Sciver, Robert E. Van

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina 2 and sina 3 alleles, providing us with an opportunity to study sina loss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) Dm SINA and human SIAH. The altered Dm SINA/hSIAH expression under the control of sev ‐, GMR ‐, and dpp ‐ and salivary gland ‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of Dm SINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINA WT/DN /SIAH WT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future. Support or Funding Information Extramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA’s importance is its high degree of evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung, and breast cancers. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 83.

Abstract SIAH is a new and potent anti-K-RAS drug target in human cancer. The oncogenic EGFR/HER2/K-RAS pathway activation is pivotal in driving uncontrolled tumor growth and systemic metastasis. Thus, counteracting ERBB/K-RAS hyperactivation in attempt to reverse malignant transformation and inhibit latent tumor growth is an important area for new therapy development against late-stage and metastatic cancer. Guided by Drosophila studies, we found that SIAH (Seven-In-Absentia Homologue) is the most downstream “gatekeeper” required for proper K-RAS signaling. Based on its extraordinarily evolutionary conservation, SIAH E3 ligase is well positioned to serve as an ideal drug target for developing new anti-K-RAS and anticancer therapy. We have shown that anti-SIAH-based therapy is indeed effective in inhibiting tumorigenesis and metastasis of pancreatic, lung and breast cancer cells in xenograft models. Importantly, we have shown that anti-SIAH-based anti-K-RAS strategy is effective against well-established, super-large and late-stage pancreatic and triple-negative breast tumors in a xenograft model in vivo. Through these studies, we have successfully identified a new oncogenic K-RAS "vulnerability," SIAH, in high-grade metastatic cancer. We aim to design and develop potent SIAH inhibitor, and translate these findings to the clinic to benefit more cancer patients with therapy-refractory, relapsed and metastatic diseases in the future. SIAH is a therapy-responsive and prognostic biomarker in human cancer: SIAH expression can be used to monitor tumor responses, and identify resistant tumor clones post-NST. SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two robust, sensitive and prognostic biomarkers to predict survival in breast cancer patients with lymph node metastases. The prognostic power of SIAH and EGFR, alone or in combination, is comparable to the clinical gold standards of clinical predictors (LN positivity, mammary tumor size, grade, stage and molecular subtypes in combination), and imaging-guided technology. A marked reduction in SIAH/EGFR expression post-NST would indicate effective therapy and increased survival, while persistent high SIAH/EGFR expression post-NST would indicate ineffective therapy and decreased survival. The therapy-induced changes in SIAH expression are prognostic in quantifying effective/ineffective therapies, differentiating partial responders, identifying resistant tumor clones, and predicting remission/relapse in breast cancer at neoadjuvant settings. The identification of therapy-responsive and prognostic biomarkers is of paramount importance to stratify patients and guide therapies in clinical oncology and personalized medicine. By validating the RAS/SIAH pathway-centered prognostic biomarkers, we hope to guide standard therapies and improve patient survival in the future. Citation Format: Amy H. Tang, Robert E. Van Sciver, Elizaveta Svyatova, Kevin Kanda, Michael P. Lee, Caroline Dasom Lee, Lauren L. Siewertsz Van Reesema, Alex C, Lafever, Amber L. Collier, Apoorva S. Iyer, L.D. Britt, Janet S. Winston, Cynthia A. Allen, David Z. Chang, Gloria M. Petersen, Richard A. Hoefer. Conquering undruggable oncogenic K-RAS-driven incurable metastatic cancer, and delivering precision medicine at neoadjuvant settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 584.

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

Abstract Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signal transduction cascade. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung and breast cancer. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and well-established Drosophila development system, we are able to study RAS activation and SINA function under normal physiological conditions. In the developing Drosophila eye, photoreceptor cells are recruited sequentially and acquire their distinctive cell fates through a series of local inductive events. The 800x cell arrays allowed us to dissect the role of SINA/SIAH downstream of RAS activation in photoreceptor cell development. To delineate SINA function, we performed an F1 modifier screen using ethyl methanesulfonate (EMS) and X-ray radiation, isolating 28 novel sina mutant alleles. These mutant alleles exhibit much stronger mutant phenotypes than those of the previously published sina2 and sina3 alleles, suggesting that the sina2 and sina3 alleles are hypomorphic alleles. Sequencing analysis of these sinamutant alleles reveals the functional roles of mutated residues and protein domains. To define SINA/SIAH functional conservation, we have generated a complete panel of transgenic fly models that express either wild-type (WT) or dominant negative (DN) SINA/SIAH. The corresponding UAS-sina/siahGOF/LOF phenotypes have been characterized using sev-, GMR-, dpp- and salivary gland-GAL4 drivers to elucidate the developmental outcomes of altered SINA/SIAH expression upon RAS activation. Ectopic expression of sinaWT/DN/siahWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum, causing PNS neurodegenerative phenotypes. Our results show that the biological functions of fly SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Mechanistic insights and regulatory principles learned from Drosophila can be directly applied to cancer biology to develop and validate next-generation anti-SIAH-based anti-K-RAS and anticancer therapy in the future. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4824. doi:10.1158/1538-7445.AM2017-4824