Abstract 4900: Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression
Authors: Birdwell Christine E. , Fiskus Warren C. , Kadia Tapan M. , Mill Christopher P. , Davis John A. , Das Kaberi , Horrigan Stephen , Bhalla Kapil N. 
Published: April 2023

Abstract
EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc-finger transcription factor. Chromosome translocation t(3;3) or inv(3) at 3q26 repositions the enhancer (E) of the tumor suppressor GATA2 to induce EVI1 over-expression, while concomitantly repressing GATA2. EVI1 over-expression promotes self-renewal and blocks differentiation of leukemia stem-progenitor cells (LSCs) and causes therapy refractoriness and inferior overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor (BETi) is active against AML cells with inv(3)/t(3;3). BETi treatment also represses EVI1 and its targets c-Myc and Bcl-xL. We recently reported that tegavivint (TV) (BC-2059), a disruptor of the nuclear TBL1/R1-β-catenin-TCF7L2 complex represses c-Myc, cyclin D1 and Survivin and inhibits growth and survival of AML LSCs. In present studies, a CRISPR screen in UCSD-AML1 cells (with 3q26 lesion and EVI1 overexpression) with gRNAs targeting epigenetic regulators highlighted BRD4 and p300 as dependencies. Treatment with BETi, e.g., OTX015, dose-dependently induced apoptosis of AML cell lines and patient-derived (PD) AML cells (AML191 and AML194) with inv(3) or t(3;3). OTX015 treatment repressed c-Myc, c-Myb, CDK4, MECOM, and MCL1, while inducing HEXIM1 and cleaved PARP levels. TV treatment also dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions, associated with depletion of nuclear β-catenin, EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but increase in p21, CD11b, BIM and cleaved PARP levels. Hi-ChIP with H3K27Ac antibody demonstrated that TV treatment abolished chromatin loops between MYC and its Es within the super-E. RNA-Seq analysis showed negative enrichment (log2-fold) of gene sets of MYC, E2F and WNT targets, DNA replication/repair and reduction of the 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization of EVI1 and β-catenin with TBL1, also confirmed by the Proximity Ligation Assay. CyTOF analysis confirmed that TV treatment reduced EVI1, c-Myc, RUNX1, β-catenin, Bcl-xL, BCL2, MCL1 and Ki67 but increased cleaved PARP levels in the phenotypically characterized LSCs (with high expression of CLEC12A, CD123, CD244 and CD99) harboring inv(3) with EVI1 overexpression. Co-treatment with OTX015 and TV or the p300 inhibitor GNE-049 synergistically induced in vitro apoptosis in the AML cell lines and the PD AML cells with 3q26.2 lesions. Notably, in the flank-implanted PDX of AML191 or tail-vein infused PDX of AML242 models in NSG mice, treatment with OTX015 and TV, compared to vehicle control or each drug alone, significantly reduced AML growth and improved survival, without any host toxicity. These findings highlight the promising pre-clinical efficacy of novel BETi-based combinations against AML models harboring 3q26 lesions and EVI1 overexpression.
Citation Format: Christine E. Birdwell, Warren C. Fiskus, Tapan M. Kadia, Christopher P. Mill, John A. Davis, Kaberi Das, Stephen Horrigan, Kapil N. Bhalla. Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4900.