Abstract 3884: Novel RUNX1 targeted therapy for AML expressing mutant RUNX1

RUNX1 is a master-regulator transcription factor involved in normal and malignant hematopoiesis. In addition to chromosomal translocations involving the RUNX1 locus, somatic, heterozygous RUNX1 mutations commonly occur in MDS, CMML, as well as in secondary (s) or de novo AML, which confer higher risk, shorter latency to AML transformation and poorer outcome in AML. We demonstrate here for the first time that stable or inducible shRNA-mediated knockdown of RUNX1 induced colony growth inhibition and greater apoptosis of AML cells expressing mutant (mt) RUNX1 versus those expressing wild-type (WT) RUNX1. Knockdown of RUNX1 also inhibited in vivo AML growth and significantly improved survival of immune-depleted mice engrafted with AML cells expressing mtRUNX1. RUNX1 transcription is driven by a BET protein BRD4-occupied super-enhancer in the first intron of the RUNX1 gene. Our findings also demonstrate that shRNA-mediated knockdown of BRD4, or treatment with BET protein (BETP) inhibitor (BETi), or BETP degrader PROTAC, but not their inactive enantiomer or congener, resulted in depletion of RUNX1 and induction of apoptosis in AML cells expressing mtRUNX1. Notably, following CRISPR-mediated editing of the +24 kb enhancer of RUNX1, the surviving clones exhibited markedly attenuated RUNX1 expression and stunted in vitro growth. Another goal of present studies was to find novel agents with an expression signature mimicking the expression signature of RUNX1 knockdown, but without the capacity to exert genotoxic effects on hematopoietic progenitors. To achieve this objective, we performed LINCS (Library of Integrated Network-based Cellular Signatures)-CMap (connectivity mapping) analysis querying with the RNA-seq signature of RUNX1 knockdown by shRNA to RUNX1. The expression-mimickers (EMs) uncovered as top hits included not only agents that have been documented to be BETis, but also agents that engage targets proven to be therapeutically effective when subjected to targeted-inhibition. Importantly, EM hits also included non-genotoxic anticancer agents, such as narciclasine (natural plant alkaloid), fenbendazole (benzimidazole anthelmintic) and cinobufagin (bufanolide steroid). Treatment with these agents depleted RUNX1 levels and dose-dependently induced loss of viability, as well as reduced AML burden and significantly improved survival of mice engrafted with AML cells expressing somatic mtRUNX1. These EMs also induced lethality of HPCs from FPD/MM (familial platelet disorder with myeloid malignancy) due to germline mtRUNX1 with co-occurring leukemogenic somatic mutations, while relatively sparing FPD HPCs without co-occurring leukemogenic mutations or normal HPCs. Collectively, these findings demonstrate BETP antagonists, as well as uncovered novel agents, as targeted, effective agents that deplete RUNX1 levels and induce in vitro and in vivo efficacy against AML expressing somatic or germline mtRUNX1. Citation Format: Christopher P. Mill, Courtney D. DiNardo, Warren Fiskus, Dyana T. Saenz, Agnieszka J. Nowak, Baohua Sun, David N. Saenz, Steven M. Kornblau, Kapil N. Bhalla. Novel RUNX1 targeted therapy for AML expressing mutant RUNX1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3884.