Abstract 2648: Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression
Authors: Birdwell Christine , Fiskus Warren C. , Mill Christopher P. , Davis John A. , Jin Qi , DiNardo Courtney D. , Takahashi Koichi , Horrigan Stephen , Kadia Tapan M. , Daver Naval , Bhalla Kapil N. 
Published: June 2022

Abstract
The EVI1 gene is located on 3q26.2 and encodes a zinc fingers-containing transcription factor. Nuclear β-catenin-TBL1/R1-TCF7L2 axis activity and EVI1 expression characterize leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. EVI1 is overexpressed in AML with chromosome translocation t(3;3) or inv(3) at 3q26, where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression confers poor response to therapy and inferior relapse-free and overall survival in AML. Tegavivint (BC-2059, Iterion) targets TBL1/R1 and disrupts its binding to β-catenin and TCF7L2, repressing MYC, cyclin D1 and Survivin, leading to apoptosis of AML cells. Here, we determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines (UCSD-AML1, MUTZ3 and OCI-AML20) and patient-derived (PD) AML cells (AML191 and AML194) with 3q26.2 lesions with/without monosomy 7. This was associated with attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, by reducing BRD4 occupancy at GATA2 enhancer and repressing EVI1, the pan-BET protein inhibitor OTX015 (100-1000 nM) also dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of inflammatory response, TNFα and interferon signaling, TGFβ and apoptosis signaling, but negative enrichment of gene sets of c-Myc, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells harboring t(3:3)/inv(3) and EVI1 overexpression (with high expression of CLEC12A, CD123, CD244, CD99). In vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (determined by SynergyFinder) of AML cell lines and the PD AML cells. In the tail-vein infused and engrafted PD AML191 cell model in NSG mice, treatment with TV and/or venetoclax or OTX015 for 6 weeks significantly reduced AML burden and improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. These findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 axis combined with BET protein or BCL2 inhibition is effective therapy against AML models harboring 3q26 lesions and EVI1 overexpression.
Citation Format: Christine Birdwell, Warren C. Fiskus, Christopher P. Mill, John A. Davis, Qi Jin, Courtney D. DiNardo, Koichi Takahashi, Stephen Horrigan, Tapan M. Kadia, Naval Daver, Kapil N. Bhalla. Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2648.