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Immunomodulatory agents (IMiDs) and cereblon E3 ligase modulators (CELMoDs) are a cornerstone of Multiple Myeloma (MM) treatment. They bind to the cereblon (CRBN) component of the CRL4 E3 ubiquitin ligase complex and designate a new set of substrates for proteasomal degradation. Most patients initially respond well to IMiD/CELMoD therapy but over time will become resistant. Understanding resistance mechanisms will enable the development of new targeted treatment strategies to overcome this pressing clinical challenge. Acquired IMiD/CELMoD resistant human MM cell lines were generated by treating IMiD/CELMoD sensitive MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Cell lines were characterized by whole exome sequencing, RNA-Seq and proteomics. All resistant lines had reduced CRBN expression and some had CRBN mutations, reflecting patient data. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s. Gene effect scores were calculated with the Chronos algorithm and compared to parental MM1s using data from the Broad Institute DepMap portal. Potential new dependencies in the resistant setting (defined as gene effect score ←1 in resistant cells and >-0.5 in parental cells) were identified in 47 genes including SETD2, a histone 3 lysine 36 methyltransferase (H3K36me2 → H3K36me3). The specific SETD2 inhibitor EPZ-719 reduced viability to a greater extent in the IMiD/CELMoD resistant lines compared to their controls in a 14-day trypan blue exclusion assay. This was most pronounced in the Pom- and Iber-resistant H929 lines. For example in Iber-resistant H929 compared to its control line the viability with EPZ-719 was 13% vs 66% at 1µM, 5% vs 46% at 5µM and 2% vs 45% at 10µM, p To investigate whether this novel dependency was related to reduced CRBN expression a CRBN knockout MM1s cell line was generated using CRISPR/Cas9. These cells showed a markedly greater reduction in viability with EPZ-719 compared to control MM1s (viability 20% vs 32% at 1µM, 8% vs 25% at 5µM and 2% vs 18% at 10µM, p A reduction in H3K36me3 was seen on immunoblotting after EPZ-719 incubation across all cell lines suggesting an on-target effect. ChIP-Seq and RNA-Seq experiments are underway to identify downstream mechanisms. In conclusion, SETD2 inhibition is more active in IMiD/CELMoD-resistant cell lines compared to sensitive counterparts. Rapid clinical translation of these findings would be possible as a SETD2 inhibitor is currently in a phase 1 trial which includes MM patients. Previous pre-clinical data suggests the t(4;14) MM subgroup may be more sensitive to SETD2 inhibition but our data highlights a novel vulnerability in the IMiD/CELMoD resistant setting that should be explored further. Citation Format: Sarah Anne Bird, Marco Licciardello, Yakinthi Chrisochoidou, James Smith, Amy Barber, Jack Cheung, Yura Grabovska, Shannon Martin, Fernando Sialana, Harvey Che, Habib Bouguenina, Benjamin Bellenie, Brian Walker, Paul Clarke, Charlotte Pawlyn. SETD2 is a novel and druggable dependency in IMiD/CELMoD resistant multiple myeloma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3879. |
