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

PURPOSE: Menin is a scaffold protein that interacts with oncogenic histone-lysine-N-methyltransferase MLL1 (KMT2A)-fusion protein (FP) complex in MLL-rearranged (MLL-r) AML. Menin inhibitors that evict menin from this interaction have been shown to be active against MLL-r and NPM1^MT leukemia by modulating the expression of the leukemogenic homeobox A9 (HOXA9) gene and its co-factor, MEIS1. Recent evidence showed that menin inhibitors can activate a tumor-suppressive network via a non-canonical transcriptional program through UTX. On the other hand, selective inhibitor of nuclear export (SINE) against XPO1/CRM1 has been shown to have robust anti-leukemia activity via enrichment of tumor suppressor proteins in the nucleus. In the present study, we hypothesized that menin and nuclear export inhibition would synergistically suppress AML cell proliferation and possibly sensitize menin inhibitor-resistant cells. We have used the clinical-stage menin inhibitor ziftomenib and SINE compound 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 was detected using quantitative real-time PCR and western blotting. Flow cytometric analysis was performed to detect cell death and cell cycle status. We have performed the whole transcriptomic analysis via the RNA sequencing approach.

RESULTS: The clinical candidate drug ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11 and MOLM13) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor stem 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, selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination enhanced menin down-expression, suggesting a molecular basis for the synergy between the two compounds. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. The combination showed increased CD11b, a marker of monocytic differentiation in MV4-11 treated cells. The combination also significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. Compared to controls, ziftomenib and selinexor-treated samples individually showed increased apoptotic and decreased G2/M populations, significantly enhanced in the combination. We observed differential expression of certain mRNAs and micro RNAs (miRs) in MV4-11 treated cells. The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by ziftomenib and selinexor, which are enhanced further in the combination group. 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. MV4-11 cell line-derived xenografts in NSG mice showed a significant difference in survival in the combination arm compared to any single agent. Metronomic dosing also showed robust improvement in survival in the combination arm. Patient-derived xenografted NSG mice model using human primary MLLr re-transplantable leukemia cells showed improved survival in the combination arm [Fig. 1B].

CONCLUSION: These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export is a viable strategy for the treatment of MLL-r AML. Ongoing experiments with functional proteomic analysis will be presented at the ASH meeting.