Abstract 4739: Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma

Abstract

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.