Abstract PR04: Degradation of leukemia oncogenes: A novel approach to therapy of leukemia

Abstract

Abstract AML is a heterogeneous group of malignancies with significant variability in phenotype and mechanisms of leukemogenesis. Despite our increased understanding of the genetic abnormalities that drive AML, very few targeted therapeutic agents have yet been developed. The prevailing approach to drugging oncoproteins is direct targeting of the mutant protein with a small molecule or antibody. Unfortunately, this strategy has not proven particularly fruitful in the case of AML as many mutant driver proteins are “undruggable” and the targetable enzymes (i.e., oncogenic FLT3) rapidly acquire resistance to inhibitors in the clinic. We are pursuing a novel and innovative approach that focuses on promoting degradation of AML oncoproteins through small-molecule inhibition of the cellular machinery that regulates turnover. Many oncoproteins are tagged for degradation by the proteasome or lysosome by post-translational addition of polyubiquitin chains. The process is, however, highly dynamic and reversible by deubiquitylating enzymes (DUBs), which cleave ubiquitin from substrate proteins. A subset of AML mutant drivers, including RAS, FLT3, and PML-RARα, have been found to undergo ubiquitin-mediated degradation; however, the DUB enzymes stabilizing them are unknown. In order to identify novel targets and compounds that regulate protein homeostasis of leukemic oncoproteins, we employed whole-cell phenotypic screens of most reported small-molecule DUB inhibitors, annotated for inhibitory activity across a broad panel of DUBS, using oncogene-dependent and control cell lines followed by hit validation and target deconvolution. Using this approach, we identified inhibitors, and candidate DUBs, involved in regulation of protein levels of mutant FLT3, mutant JAK2, mutant KRAS, and PML-RARα;. Follow-up work that focused on FLT3-ITD identified USP10 as a DUB that stabilizes the oncoprotein via removal of a degradative ubiquitin tag. Furthermore, we show that pharmacologic inhibition of USP10 promotes degradation of FLT3-ITD but not wild-type (wt) FLT3, leads to selective killing of oncogenic FLT3-expressing AML cells in vitro and in vivo, and overrides resistance to FLT3 kinase inhibitors caused by tyrosine kinase domain (TKD) mutations and other mechanisms. Our studies hence validate USP10 as a therapeutic target for FLT3 mutant-positive AML. Building on this work, we executed a medicinal chemistry optimization effort that has yielded USP10 inhibitors with improved potency, and selectivity and mechanism studies have yielded insights into the origin of specificity for mutant versus wt FLT3. Furthermore, we have validated multiple DUB inhibitors that promote selective degradation of V617F JAK2 and intriguingly, identified compounds efficacious, in terms of inhibition of proliferation, across multiple genetic backgrounds. To the best of our knowledge, this is the first identification of a novel DUB substrate using a DUB-targeting small-molecule library screen and the first demonstration of stabilization of a mutant driver oncoprotein in AML by a DUB enzyme. DUB inhibitors have the potential to eliminate oncoproteins, but it is early yet in the field. The first DUB inhibitor clinical trial is starting this year for multiple myeloma. Our work establishing the therapeutic potential of DUB inhibitors for oncogenes including PML-RARα; and constitutively activated FLT3, JAK2, and RAS, as well as potentially other oncoprotein-driven AML patient populations, will lay the groundwork for development of first-in-class drugs for AML. This abstract is also being presented as Poster 10. Citation Format: Sara Buhrlage, Ellen Weisberg, Ilaria Lamberto, Jing Yang, Nathan Schauer, Martin Sattler, Atushi Nonami, Amanda Christie, David Weinstock, Stella Ritorto, Virginia DeCesare, Matthias Trost, Richard Stone, Nathanael Gray, James Griffin. Degradation of leukemia oncogenes: A novel approach to therapy of leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR04.