Abstract A19: Conditional inactivation of SHOC2 in adult mice to study its role in tissue homeostasis

Abstract

Abstract The RAS-RAF-MEK-ERK signaling pathway plays an important role throughout mammalian development, from embryogenesis to tissue-specific cellular homeostasis, and its aberrant activation is a major driver of human cancer. Mouse models have highly improved our current understanding of the RAS-ERK pathway in physiologic and pathologic contexts. RAF activation is a complex process that involves multiple regulatory steps in addition to RAS binding. Key among them is the dephosphorylation of a conserved inhibitory site (S259-RAF1, S365-BRAF) by a phosphatase complex comprising SHOC2, MRAS and PP1 (SHOC2 complex). Work from our lab has proposed the SHOC2 complex has properties of an attractive therapeutic target against RAS-driven tumors that may help overcome the main problems associated with pharmacologic inhibition of the pathway in the clinic, namely, drug resistance and toxicity. In order to study the role of SHOC2 in vivo/in tissue homeostasis we have generated 2 mouse models of conditional SHOC2 inactivation. A knock-out model (KO) was generated by flanking exon 4 with loxP sites. A knock-in (KI) model was generated using a minigene strategy, where wild-type SHOC2 is expressed in a cDNA configuration under its endogenous promoter and replaced after Cre-mediated recombination by a mutant SHOC2 D175N allele that is defective for interaction with MRAS and PP1. SHOC2 KO and KI mice are embryonic lethal, indicating SHOC2 function is required during mouse development. To study SHOC2 function in adult tissue homeostasis, SHOC2 KO and KI mice were crossed with animals carrying an inducible ubiquitously expressed CreERT2 recombinase (R26-CreERT2). Treatment with tamoxifen leads to efficient recombination (>80%) in all tissues examined except brain. Significantly, SHOC2 inactivation is tolerated well in the short term as mice appear normal up to ~10 weeks post-treatment. However, at later times KO female mice start to lose weight and have to be sacrificed after ~14 weeks. Male KO mice do not lose weight but instead develop skin lesions and have to be culled at ~15 weeks. Postmortem studies show both sexes have splenomegaly and swollen lymph nodes consistent with an immune phenotype that is under study. Male KO mice also have swollen bladders, suggesting a sexually dymorphic role for SHOC2 in urinary function. Our mouse studies reveal that SHOC2 inactivation in adult mice appears well tolerated in the short term and thus help validate SHOC2 as a good therapeutic target. However, our studies also reveal new roles for SHOC2 in tissue homeostasis and suggest pleiotropic phenotypes likely to emerge after sustained inhibition. Citation Format: Sibel Sari, Isabel Boned del Rio, Greg Jones, Jake Henry, Maria De Las Nieves Amalia Peltzer, York Posor, Sergio Quezada, Pablo Rodriguez-Viciana. Conditional inactivation of SHOC2 in adult mice to study its role in tissue homeostasis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A19.