Project 3: Therapeutic inhibition of splicing through inhibition of protein arginine methylation in leukemia

摘要

ABSTRACT Leukemias often display genetic alterations that result in dysregulation of the epigenome. To identify potential epigenetic vulnerabilities, we recently performed a paired in vitro and in vivo shRNA screen in a number of acute leukemia cell lines. The results showed that myeloid as well as lymphoid leukemia cells are preferentially dependent on protein arginine methyltransferase (PRMTs), a family of enzymes that dimethylate arginine residues of many proteins. A number of prior studies have identified PRMT5 as a promising therapeutic target in cancer, which has led to an ongoing phase I clinical trial of a PRMT5 inhibitor for patients with refractory solid tumors and Non-Hodgkin's lymphoma. However, which substrates of PRMTs are most critical for anti- cancer effects of PRMT inhibition remains unknown and biomarkers predicting response to PRMT inhibition are greatly needed. Toward understanding the anticancer effects of PRMT inhibitors, we have also found that blocking PRMT function perturbs RNA splicing, and that inhibiting either symmetric (mediated by PRMT5) or asymmetric dimethyl arginine methylation (by Type I PRMTs) results in strong preferential killing of spliceosomal mutant leukemias over their wild-type (WT) counterparts. Moreover, we have observed synergistic effects of combining both type I with type II PRMT inhibition and/or inhibition of core spliceosome function. We therefore hypothesize that the main cytotoxic effect of PRMT inhibition results from modulation of splicing. We will examine our hypothesis by characterizing the effects of inhibiting type I PRMT or type II PRMTs on pre-mRNA splicing, gene expression, and the methyl-arginine proteome in WT or spliceosomal-mutant leukemia cells (Aim 1). We will also evalute whether combining inhibitors of type I PRMTs, type II PRMTs, and the splicing factor SF3b enhances toxicity to myeloid and lymphoid leukemia cells, and the relationship between these inhibitors' efficacy and mutations in various splicing factor genes (Aim 2). Finally, we will identiy biomarkers of efficacy of PRMT5 inhibition using samples from a phase I/II trial of GSK's small molecule PRMT5 antagonist (Aim 3) for the treatment of patients with refractory AML, CMML, and MDS. This project stands to greatly improve understanding of the molecular basis for the efficacy of PRMT inhibitors in cancer and to advance these drugs toward clinical trials in leukemia.