Pre-Clinical Efficacy of Targeting TBL1/R-β-Catenin-TCF7L2 Axis Against AML with 3q26 Lesions and EVI1 Overexpression

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 < 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