CXXC1 is required for IRF4 chromatin binding and expression and regulates cellular fitness in lenalidomide-sensitive and -resistant myeloma cells Free

IRF4 is a plasma cell lineage-defining transcription factor that orchestrates important aspects of the oncogenic transcriptome in multiple myeloma (MM) largely through the deregulated IKZF1/3-IRF4-MYC axis. This transcription factor feed-forward loop is also critical for response and resistance to immunomodulatory drugs (IMiDs), a cornerstone of MM treatment. IMiD-induced degradation of IKZF1 and IKZF3 leads to reduced expression of IRF4 and MYC and myeloma cell death. Conversely, resistance to IMiDs, often due to impaired IKZF1/IKZF3 degradation, converges to sustained IRF4 expression and transcriptional activity. Therefore, a more in-depth understanding of the chromatin-based mechanisms of IRF4 activity in myeloma cells would inform its direct or indirect therapeutic targeting in both IMiD-sensitive and -resistant MM.

We previously found that the chromatin factor CXXC1 is most highly expressed in myeloma cells as compared to other hematologic and solid tumor cells while chromatin accessibility followed by footprinting analysis suggested higher frequency of CXXC1 chromatin binding in myeloma as compared to normal plasma cells. CXXC1 binds unmethylated CpG islands and is part of the COMPASS complex that catalyses histone H3K4me3, a histone mark linked to transcriptional activation.

Based on these observations, we investigated the hypothesis that CXXC1 regulates important oncogenic transcriptional programmes in MM.

We found that in different myeloma cells, including the MAF-translocated MM1.S myeloma cell line, shRNA-mediated CXXC1 knock down or degron-mediated CXXC1 acute protein depletion are toxic to myeloma cells. Transcriptome analysis of CXXC1-depleted MM1.S cells followed by pathway enrichment analysis and relevant cellular assays suggest that CXXC1 activates gene programs required for cell proliferation, glycolytic metabolism, unfolded protein response and DNA damage response.

Proteomics analysis involving immunoprecipitation of CXXC1 followed by mass spectrometry in MM1.S cells confirmed association of CXXC1 with several components of the COMPASS complex, including the SETD1A/B histone methyltransferases. Notably, it also identified IRF4 and IKZF3 (Aiolos) as direct CXXC1 interactors. Consistent with these findings, ChIP-seq in MM1.S and primary myeloma cells showed extensive co-binding of CXXC1 with IRF4, IKZF3 and IKZF1 at promoters and enhancers of key genes, including MYC. ChIP-seq after degron-induced depletion of CXXC1 showed drastic reduction of IRF4 chromatin binding, correlating with reduced CXXC1 occupancy.

No changes in long range chromatin interactions were observed by Hi-ChIP assay upon CXXC1 depletion, suggesting that CXXC1 is not required for 3D chromatin organisation.

Further epigenetic profiling showed that while H3K27ac abundance was preserved, there was a notable contraction of the size and/or reduction of the intensity of signal of H3K4me3 broad domains, previously linked to transcriptional activation of lineage identity genes. Indeed, such broad domain changes were found at the regulatory areas of several plasma cell lineage-defining genes such as IRF4, PRDM1 and XBP1.

To explore the role of CXXC1 in IMiDs resistance, we generated two independent lenalidomide (Len) resistant lines derived from the MM1S CXXC1-degron cells. As expected, upon Len treatment, IRF4 and MYC expression as assessed by western blotting decreased in Len-sensitive cells but was sustained in Len-resistant cells . Nevertheless, degron-mediated CXXC1 depletion was equally toxic to both Len-sensitive and -resistant cells lines and was associated with and extensive loss of IRF4 as well as CXXC1 chromatin binding in both Len-sensitive and Len-resistant cells. Transcriptome analysis of the same cells showed significant downregulation of IRF4 and MYC and of genes involved in mTORC1 signalling, MYC targets, cell cycle, DNA damage and unfolded protein response .

We conclude that in myeloma cells, CXXC1 is required for IRF4 binding to chromatin and their co-binding regulates essential, oncogenic transcriptional programmes including expression of IRF4 itself. This dependency of IRF4 chromatin binding on CXXC1 extends to include Len resistant myeloma cells thus providing proof-of-principle that CXXC1, and likely the COMPASS complex, constitute a novel therapeutic target for Len-sensitive and resistant MM.