Abstract 6382: DNA damage repair pathways enable a drug-tolerant persister state in RET-fusion NSCLC and precedes TKI resistance

RET-fusion-driven lung adenocarcinomas (LUADs) are a rare and particularly aggressive subtype of lung cancer, often affecting young, non-smoker populations. While selective RET-TKI therapies are initially highly effective, they often fail to provide durable responses, with resistance mechanisms emerging over time. Notably, approximately 40% of patients exhibit resistance mechanisms that remain unidentified. To uncover these unknown mechanisms, we performed a comprehensive multi-omic analysis of clinical samples from patients with RET-fusion lung cancers before and after RET-TKI treatment. RNA sequencing analysis revealed inactivation of RB1 and upregulation of DNA damage repair (DDR) pathways in resistant tumors relative to TKI-sensitive samples. Accordingly, methylome analysis confirmed hypomethylation of E2F targets and DDR pathways, suggesting that their transcriptomic upregulation is mainly driven by epigenetic changes. We hypothesized that DDR pathway upregulation was a result of inhibition of Rb1 during TKI treatment. To validate this, we knocked down Rb1 or overexpressed of E2F, and confirmed upregulation of key DDR genes such as BRCA1, MSH6, and Rad18 in RET cell lines. To validate the role of DDR pathways in resistance, we established isogenic cell lines by overexpressing or knocking out BRCA1, MSH6, and Rad18, and conducted drug-tolerant persister (DTP) assays. Genetic inhibition of each of these DDR proteins significantly reduced the generation of DTPs after high dose treatment with the RET-TKI selpercatinib (4.5 uM) while overexpression increased the number of DTPs. We next evaluated the inhibition of XPO1, an exportin protein linked to DDR pathway activation, as a strategy to target DTPs. Treatment with selinexor, an FDA-approved XPO1 inhibitor, in combination with selpercatinib strongly reduced the number of DTPs in vitro, and overexpression of the DDR genes BRCA1, MSH6, and Rad18 rescued this effect. In vivo experiments using RET-driven patient-derived xenograft (PDX) models confirmed that treatment with combination of selpercatinib with selinexor significantly delayed tumor relapse in TKI-sensitive PDX models (n=4) and resensitized resistant PDX tumors to selpercatinib (n=2). Our research highlights DDR pathways as a critical mechanism of resistance via Rb1 inactivation in TKI-treated RET-driven lung cancers and identifies XPO1 inhibition as a promising therapeutic strategy to overcome this resistance. Finally, we also found upregulation of DDR proteins upon treatment with lorlatinib and osimertinib in ALK and EGFR driven LUAD PDXs, respectively, suggesting that targeting DDR pathways could be an effective strategy against resistance in a pan-TKI setting across other RTK-driven LUADs. Citation Format: Fathema Z. Uddin, Samuel Tischfield, Romel Somwar, Alexander Lim, Hyung Jun Woo, Christina Falcon, Barbara P. Mello, Dennis Kinyua, Harsha Sridhar, Parvathy Manoj, Hong Zhong, Juan Qiu, Elisa de Stanchina, Mark Donoghue, Mark Ladanyi, Esther Redin, Charles M. Rudin, Alvaro Quintanal-Villalonga, Alexander Drilon. DNA damage repair pathways enable a drug-tolerant persister state in RET-fusion NSCLC and precedes TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6382.