PHF19 inhibition increases IMiDs sensitivity by epigenetically regulating IRF4 and MYC in multiple myeloma

Abstract Immunomodulatory drugs (IMiDs) are a cornerstone of multiple myeloma (MM) therapy. However, drug resistance remains a major obstacle to cure the disease. Epigenetic dysregulation has been linked to disease progression and drug resistance, prompting the exploration of epigenetic-targeting drugs. Identifying new therapeutic targets is critical to overcoming treatment resistance and improving patient outcomes. We performed RNA sequencing (RNA-seq) on primary CD138+ bone marrow mononuclear cells (BMMCs) from MM patients with differential responses to IMiD-based combination immunotherapy. Analysis revealed that the E2F pathway, G2M pathway and MYC pathway were significantly activated in non-responders (n=8) compared to responders (n=8). By integrating our dataset with upregulated genes from D-KRd non-responders (Nat Med. 2021), we identified the epigenetic regulator PHF19 as the top correlated gene. PHF19 knockdown (KD) impaired MM cell proliferation, induced cell cycle arrest, and promoted apoptosis. RNA-seq of PHF19-KD cells showed significant downregulation of MYC targets, E2F signaling, G2M pathway, and IRF4-dependent pathways (as defined by the Shaffer signature), partially recapitulating the transcriptional profile of non-responders mentioned above. Consistent with this, RT-qPCR and Western blot confirmed reductions in IRF4 and MYC at both mRNA and protein levels upon PHF19 depletion. To elucidate PHF19's mechanistic role, we performed co-immunoprecipitation mass spectrometry (Co-IP/MS), which revealed PHF19 interactions with PRC2 complex components and unexpectedly, mRNA metabolic regulators. Subsequent ATAC-seq in PHF19-KD cells demonstrated widespread reductions in chromatin accessibility at multiple gene loci. Integrated RNA-seq/ATAC-seq analysis identified 391 overlapping genes enriched in cell cycle regulation, including IRF4 and MYC, suggesting that PHF19 sustains their expression by modulating transcriptional accessibility. Additionally, mRNA stability assays using actinomycin D revealed that IRF4 and MYC transcripts were destabilized in PHF19-KD cells, implicating PHF19 in post-transcriptional regulation. Our group previously linked PHF19 to resistance to proteasome inhibitors (PIs) and anti-CD38 monoclonal antibodies. Here, we assessed IMiD sensitivity via CCK-8 proliferation assays and flow cytometry-based apoptosis measurements. Strikingly, PHF19 depletion sensitized MM cells to IMiDs. Mechanistically, IMiDs treatment further suppressed IRF4 and MYC in PHF19-KD cells, whereas their levels remained stable in controls. PHF19 is overexpressed in immunotherapy non-responders and drives resistance by sustaining IRF4/MYC expression through dual epigenetic (chromatin remodeling) and post-transcriptional (mRNA stabilization) mechanisms. Targeting the PHF19-IRF4-MYC axis enhances IMiD sensitivity, positioning PHF19 as a promising epigenetic target to counteract myeloma progression and therapy resistance.