Targeting the compass complex to overcome resistance in KMT2A-rearranged Acute Myeloid Leukemia

Abstract

Abstract Introduction Regulation of gene expression by histone modification is critical in drug resistance and progression. KMT2A-rearranged (KMT2A-r) AML can activate aberrant leukemogenic HOXA and MEIS1 gene signatures through its interaction with menin. KMT2A is part of COMPASS complex along with ASH2L, WDR5, RBBP5, and DPY30, sometimes called 'WRAD’ complex, and functions as writers and readers to catalyze H3K4 methylation and drives transcription of leukemogenic genes such as HOXA9 and MEIS1. ASH2L is part of the core complex and inherently enhances the enzymatic activity amongst the other scaffolding proteins. We hypothesized that ASH2L (a histone methyltransferase writer) and WDR5 (a chromatin reader) constitute an escape, non-canonical resistance mechanism that rewires the leukemogenic transcription program even in the presence of menin inhibitor KMT2A-r AML cells. Methods To this note, we aimed to establish an in-vitro resistant model in KMT2Ar leukemia cell lines (MV4-11 and MOLM-13) by chronically exposing them to sequentially escalating, sub-therapeutic doses of a menin inhibitor (ziftomenib, Selleckchem, cat.no:E1290). COMPASS complex gene and downstream transcripts and protein expressions were detected by quantitative RT-qPCR and western blotting. SiRNA-mediated knockdown (kd) of ASH2L and WDR5wasperformed to investigate their functional role in resistance, and western blotting was used to confirm protein depletion and assess downstream effects on MLL1/COMPASS components. An ATP-based cell proliferation assay was performed to assess cell viability and growth inhibition following SiRNA KD and by using WDR5 inhibitor (OICR-9429, Selleckchem, cat.no:S7833) to pharmacologically target the complex. ChIP-qPCR was performed to determine whether resistance is mediated by altered chromatin occupancy by alternate COMPASS components to the HOXA9 promoter in the resistant models. Results Incremental exposure of MV4-11 cells to a menin inhibitor (ziftomenib) over six months resulted in a progressive increase in IC₅₀ from baseline, with the cells maintaining proliferative capacity even at continued exposure of 60 nM of the inhibitor. Cell line authentication using short-tandem-repeats verified that the resistant subline was genetically identical to the parental MV4-11 cell line, and samples were processed for sequencing of the MEN1-binding pocket to screen for menin inhibitor resistance-associated mutations. RT-qPCR and immunobloting exposed clear transcriptional and protein level differences in the resistant and sensitive AML cells. Sensitive cells retained low HOXA10 and MEIS1/2 expression whereas resistant cells exhibited reduced menin levels but increased ASH2L, WDR5, and HOXA9 expression, suggesting that HOXA9 upregulation may serve as a potential escape mechanism driving resistance. SiRNA knockdown of ASH2L and WDR5 in resistant AML cells led to a marked decrease in leukemogenic transcripts, including HOXA9 and MEIS1, as confirmed by immunoblotting. In parallel, we generated dose-response curves for WDR5 dependency in sensitive (IC50 for ziftomenib, 14nm) and menin-inhibitor-resistant MV4-11 cells (IC50 for ziftomenib, 65nm), and notably, treatment with a WDR5 inhibitor restored the IC₅₀ of resistant cells to levels comparable to the sensitive parental line (IC50 for WDR5 inhibitor 12nm). ChIP-qPCR with ASH2L immunoprecipitation in menin-inhibitor-resistant cell line showed markedly increased ASH2L occupancy at HOXA9 promoter, suggesting that higher occupancy may contribute to resistance to menin inhibition. Ongoing work examines selective ASH2L inhibition—alone or combined with menin and WDR5 blockade—to overcome resistance in KMT2A-r AML. Genome-wide ChIP-seq to map ASH2L and WDR5 redistribution at promoters/enhancers, extending our HOXA9 data. TurboID proximity labeling to compare protein-interaction networks in sensitive versus resistant cells to uncover COMPASS partners. CRISPR knockdowns and patient cells will be tested in xenografts for therapeutic response. Conclusion ASH2L and WDR5 are critical mediators of COMPASS-dependent therapy resistance in KMT2A-r AML. Targeting this complex, particularly ASH2L and WDR5, represents a promising strategy to possibly reverse resistance and improve therapeutic outcomes in resistant KMT2A-r AML.Our preliminary data indicate other components of the COMPASS complex to possibly cause resistance in KMT2A-r AML, and further validations are in progress.