Assistant Professor • SOM: HMO: Hematology Emory University Atlanta, Georgia, United States
Introduction: Resistance to IMiDs is a major problem in the treatment of MM and is explained in only a fraction of cases by alterations of the CRBN pathway that reduce the ability of IMiDs to deplete IKZF1 and IKZF3. Here, we show that IMiDs synergize with EP300 inhibitors (EP300i) but that transcriptional heterogeneity can overcome IKZF1, IKZF3, and EP300 dependency to maintain MYC and IRF4 expression and mediate IMiD resistance.
Methods: Human myeloma cell lines (HMCL) were treated with pomalidomide (POM) and EP300i (GNE781 and CCS1477) and proliferation was measured by cell count. IKZF1, IKZF3, MYC, and IRF4 protein levels were measured by Western blot. Vk*MYC;hCRBN mice were treated with POM (50 mpk) and/or G781 (15 or 3.75 mpk) for two weeks and survival was measured. shRNA knockdown and doxycycline-inducible lentiviral overexpression were used for functional characterization of BATF proteins. Analysis of CoMMpass used IA17 with RNAseq from Genomic Data Commons.
Results: Treatment with POM resulted in a universal degradation of IKZF1 and IKZF3 across 48 HMCL, but this did not correlate with effects on proliferation, which was more closely linked with MYC and IRF4 downregulation. MYC and particularly IRF4 downregulation could also be achieved with EP300i, which showed single-agent activity in a clinically predictive murine model of MM but had a narrow therapeutic index. POM+EP300i resulted in further downregulation of IRF4 and MYC, induced synergistic responses in HMCL, was tolerable in mice with murine MM, and significantly extended their survival. ChIPseq showed that 79% of EP300 binding sites were also bound by IKZF1. HMCL resistant to POM+EP300i were defined by high expression of ETS and BATF AP-1 factors. When expressed, HMCL were dependent upon BATF, BATF2, and BATF3, and their knock-down induced sensitization to POM+EP300i while their overexpression promoted resistance and maintained IRF4 and MYC expression. Mechanistically, BATF2, IRF4, and IKZF1 bound the same sites in the IGH and DUSP22/IRF4 super-enhancers suggesting BATF factors bind with IRF4 at AP-1:IRF composite elements (AICE). Overexpression of a BATFH55Q, which cannot interact with IRF4, failed to induce resistance to POM+EP300i. Finally, BATF expression was associated with reduced PFS and upregulated upon relapse in patients treated with IMiDs in CoMMpass.
Conclusions: These data indicate that MYC and IRF4 downregulation are critical events for IMiD responses, which can be augmented through combined EP300i. The overlap of P300 and IKZF1 binding sites provides a molecular explanation for the observed synergy between POM and EP300i. These data provide a strong preclinical rationale for the ongoing clinical trial of POM+CCS1477 (NCT04068597). Finally, the expression of BATF AP-1 factors can mediate IMiD resistance by alleviating IKZF1 and IKZF3 dependency, likely through cooperative binding with IRF4 at essential myeloma super-enhancers that maintain MYC and IRF4 expression.
