Inhibiting FOXM1 Sensitizes Myeloma Cells to BCL2 Inhibitor Venetoclax By Repressing MYC Pathway

Abstract

Background: Despite improved 5-year survival in multiple myeloma (MM), relapsed and/or refractory multiple myeloma (RRMM) remains a big challenge. Forkhead box transcription factor FOXM1 is a key regulator of metabolism and cell cycle progression in RRMM. FOXM1 is highly expressed in OPM2 and Delta47 cells compared to 9 other myeloma cell lines. Inhibiting FOXM1 function by deleting the FOXM1 gene or using the small-molecule FOXM1 inhibitor, NB73, suppresses OPM2 and Delta47 cells in vitro and in vivo. We hypothesized that FOXM1-targeted inhibition of RRMM may be deepened by combining NB73 with established or candidate myeloma drugs. Materials and methods: We used 3 tools to develop FOXM1-targeted combinatorial therapies by: (1) combining NB73 with 6 widely used myeloma drugs; (2) repurposing screens of cancer drugs in FOXM1-knockout (FOXM1 KO) vs FOXM1-proficient (FOXM1 WT) OPM2 cells; and (3) combining NB73 with compounds recommended by CMap analysisof FOXM1 KO vs FOXM1 WTcells. RNA seq analysis was used to elucidate pathways underlying drug synergy. Results: We adopted ZIP drug synergy scoring assay to examine the interactions between NB73 and the 6 myeloma drugs in MM cells. ZIP score takes the merits of both Loewe score that appraises drugs targeting the same pathway and Bliss score that appraises drugs targeting different pathways. Venetoclax, a BCL2 inhibitor treating MM with t(11;14) translocation, synergized with NB73 in killing OPM2 and Delta47 cells. In contrast, taking advantage of FOXM1 WT and FOXM1 KO MM cells, we conducted repurposing screens of FDA-approved anti-cancer drug library (NCI AOD X) in these cells. We identified Dasatinib and Panobinostat that killed FOXM1 KO OPM2 cells much more than FOXM1 WT cells. We also identified 3 drugs from the repurposing screens in FOXM1 WT and FOXM1 KO Delta47 cells. Thirdly, CMap, a large-scale compendium of functional perturbations in cultured human cells coupled to a gene expression read-out, implicated a synergy between NB73 and Thapsigargin that was verified by ZIP drug synergy scoring assay. Collectively, we have identified a total of 7 drugs (6 FDA-approved) synergizing with NB73 in killing OPM2 and/or Delta47 cells in which FOXM1 is highly expressed. Since Venetoclax is a current myeloma drug, we have focused on how inhibiting FOXM1 sensitizes MM cells to Venetoclax. Firstly, we showed the synergy between NB73 and Venetoclax in inducing apoptosis in OPM2 and Delta47 cells. We used the experimental condition in apoptosis assay to treat OPM2 cells for RNA sequencing study. Principal Component Analysis of RNA seq data indicated that the clusters of NB73 and the combo were well separated from each other and from the clusters of DMSO and Venetoclax. Gene Set Enrichment Analysis of these 4 groups presented many changes in tumor-associated pathways. Pathways regulated by a single drug ( e.g. MYC, p53, IFN-α) were further aggravated by the combo. Pathways ( e.g. Apoptosis) were not enriched by NB73 or Venetoclax, but enriched by the combo. Hyperactivation of MYC pathway is prevalent in MM. The combo significantly repressed MYC, PLK1, CCNA2 and CDC20 in MYC pathway through several mechanisms. For example, both NB73 and Venetoclax decreased MYC RNA, and the combo further lowered MYC, showing a “sum-up” effect. In contrast, the single drug lightly increased CDC20, but the combo significantly decreased it, presenting a “ de novo” effect. Since these 4 genes are important tumor promoters and inhibiting any one markedly represses tumor growth, we are addressing how the combo inhibits them. Ongoing experiments: We transplanted OPM2 cells into NSG mice and established the MM xenograft for in vivo test. NB73 is being given via sub-Q and Venetoclax is being given via oral gavage (n=5/group, 4 groups). The tumor burden will be monitored by live imaging every 2 weeks because OPM2 cells express Luciferase and the survival curve will be drawn. We are recruiting MM patients for bone marrow cells in order to develop a simplified protocol of culturing MM cells ex vivo that will be used to test new drug combinations targeting FOXM1 or other molecular vulnerabilities in MM. Conclusion: We identified FOXM1-targeted therapies consisting of novel FOXM1 inhibitor NB73 and FDA-approved drugs in vitro. We are addressing how the combo represses MYC pathway to sensitize MM cells to BCL2 inhibitor Venetoclax.