IRF4 maintenance in multiple myeloma is conserved in de novo-derived inobrodib resistance, conferring cross resistance to immunomodulatory drugs

Abstract Multiple myeloma (MM) is the second most common hematological malignancy. It is marked by widespread transcriptional dysregulation and is highly dependent on key transcription factors such as IRF4, MYC, IKFZ1/3 and the lysine acetyltransferases P300/CBP. Several therapeutic approaches target these factors, including immunomodulatory drugs (IMiDs), which degrade IKZF1/3 to downregulate IRF4 and MYC. Despite advances in treatment strategies, relapse is inevitable. Moreover, relapsed patients become refractory to their previous treatments; resistance has been demonstrated to arise through genetic and epigenetic mechanisms. This highlights the importance of developing new myeloma drugs to better treat relapse/refractory patients. Inobrodib is a P300/CBP bromodomain inhibitor that is currently in a phase I/IIa clinical trial for relapsed/refractory MM (NCT04068597). Initial findings indicate that pomalidomide-refractory patients are resensitized when co-treated with inobrodib. Aside from its favorable therapeutic profile, it is also well-tolerated in patients. Its success appears to pivot on its ability to downregulate IRF4 and MYC, causing cell cycle arrest in MM cell lines. Considering the incurable nature of MM, we hypothesized that patients successfully treated with inobrodib will inevitably relapse and become refractory. Hence, we aimed to model inobrodib resistance mechanisms and identify novel vulnerabilities that may be harnessed as potential treatments for inobrodib-resistant patients, prior to their emergence in clinic. We evolved inobrodib resistance in three MM cell lines (KMS12, H929 and JJN3) representing the most common translocation subtypes [t(11;14), t(4;14) and t(14;16), respectively] and obtained clonal populations for analysis. These cells also demonstrated resistance against the acetyltransferase inhibitor A485, indicating a general loss of dependence on P300/CBP. We captured transcriptional and epigenetic changes in response to inobrodib through RNA-seq, ChIP-seq and ATAC-seq in sensitive and resistant cell lines to elucidate resistance mechanisms. We observed widespread transcriptional differences between sensitive and resistant cells, along with genome-wide redistribution of H3K27ac, P300 and IRF4 binding, and differential responses to inobrodib treatment. By comparing clonal populations of resistant cells, we found evidence of at least four distinct resistance pathways. However, maintenance of IRF4 expression following inobrodib treatment was a common feature in all resistant clones. Surprisingly, despite no previous exposure to IMiDs, the inobrodib-resistant cells also displayed resistance towards pomalidomide. However, combination treatment with pomalidomide and inobrodib was able to drive IRF4 downregulation, resensitizing inobrodib-resistant cells to treatment. Confirming the importance of IRF4 in resistance, we found that exogenous IRF4 expression in sensitive cell lines increased their tolerance to both inobrodib and pomalidomide. Together, these findings implicate IRF4 as a common driver of inobrodib and IMiD resistance. Despite identifying multiple pathways to inobrodib resistance, we found that IRF4 maintenance in the presence of inobrodib is a shared feature. This ability likely explains inobrodib and IMiD cross-resistance in these cells. Given that both pomalidomide and inobrodib act via IRF4 downregulation, combination treatment appears to be an effective strategy to target patients refractory against either drug. Additionally, we propose that direct targeting of IRF4 activity may be a fruitful avenue for further investigation where IMiD and inobrodib treatment is unsuccessful.