Assistant Professor • SOM: HMO: Hematology Emory University Atlanta, Georgia, United States
Introduction: Immunomodulatory Drugs (IMiDs) improve outcomes in multiple myeloma (MM), in part, by targeting IKZF1 and IKZF3 for degradation. Almost all MM patients receive IMiDs as front-line therapy, and they eventually develop resistance. Downregulation and mutations have been reported in CRBN, the direct binding target of IMiDs, but these only account for a small fraction of IMiD-resistant cases. Here, we show that the ETS factor ETV4 binds the same elements as IKZF1 to maintain enhancer function, MYC expression, and IMiD resistance.
Methods: Myeloma cells were treated with 10 uM lenalidomide (Len) and responses were measured by MTT assays, RNAseq, and ChIPseq. ETV4 ablation was performed by CRISPR/Cas9. Computational analyses used R/Bioconductor and CoMMpass data from Genomic Data Commons with IA19 outcome data.
Results: Len treatment of 12 myeloma cell lines resulted in IKZF3 degradation in all cases, but MYC downregulation only occurred in cells where growth was inhibited. ChIP-seq showed IKZF1 bound both proximal and telomeric distal MYC enhancers as well as immunoglobulin (Ig) super-enhancers, that are often translocated to MYC and other oncogenes. However, both IMiD-sensitive (MM1S) and resistant (RPMI8226) myeloma cells showed IKZF1 binding to these elements. Similarly, Len treatment resulted in the depletion of chromatin-bound IKZF1 in both MM1S and RPMI8226 at DUSP22/IRF4, MYC, and Ig super-enhancers. In contrast, the transcriptional co-activators P300 and BRD4 were depleted from chromatin in MM1S upon Len treatment but remained unchanged in RPMI8226 despite IKZF1 depletion, suggesting other factors may be substituting for IKZF1 to maintain RPMI8226 enhancer function. Bioinformatic analyses identified expression of the ETS factor ETV4 as associated with IMiD response in myeloma cell lines and enrichment of the ETV4 consensus binding motif “AGGAAG” at IKZF1-bound regions. ETV4 ChIP-seq showed a 79% overlap with IKZF1-bound regions including those at the IRF4/DUSP22, MYC, and Ig super-enhancers. CRISPR/Cas9-mediated ablation of ETV4 in RPMI8226 resulted in Len downregulating MYC, and reducing RPMI8226 cell growth. Finally, ETV4 expression was found in 10% of newly-diagnosed patients from the CoMMpass and POLLUX trials and each study showed ETV4 expression was associated with poor progression-free survival and upregulated upon relapse.
Conclusions: These data indicate that the downregulation of MYC is a “bottleneck” in IMiD responses and that this requires enhancer decommissioning. Depletion of chromatin-bound IKZF1 is not a differentiating factor in IMiD responses. Rather, the determining factor is whether or not enhancer function is dependent upon IKZF1 binding resulting in MYC downregulation and IMiD-mediated growth inhibition. In this regard, we have identified ETV4 transcriptional plasticity in 10% of newly diagnosed myeloma as a mediator of IMiD resistance. These data provide a rationale for targeting enhancer co-factors (e.g. P300) in combination with IMiDs.
