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

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.

Grant Number: 5P50CA254838-05
NIH Institute/Center: NIH
Principal Investigator: Omar Abdel-Wahab