Abstract Background: Mezigdomide (Mezi, CC-92480) is a novel CRBN E3 ligase modulator (CELMoD) agent, that induces rapid and deep degradation of Ikaros and Aiolos, two transcription factors essential for multiple myeloma (MM). Elevated expression of PRC2 complex members, including the histone methyltransferase EZH2, is associated with inferior overall survival in MM. A recent clinical study (NCT05372354) demonstrated promising efficacy using a combination of Mezi, dexamethasone, and Tazemetostat (TAZ), an EZH2 inhibitor, in heavily pretreated MM patients. However, the molecular mechanisms underlying the impressive clinical activity remain poorly understood. Here, we employed integrated epigenomic and transcriptomic analyses to elucidate cell intrinsic mechanisms of the combination regimen. Methods: We evaluated the pharmacological effects of Mezi and TAZ, alone and in combination, across multiple MM cell line models. Utilizing multi-omics approaches—including RNA-sequencing, Chromatin Immunoprecipitation sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)—we analyzed differential impacts between single and combination treatments across three MM cell lines at 36 and 72 hours post compound treatment. CRISPR-Cas9 technology was used to generate FoxO3 knockout cell lines to assess the contribution of FoxO signaling to the observed preclinical synergy. Results: Pharmacologic validation through longitudinal INCUCYTE measurements confirmed that combining Mezi with TAZ substantially reduced cell proliferation and viability compared to either agent alone. RNA-sequencing analysis revealed enrichment of upregulated apoptotic and downregulated cell cycle-associated gene clusters. Notably, gene set variation analysis demonstrated significant upregulation of the Forkhead box O (FoxO) transcription factor (TF) signatures related to cell cycle and cell death, with ATAC-seq confirming corresponding increases in chromatin accessibility at these loci. FoxO TFs act as tumor suppressors in a context-dependent manner. Functionally, Cas9-mediated FoxO3 knockout in AMO1 and KMS12BM cell lines partially reduced the growth inhibitory effects of the combination compared to non-targeting controls (AMO1: sgNT: 78% vs sgFoxO3: 53% confluence; KMS12BM: sgNT: 35% vs sgFoxO3: 24% confluence at day 5) highlighting the potential role of FoxO signaling in mediating therapeutic synergy. Mechanistically, combined treatment significantly decreased inhibitory phosphorylation of FoxO1 (Ser256) and FoxO3 (Ser318/321) by 50% and 70%, respectively, relative to baseline. This dephosphorylation promoted nuclear retention of FoxO3, suggesting direct transcriptional regulation of the downstream BH3-only pro-apoptotic genes, such as BMF and BIM,both of which were found to be upregulated in gene expression profiling. ChIP-seq analysis revealed co-occupancy of Aiolos/Ikaros and H3K27me3 repressive marks at the BMF locus in vehicle treated cells. The Mezi-TAZ combination significantly reduced both Aiolos/Ikaros binding and H3K27me3 deposition at this locus, correlating with increased BMF expression and enhanced apoptotic signaling. Concordantly, immuno-blot analysis confirmed that FoxO3 knockout in AMO1 greatly reduced the combination-induced upregulation of BMF and BIM proteins. Conclusions: Our preliminary findings reveal a novel mechanism where EZH2 inhibition enhances CELMoD efficacy by coordinating chromatin remodeling and transcriptional reprogramming in MM cells leading to synergistic anti-proliferative activity. We propose that TAZ treatment increases chromatin accessibility by reducing H3K27me3 repressive marks, thereby facilitating FoxO-driven tumor suppressor programs concurrent with Aiolos/Ikaros degradation by Mezi. This dual-targeted approach represents a rational and promising strategy to overcome resistance mechanisms in MM. The interplay between H3K27me3 modulation, Aiolos/Ikaros degradation, and FoxO activation offers mechanistic insight for next-generation combination therapies. Further investigation of additional FoxO pathway mediators and effectors is warranted to better define this synergistic therapeutic axis in MM.
Anita K. Gandhi (Mon,) studied this question.