Abstract 5248: The use of reverse phase protein array (RPPA) to identify new tumor vulnerability and actionable drug targets against human cancer cell lines

Abstract

Abstract Introduction: The EGFR/K-RAS pathway is a major tumor driver whose hyperactivation is associated with malignant tumor growth, multidrug resistance, early tumor relapse, chemo-resistance, and metastasis. Despite more than 40 years of research, oncogenic K-RAS activation remains an undruggable target in clinical oncology. Seven-In-Absentia (SINA) homologues (SIAH) are conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/K-RAS pathway. Our prior studies have shown that tumor growth was abolished in human tumor cell lines following SIAH inhibition; however, the underpinning molecular mechanisms that give rise to this anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific Objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding growth, we conducted reverse-phase protein array (RPPA)-based kinomic analysis to discover how major cancer signaling pathways and K-RAS-dependent signaling networks are remodeled in response to SIAH2 inhibition. Ongoing studies aim to identify and validate dysregulated phospho-proteins identified by RPPA-based kinomic analysis. Methods: 300 proteins/phosphoproteins were quantitatively measured by the RPPA platform to identify new tumor vulnerabilities, actionable targets, and compensatory signaling network alterations in response to anti-SIAH targeted therapies in five cancer cell lines in triplicate. Doxycycline (DOX)-inducible Tet-ON MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A459 cell lines were generated from a single cell, and DOX-induced SIAH2PD expression was confirmed. Reverse Phase Protein Array (RPPA) was conducted to quantify fold-changes of proteins whose expression was significantly (p<0.05) altered in response to SIAH inhibition. Ongoing Western Blot and Immunofluorescence (IF) studies are being used to validate our key RPPA findings. Results: Supported by statistical analyses, we identified 6 unique proteins that were significantly up-or down-regulated in response to SIAH inhibition. These proteins play a role in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, immune function, and cell attachment pathways. Thus, our data provide additional evidence supporting our tumor eradication phenotype observed in these human cancer cell lines lacking functional SIAH. Western Blot and IF validation studies have yielded further supporting evidence to confirm our RPPA results. Conclusion: Validation studies are being conducted to gain insight into pathway alterations and dynamic rewiring in order to uncover SIAH as a major tumor vulnerability in multiple malignant cancers. Our data supports the explanation as to why anti-SIAH targeted therapy works so effectively to shut down malignant tumor growth, as reported in the literature. Citation Format: Andrew Howell, Julia Wulfkhule, Rosa Gallagher, Jonathan Baker, Ashleigh Hannah, Emanuel F. Petricoin, Amy H. Tang. The use of reverse phase protein array (RPPA) to identify new tumor vulnerability and actionable drug targets against human cancer cell lines. [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 5248.