Abstract LB-081: Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation
Authors: Mill Christopher P. , DiNardo Courtney , Fiskus Warren C. , Saenz Dyana T. , Sun Baohua , Nowak Agnieszka J. , Kim Misun , Routbort Mark , Takahashi Koichi , Bhalla Kapil N. 
Published: July 2017

Abstract
RUNX1 is a transcription factor involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 (mtRUNX1) have been documented in de novo and secondary AML (10%), MDS (~10%) and CMML (up to 37%). Germ-line, mono-allelic mutations and deletions in RUNX1 cause the highly-penetrant (~40%) autosomal dominant, Familial Platelet Disorder with a propensity to Myeloid Malignancy (FPD-MM). The majority of mtRUNX1 are missense mutations, large deletions or truncation-mutations in the DNA-binding ‘RUNT’ homology domain, or in the C-terminal transactivation domain. These mutations behave mostly as loss of function mutations, associated with relative resistance to standard chemotherapy and an unfavorable prognosis in AML. This highlights an unmet need to develop and test novel targeted therapies for AML due to germ-line or somatic mtRUNX1. In the present studies, we demonstrate that shRNA-mediated knockdown of mutant and wild-type RUNX1 repressed its target genes MYC, PU.1 and MPO (myeloperoxidase), as well as inhibited growth and induced apoptosis of AML cells expressing mtRUNX1 (OCI-AML5 and Mono-Mac-1). Ex vivo depletion of RUNX1 abrogated the leukemia initiating potential of OCI-AML5 cells. After engraftment, tetracycline-inducible shRNA-mediated in vivo knockdown of RUNX1, as compared to the non-induced controls, enhanced survival of the immune-depleted (NSG) mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by the BET protein BRD4-occupied super enhancer in the first intron of the RUNX1 gene. A heat map of Hi-C interaction scores within the RUNX1 TAD (topology-associated domain) showed triangle-shaped regions of high interaction, with CTCF binding sites defining the TAD boundaries, anchoring a loop that separates the RUNX1 TAD from other TADs. Consistent with this, our findings show that, shRNA-mediated depletion of BRD4 or treatment with BET protein (BETP) inhibitor (BETi) OTX015 reduced BRD4 occupancy at the enhancer and promoter of RUNX1. This was associated with depletion of RUNX1 and its target-gene expressions and apoptosis of cultured and patient-derived (PD), primary AML blast progenitor cells (BPCs). Additionally, treatment of NSG mice engrafted with luciferase-transduced OCI-AML5 cells with OTX015 (50 mg/kg/day X 5 days, for 3 weeks) reduced the AML burden and significantly improved their survival (p &lt; 0.01). Co-treatment with the BETi and BCL2-antagonist venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and PD AML BPCs (combination index values of &lt; 1.0). Results describing the in vivo efficacy of BETi and BETP-PROTAC (proteolysis targeting chimera), alone and in combinations against AML expressing mtRUNX1 will be updated at the AACR meetings. Collectively, these findings highlight novel and effective, mechanistically-targeted, single agent or combination, BETP antagonist-based therapy of AML expressing mtRUNX1.
Citation Format: Christopher P. Mill, Courtney DiNardo, Warren C. Fiskus, Dyana T. Saenz, Baohua Sun, Agnieszka J. Nowak, Misun Kim, Mark Routbort, Koichi Takahashi, Kapil N. Bhalla. Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2017-LB-081