gene modulation via histone methylation and has been shown to interact with JAK2.³ Human studies have shown that the JAK2 V617F mutation binds with a higher affinity to PRMT5 than the wild type JAK2, resulting in increased PRMT5 phosphorylation and inhibition of its methylation activity.^1,2 A similar study also showed that this JAK2 V617F-mediated PRMT5 inhibition alters the negative regulation on myeloid cell proliferation and erythroid differentiation thereby potentially contributing to MPN pathology. Moreover, PMRT5 seems to be overexpressed in patients with myeloproliferative neoplasms (MPNs) and especially in those with polycythemia vera (PV).⁴

All these findings indicate that PRMT5 is a promising therapeutic target for MPN. To investigate this further, Friederike Pastore presented the results of their preclinical study³ during the 61^st American Society of Hematology  Annual Meeting & Exposition (ASH 2019). In their study, they assessed the efficacy of the selective PRMT5 inhibitor, C220, on MPN cells lines, primary human MPN cells and murine mouse models.³ The current article is based on data presented at ASH and may supersede the data in the published abstract.

Study design

MPN cell lines & primary CD34⁺ cells

• To test the effects and efficacy of the PRMT5 inhibitor, C220 (PRMT5i) the following cell lines were used:
  • The BaF3 cell line transduced with a JAK2 V617F or wild type vector, accordingly
  • The JAK2 V617F harbouring MPN cell lines: SET2, HEL and UKE1
• Human primary CD34⁺ cells from patients with PV or from patients with primary myelofibrosis (MF) were also used in the initial PRMT5i efficacy and sensitivity experiments

Murine MPN models

• PV mouse model JAK2 V617F characterised by splenomegaly, high hematocrit and leucocytosis
• Essential thrombocythemia (ET)/MF MPLW515L mouse model characterised by splenomegaly, leucocytosis, thrombocytosis and extensive bone marrow (BM) fibrosis

Results

In vitro results

• To assess the cellular activity of the potent, highly selective PRMT5i, dose-response viability curves were compared between BaF3 wild type cells and BaF3 cells transduced with the JAK2 V617F mutation:
  • C220 for 6 days inhibited proliferation at lower doses in JAK2 V617F BaF3 cells (IC50: 4.8 nM) compared to wild type BaF3 cells (IC50: 11.9 nM)
• Dose-dependent reduction in proliferation was also seen in the following JAK2 V617F cell lines:
  • SET2 (IC50: 5.2 nM)
  • UKE1 (IC50: 2.2 nM)
  • HEL (IC50: 11.8 nM)
• C220 was successful in reducing in a dose-dependent manner the proliferation of human primary CD34⁺ cells from PV (IC50: 8.4 nM) or PMF patients (IC50: 13.5 nM)
• In SET2 cell lines, C220 showed potent on target efficacy by reducing levels of symmetric dimethylarginine (SDMA) on histone H4, in a dose dependent fashion

In vivo results

• C220 efficacy was further assessed in the PV mouse model. Oral C220 at 15 mg/kg once daily (N = 21) resulted in:
  • Significantly reduced leucocytosis, elevated hematocrit, as well as splenomegaly and increased liver weights
  • Improved histological architecture of the spleen and bone marrow as assessed by immunohistochemistry
• In the ET/MF mouse model, oral C220 at 15 mg/kg once daily (N = 11) resulted in:
  • Significantly reduced leucocytosis, thrombocytosis as well as splenomegaly, and enhanced liver weights
  • Reduced levels of BM and splenic fibrosis as seen by immunohistochemistry

RNA sequencing

• To identify the pathways affected by PRMT5i in MPN, the authors used RNA sequencing on JAK2 V617 PV mutant cells. A total of 452 genes were identified, which were altered in JAK2 V617 PV cells and could be downregulated by C220. These belonged in the following top enriched pathways:
• Interferon gamma (IFN-γ) response
• E2F targets
• IFN-α response
• Mammalian target of rapamycin complex 1 (mTORC1) signalling
• The authors focused on E2F targets (which are not affected by JAK2 inhibition) and hypothesized that E2F transcription factor 1 (E2F1) could be a methylation target of PRMT5 in MPN cells
  • co-immunoprecipitation assays in SET2 and UKE1 cells showed that E2F1 and PRMT5 do interact with each other
  • In vitro PRMT5 inhibition in SET2 cells, significantly reduced the methylation status of E2F1 and facilitated its interaction with its partner, the retinoblastoma protein (Rb), which together promote inhibition of E2F target gene expression and cell growth
• In vitro C220 for 6 days led to an increase in DNA damage and promoted cell cycle arrest and apoptosis

Combination treatment with PRMT5i and JAK inhibitor

• C220 in combination with the JAK inhibitor (JAKi) ruxolitinib was efficacious and well tolerated in both the PV and the ET/MF mouse models
• After 4 weeks of combination treatment and when compared to the vehicle control(C220 or ruxolitinib alone), the PV mice showed:
  • Significantly enhanced reduction in hematocrit, white blood cells (WBCs) and spleen weight
  • Significantly enhanced reduction in megakaryocytic and erythroid progenitor cells
• Significant decrease in pro-inflammatory cytokines, in particular LIX (the murine analog of human IL-8), was also observed in both mouse models after combination treatment with PRMT5i and ruxolitinib when compared to vehicle control, ruxolitinib alone or C220 alone
• SET2 cell clones that were selected for their resistance to ruxolitinib treatment maintained their sensitivity to PRMT5 inhibition by C220, indicating a mechanism of PRMT5 inhibition that is succinct to JAK2 inhibition

Conclusions

The results of this preclinical study show that PRMT5 is a promising target for the treatment of MPN. The authors showed that PRMT5 interacts with E2F1, and that PRMT5i reduces the expression of E2F1 induced genes involved in cell proliferation in MPN cell line models. In vivo PRMT5i led to significant MPN symptom alleviation and hematological responses that could be further enhanced when used in combination with ruxolitinib. These results warrant the clinical evaluation of PRMT5i in patients with MPN. A phase I trial is currently underway evaluating the PRMT5 inhibitor, PRT543, in patients with advanced cancers, including MF.