Abstract 5762: RPPA kinomic profiling of a tumor eradication phenotype induced by SIAH inhibition, a major tumor vulnerability in the EGFR/HER2/RAS signaling pathway, in late-stage and metastatic human cancer cell lines

Abstract Introduction: Persistent activation of the EGFR/K-RAS pathway is a major driver of tumor malignancy. Therapies attempting to contravene intertwined RAS effector pathways have yet to achieve durable antitumor efficacy. Seven in absentia homologues (SIAH1 and SIAH2) are RING-domain E3 ubiquitin ligases that function as the most downstream signaling gatekeeper of the EGFR/K-RAS pathway. We showed that SIAH inhibition led to tumor eradication against late-stage human cancer cell lines in xenograft mouse models. We propose that SIAH is a major tumor vulnerability whose complete inhibition impedes EGFR/K-RAS pathway activation driving cancer progression. We aim to elucidate the molecular mechanisms underpinning the curative efficacy of our anti-SIAH strategy. Methods: Reverse phase protein arrays (RPPA) and principal component analysis (PCA) were used to quantify the changes of 300 proteins/phospho-proteins that were significantly up- or down-regulated in response to SIAH blockade. Independent RPPA assays were performed in triplicate on doxycycline (DOX)-inducible MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A549 cell lines in which our SIAH inhibitor, SIAH2 PD, expression was controlled by a Tet-ON/OFF system. Four experimental conditions were used: Tet-ON control cells without DOX (group A) and with DOX induction (group B), Tet-ON-SIAH2 PD cancer cells without DOX (no SIAH2 PD expression in group C) and with DOX induction (SIAH2 PD expression in group D). The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated in a pairwise comparison after normalization to GAPDH. Western immunoblotting (WB), immunofluorescence staining (IF), and flow cytometry of the Tet-ON/OFF cancer cells and protein extracts were analyzed at 3-, 5-, and 7-days (+/-) DOX induction in triplicate to validate the results. SIAH2 PD-induced protein expression alterations were normalized to β-Actin or α-Tubulin, and the standardized data were quantified. Statistical analyses were performed by one-way ANOVA using GraphPad Prism software. Results: We identified the altered expression of NFκB, Caspase-3, Caspase-7, and PARP in response to SIAH2 blockade, with independent confirmation by RPPA, WB, and flow cytometry, supporting their roles as a mechanistic readout of SIAH inhibition in human cancer cells. Conclusion: RPPA cancer pathway mapping provides molecular insights into cancer network rewiring in response to SIAH blockade. This study supports our innovative strategy to design anti-SIAH-based, anti-EGFR/K-RAS targeted therapies to control and eradicate late-stage and incurable human cancers in the future. Citation Format: Jonathan M. Baker, Andrew Howell, Daniel J. McWilliams, Ashleigh E. Hannah, Julia Wulfkuhle, Rosa Gallagher, Emanuel Petricoin III, Amy H. Tang. RPPA kinomic profiling of a tumor eradication phenotype induced by SIAH inhibition, a major tumor vulnerability in the EGFR/HER2/RAS signaling pathway, in late-stage and metastatic human cancer cell lines [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 5762.