Novel Combination of Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit MLL-r AML

Abstract

PURPOSE: Menin is a scaffold protein that tethers histone-lysine-N-methyltransferase MLL1 (KMT2A) to chromatin by binding to the menin binding domain (MBD) of KMT2A. The N-terminus of the KMT2A gene recombines with a range of genes to form more than 80 fusion proteins (FP) in MLL-rearranged (MLL-r) AML. In myeloid stem progenitor cells, the menin-KMT2A complex modulates the expression of the leukemogenic homeobox A9 (HOXA9) gene, as well as its co-factor, MEIS1. Menin inhibitors evict menin from the chromatin and reduce KMT2A and KMT2A-FP binding to their targets in MLL-r leukemia, breaking the differentiation block and inducing tumor regression. Aberrant nuclear export is common in cancer, resulting in anomalous localization of various proteins, including tumor suppressor proteins (TSPs). Blocking XPO1/CRM1 using a selective inhibitor of nuclear export (SINE) compound selinexor has been shown to have robust anti-leukemia activity. In the present study, we hypothesized that inhibition of menin and nuclear export would synergistically suppress AML cell proliferation and survival. We have used the clinical-stage menin inhibitor ziftomenib and selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. METHODS: To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression and cell death were detected using quantitative real-time PCR, western blotting, and annexin-V/PI fluorescence-activated cell sorting, respectively. RESULTS: The clinical candidate ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11, MOLM13, and SEM) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, the nuclear export inhibitor selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination of menin and XPO1 inhibitors enhanced menin down expression, suggesting a molecular basis for the synergy between the two compounds [Fig 1.B]. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. This combination significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. We have performed total RNA sequencing and observed differential expression of certain mRNAs and micro RNAs (miRs). The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by KO539 and Selinexor, which are enhanced further by combination treatment. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. CONCLUSION: These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export could be a viable strategy for the treatment of MLL-r AML. Ongoing experiments with CDX and PDX using MLL-r models and functional analysis using total transcriptome sequencing and proteomics will be presented at the ASH meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal