Background: Rhabdoid tumors harbor wild-type TP53, making MDM2 inhibition an attractive therapeutic strategy. However, intrinsic resistance limits single-agent efficacy, underscoring the need to identify rational combination partners. Methods: We conducted an unbiased single-point cell viability screen of the St. Jude cereblon-targeting molecular glue compound library in resistant Rhabdoid tumor cell lines in the presence or absence of the MDM2 inhibitor Brigimadlin. Synergistic hits were validated in dose-response. Lead compound selection was guided by analogue profiling, TMT-based quantitative proteomics, and molecular docking. Mechanistic characterization included live-cell imaging (Cellcyte), flow cytometry, and Western blot analysis across three resistant Rhabdoid cell lines (A204, BT16, BT12). Pharmacokinetic, Pharmacodynamic, and tolerability studies were conducted in vivo. Results: The screen identified multiple synergistic hits, all converging on GSPT1 degradation, implicating the GSPT1–MDM2i axis as a synthetic vulnerability in Rhabdoid tumor. Dose-response validation and analogue benchmarking identified SJ001010017 (SJ017) as a superior GSPT1 degrader. TMT proteomics confirmed on-target specificity, with Lenalidomide competition establishing cereblon engagement. SJ017 selectively degraded GSPT1 without affecting CK1α or IKZF1, and outperformed a previously reported selective GSTP1, SJ986, degrader across all three resistant cell lines. Molecular docking provided a structural basis for its enhanced potency. In A204 and BT16 models, combination treatment reduced MCL1 levels and induced apoptosis as confirmed by caspase-3 and PARP cleavage. Interestingly, BT12 exhibited MCL1-independent cell death, and was associated with reduced NOXA expression and elevated GCN2 activity, suggesting ISRmediated cell death mechanism. In vivo pharmacodynamic and toxicity studies demonstrated effective, well-tolerated GSPT1 degradation, supporting initiation of an efficacy study combining SJ001010017 with Brigimadlin. Conclusions: GSPT1 degradation is a potent and selective strategy to sensitize resistant rhabdoid tumor to MDM2 inhibition. SJ017 represents a promising clinical candidate, with ongoing in vivo studies evaluating combination efficacy. The MCL1-independent resistance in BT12 implicates GCN2/ISR signaling as a potential mediator of residual resistance, warranting further investigation.

Updating soon...