Abstract 802: Targeted inhibition of KDM1A and BET proteins or DNMT1 exerts synergistic lethal activity against human AML blast progenitor cells
Authors: Fiskus Warren C. , Mill Christopher P. , Saenz Dyana T. , Nowak Agnieszka J. , Sun Baohua , Saenz David N. , Kornblau Steven M. , Sharma Sunil , Bhalla Kapil N. 
Published: August 2018

KDM1A is an FAD-dependent amine oxidase that demethylates H3K4Me1/2 chromatin, which regulates enhancer maintenance and transcription in stem/progenitor cells. GFI1 is a zinc-finger transcriptional repressor that binds and recruits KDM1A-CoREST-HDAC complex to repress loci regulating lineage differentiation in hematopoiesis. In the present studies, we evaluated the anti-AML efficacy of KDM1A inhibition alone and in combination with BET protein inhibitor (BETi) or DNMT1 inhibitor. Tet-inducible shRNA to KDM1A depleted mRNA and protein levels of KDM1A, repressed c-Myc, de-repressed p21, CD11b, CD86 and CEBPα, as well as inhibited colony growth and modestly induced lethality in genetically diverse cultured AML cell lines, including MOLM13 and OCI-AML5 cells. Following knockdown of KDM1A in the AML cells, RNA-seq followed by gene-set enrichment analysis linked mRNA perturbations to pathways involving signal transduction, metabolism, RNA POL II transcription and its regulation. Treatment with the reversible KDM1A inhibitor SP2577 or the irreversible inhibitor ORY-1001 also inhibited cell proliferation and clonogenic survival, associated with induction of genes involved in myeloid differentiation, of AML OCI-AML5, MOLM13, THP1 and MV4-11 cells. KDM1A inhibitors also induced loss of cell viability in patient-derived CD34+ AML BPCs as well as in CD34+CD38- Lin- AML stem cells. SP2577 or ORY-1001 also disrupted binding of KDM1A with Co-REST in OCI-AML5, MOLM13 and THP1 cells. Utilizing CRIPSR/Cas9 technology, we gene-edited KDM1A in OCI-AML3 cells. Surviving clones exhibited ~50% KDM1A as well as decreased c-Myc and DNMT1 expression compared to control OCI-AML3 cells. By utilizing RNA-seq mRNA signatures from KDM1A-depleted AML cells, we also queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. Among the top hits were pan-histone deacetylase inhibitors (HDIs), which we have previously reported to exert in vitro synergistic lethality in combination with KDM1A inhibitor (Leukemia 2014;28:2155-64). Utilizing publicly available chIP-seq data with anti-BRD4, anti-H3K27Ac and RNA Pol-II antibodies, we identified that the LSD1 promoter is occupied by BRD4 in the AML MOLM14 cells. Treatment with the BETi OTX015 depleted KDM1A expression in AML cells. Co-treatment with SP2577 and OTX015 synergistically induced apoptosis of cultured and PD CD34+AML BPCs and CD34+CD38- Lin- AML stem cells (combination indices &lt; 1.0). Notably, co-treatment with KDM1A inhibitor (SP2577 or ORY-1001) and the DNMT1 inhibitor decitabine also resulted in synergistic growth inhibition and lethality of cultured and PD CD34+ AML BPCs (combination indices &lt; 1.0). These findings strongly support further in vivo testing and preclinical development of KDM1A inhibitor-based combinations with BETi and DNMT1 inhibitor against AML BPCs.

Citation Format: Warren C. Fiskus, Christopher P. Mill, Dyana T. Saenz, Agnieszka J. Nowak, Baohua Sun, David N. Saenz, Steven M. Kornblau, Sunil Sharma, Kapil N. Bhalla. Targeted inhibition of KDM1A and BET proteins or DNMT1 exerts synergistic lethal activity against human AML blast progenitor cells [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 802.