Preclinical Efficacy of Novel Drug Combination Against AML with 3q26 Lesions and EVI1 Overexpression

Abstract

The EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc finger transcription factor (TF). EVI1 protein is expressed in leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. As a result of the chromosome translocation t(3;3) or inv(3) at 3q26, the distal enhancer of the TF and tumor suppressor GATA2 is dislocated and repositioned to induce expression of EVI1, while concomitantly repressing GATA2. EVI1 overexpression results in therapy refractoriness and inferior relapse-free and overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor has activity against AML cells with inv(3)/t(3;3). Among the targets of EVI1 are the pro-growth and pro-survival proteins c-Myc and Bcl-xL, expressions of which are also regulated by enhancer activity that is undermined by BRD4 inhibition. We have recently reported that the nuclear TBL1/R1-β-catenin-TCF7L2 complex transcriptionally regulates c-Myc, cyclin D and Survivin and sustains AML stem cells, such that the undermining of this transcriptional axis by the TBL1/R1-β-catenin-binding disruptor tegavivint (BC-2059, Iterion) represses MYC, cyclin D1 and Survivin and inhibits growth and survival of AML stem-progenitor cells. Based on these observations, we interrogated the pre-clinical efficacy of pan-BET inhibitor OTX015 and/or tegavivint (TV) against AML cells with inv(3)/t(3;3) and overexpression of EVI1. Treatment with BET inhibitor OTX015 dose-dependently (100 to 1000 nM) induced apoptosis of AML cell lines (UCSD-AML1, OCI-AML20, MUTZ-3 and HNT-34) and patient-derived (PD) AML cells (AML191 and AML194) with inv(3)/t(3;3) and with or without monosomy 7. OTX015 treatment was associated with repression of c-Myc, c-Myb, CDK4, MECOM, and MCL1 as well as induction of HEXIM1 and cleaved PARP levels. We also determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions with/without monosomy 7. Furthermore, TV treatment resulted in attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, while inducing p21, CD11b, BIM and cleaved PARP levels. Next, utilizing H3K27Ac antibody Hi-ChIP (Arima Hi-ChIP), we interrogated the active gene regulatory interactions through a combined Hi-C and ChIP workflow, followed by Tn5 transposase-mediated on-bead library construction and NGS. We observed that in untreated AML191 cells numerous loop interactions were present between the super-enhancers and promoters respectively of MYC and BCL2. Following TV treatment (100 nM for 16 hours), differential loop calling revealed a loss of these interactions. RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of TGFβ, inflammatory response, TNFα and interferon signaling, and apoptosis signaling, but negative enrichment of gene sets of 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 the phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99) harboring inv(3) and EVI1 overexpression. Co-treatment with TV and OTX015 or the BCL2 inhibitor venetoclax synergistically induced in vitro apoptosis (determined by SynergyFinder 2.0) in the AML cell lines and the PD AML cells. Notably, in the flank-implanted PD AML191 or tail vein AML242 models in NSG mice, treatment with TV and OTX015 for 2 to 5 weeks, compared to vehicle control or each drug alone, significantly reduced AML growth (p < 0.01). Furthermore, in the tail-vein infused and engrafted PD AML191 PDX model, treatment with TV and OTX015 or venetoclax for 6 weeks, as compared to vehicle control or each drug alone, significantly improved overall survival of the NSG mice (p < 0.01), without inducing any toxicity. These findings highlight the promising pre-clinical efficacy of novel combinations of TV and BET protein or venetoclax against AML models harboring 3q26 lesions and EVI1 overexpression.