Pre-Clinical Efficacy of Targeting TBL1/R-β-Catenin-TCF7L2 Axis Against AML with 3q26 Lesions and EVI1 Overexpression
Authors: Birdwell Christine , Fiskus Warren C. , Mill Christopher Peter , Davis John A. , Salazar Arnold , DiNardo Courtney D. , Takahashi Koichi , Horrigan Steve , Kadia Tapan M. , Bhalla Kapil N. 
Published: November 2021

EVI1 gene maps to the MECOM locus at chromosome 3q26.2 and encodes for a zinc finger domain-containing transcriptional regulator. EVI1 supports hematopoietic stem cell self-renewal and blocks hematopoietic differentiation. EVI1 is overexpressed in up to 10% of AML, including those harboring chromosome translocation t(3;3)(q21;q26.2) or inv(3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression due to 3q26.2 lesions in MDS and AML is frequently associated with monosomy 7 and confers poor response to therapy and inferior relapse-free and overall survival. We had previously reported the pre-clinical efficacy of targeting TBL1/R1-nuclear β-catenin-TCF7L2 by tegavivint (BC-2059, Iterion Therapeutics) against AML stem/progenitor cells. This led us to interrogate the anti-AML activity of tegavivint (TV) in AML models harboring 3q26.2 lesions, where EVI1 overexpression has been documented to drive the biology of AML stem/progenitor cells. For this, we utilized AML cell lines with 3q26.2 lesions with/without monosomy 7 (UCSD-AML1, OCI-AML20, AML191 / MUTZ-3, AML194, HNT34), as well as patient-derived (PD) AML cells with 3q26 lesions with or without monosomy 7. Treatment with TV (10 to 100 nM) dose-dependently induced apoptosis in these cellular models. This was associated with attenuation of protein levels (determined by immunoblot analyses) 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, pan-BET protein inhibitor OTX015 (100 to 1000 nM) dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). Following TV treatment, RNA-Seq and gene set enrichment analysis in UCSD-AML1 and OCI-AML20 cells showed log2 fold-changes in gene expression and positive enrichment of pathway genes and/or reactomes of inflammatory response, TNFα and interferon signaling, TGFβ, NOTCH and apoptosis signaling, as well as negative enrichment of gene sets of c-Myc, E2F, G2M checkpoint, DNA replication and repair and chromosome maintenance (all with FDR q-values &lt; 0.1). QPCR analysis showed repression of EVI1, MYC and KIT, but upregulation of Axin2 mRNAs. Following TV treatment, confocal microscopy showed reduction of nuclear protein levels of EVI1 and β-catenin, as well as disrupted their co-localization with TBL1. Proximity ligation assay also demonstrated that exposure to TV significantly reduced the proximity of TBL1 and EVI1 as well as of TBL1 and β-catenin. Mass cytometry (CyTOF) analysis of patient-derived (PD) AML samples with t(3:3)/inv(3) confirmed that TV treatment attenuated protein levels of 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 (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). Consistent with effects of TV on gene-expressions that regulate cell death pathways, in vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (as determined by the SynergyFinder algorithm) of AML cell lines and PD AML cells with t(3:3)/inv(3) and EVI1 overexpression. In the in vivo HNT-34 model of flank-implanted and established tumors in athymic nude mice, treatment with TV (50 mg/kg, TIW, IP) and venetoclax (30 mg/kg, PO, daily) for 5-weeks yielded more tumor growth delay than vehicle control or TV alone. Additionally, following tail vein infusion and engraftment of luciferized AML191 cells in NSG mice, treatment with TV and/or venetoclax or OTX015 (30 mg/kg, PO, daily) was evaluated for 6 weeks. Co-treatment with TV and venetoclax or TV and OTX015 significantly reduced AML growth (determined by reduction in bioluminescence by Xenogen camera) (p &lt; 0.05), as well as improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. Collectively, these findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 by treatment with TV also inhibits EVI1 and its targets. They also demonstrate superior pre-clinical efficacy of novel TV-based combinations with BCL2 or BET protein inhibitor against AML models harboring 3q26 lesion and EVI1 overexpression.

Disclosures

DiNardo: Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy. Horrigan: Iterion Therapeutics: Current Employment. Kadia: Astellas: Other; Cure: Speakers Bureau; Genfleet: Other; Ascentage: Other; Jazz: Consultancy; Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Cellonkos: Other; Novartis: Consultancy; AstraZeneca: Other; BMS: Other: Grant/research support; Liberum: Consultancy; Amgen: Other: Grant/research support; Pulmotech: Other; Pfizer: Consultancy, Other; Genentech: Consultancy, Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support.