Abstract 1841: Investigating mechanisms of tumor eradication by blocking SIAH E3 ligase, a major tumor vulnerability, in EGFR/RAS-driven human cancer
Authors: Baker Jonathan M. , Howell Andrew , Hannah Ashleigh , Wulfkuhle Julia , Stahr Natalie , Gallagher Rosa , Petricoin Emanuel , Tang Amy 
Published: March 2024

Introduction: Hyperactivation of the EGFR/RAS pathway is a major driver of tumorigenesis and metastasis. It has been difficult to design effective therapies contravening the compensatory EGFR/RAS downstream effector pathways to achieve curative 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/RAS pathway. Our prior studies demonstrated that SIAH inhibition led to tumor eradication of multiple malignant human cancers cell lines in xenograft models. We propose that SIAH is a major tumor vulnerability/actionable drug target for inhibiting persistent EGFR/RAS pathway activation; we aim to elucidate the molecular mechanisms underpinning antitumor efficacy of our SIAH inhibitor.

Methods: Reverse phase protein arrays (RPPAs) in conjunction with Principal Component Analysis (PCA) were used to quantify fold-changes of 300 proteins/phospho-proteins that were significantly up- or down-regulated in response to SIAH inhibition (p &lt; 0.001). Independent RPPA assays were performed in triplicate on doxycycline (DOX)-inducible MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A459 cell lines in which our SIAH inhibitor, SIAH2 PD, expression was induced 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 inhibitor expression in group C) and with DOX induction (SIAH2 PD inhibitor 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 as an internal control. To validate identified targets of interest, immunoblotting of biological quadruplicate cell lysates for each respective cell line were performed for group C and D at 3-, 5-, and 7-days post DOX induction; target proteins’ expression normalized to β-actin, α-Tubulin, or GAPDH respectively, and the pathway alterations induced by SIAH2 inhibition were standardized, quantified, and analyzed.

Results: RPPA analyses identified 6 proteins (NFκB, Caspase-7, PARP, Cofilin, PD-L1, and Collagens) exhibiting differential expression in response to SIAH2 blockade. Western blot analyses independently confirmed their altered expression patterns, further supporting their roles as the biological readout of SIAH2 loss-of-function in human cancer cells.

Conclusion: The RPPA-based cancer pathway mapping provides valuable molecular insight into the antitumor efficacy and reveals a major tumor vulnerability in human cancer pathway/network rewiring when SIAH2 is blocked in late-stage malignant cells. The kinomic data support our innovative strategy to design anti-SIAH-based anti-EGFR/RAS target therapies to control and eradicate undruggable and incurable human cancers in the future.

Citation Format: Jonathan M. Baker, Andrew Howell, Ashleigh Hannah, Julia Wulfkuhle, Natalie Stahr, Rosa Gallagher, Emanuel Petricoin, Amy Tang. Investigating mechanisms of tumor eradication by blocking SIAH E3 ligase, a major tumor vulnerability, in EGFR/RAS-driven human cancer [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 1841.