MOB3A Bypasses BRAF and RAS Oncogene-Induced Senescence by Engaging the Hippo Pathway.

Oncogenic activation of the RTK-RAS-RAF-MEK-ERK pathway occurs in approximately 25% of all human cancers, yet activated RAS, BRAF, or MEK expression in primary cells leads to a prolonged and predominantly irreversible cell-cycle arrest termed oncogene-induced senescence (OIS). OIS acts as an intrinsic tumor suppressor mechanism, serving as a barrier to tumor progression. Screening a library of activated kinases and kinase-regulatory proteins we identified MOB3A, a Mps-one binder coactivator (MOB) protein family member, whose constitutive expression permits proliferation and suppresses senescence in response to oncogenic RAS and BRAF signals. MOB3A is one of seven human MOB genes, which are highly conserved from yeast to human and that function to activate the Hippo pathway kinases (MST/LATS) or NDR kinases through direct association. Here we show that within the MOB family of genes MOB3A and C are unique in their ability to allow primary cell proliferation in the face of sustained oncogene signaling. Unlike the canonical MOB1A/B proteins, MOB3A inhibits Hippo/MST/LATS signaling and constitutive MOB3A membrane localization phenocopies OIS bypass seen with elevated YAP expression. Moreover, inhibition of MOB3 family member expression results in decreased proliferation and tumor growth of cancer cell lines. Together these data identify MOB3A's role in bypass of oncogene induced senescence and its role as a Hippo pathway inhibitor. IMPLICATIONS: These results suggest that MOB3 targeting to re-engage the Hippo pathway, or direct targeting of YAP/TAZ, may be viable therapeutic strategies potential for RAS-pathway driven tumours.

Utilization of Mutational Analysis (MA) in Gastrointestinal Stromal Tumor (GIST) Management in British Columbia (BC) Between January 2008 to December 2017: a Retrospective Population-Based Study.

PURPOSE: To examine oncologists' practice pattern of ordering MA in localized and metastatic GISTs in British Columbia (BC). METHODS: Patients diagnosed with GIST from January 2008 to December 2017 in BC were identified. Chart review was performed to determine clinical characteristics and the use of MA as part of their oncologic care. RESULTS: The cohort included 411 patients: median age 64 (18-94 years), 49.1% male, primary site included stomach (53%), small intestine (32%), and others (15%). Sixty-nine percent had localized disease, while 13% presented with de novo metastatic disease and 18% had recurrent metastatic disease. MA was ordered in 41% of the patients overall, 28% in localized, and 70% in metastatic settings (63% in de novo metastasis and 78% in recurrent metastasis). Among patients with localized disease, higher MA use rates were observed among those undergoing neoadjuvant/adjuvant treatment (45%) compared to those not receiving systemic therapy (18%). While MA use rates in localized GIST did not change over time (28.5% before 2015 and 28% after 2015), MA use in metastatic disease increased from 54% before 2015 to 79% after 2015. Among all MA ordered for metastatic disease, 82.4% were ordered at the time of de novo metastatic diagnosis, and 77.4% were ordered either at the time of recurrent metastatic diagnosis or earlier when the disease was localized. CONCLUSION: MA use has remained stable for localized disease but has increased after 2015 in the metastatic setting which may be due to evolving sequencing technology, expansion of metastatic treatment options, and enhanced awareness of MA.

Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence.

Cellular senescence is characterized by a prolonged and predominantly irreversible cell-cycle arrest state, which is linked to loss of tissue function and aging in mammals. Moreover, in response to aberrant oncogenic signals such as those from oncogenic RAS or BRAF, senescence functions as an intrinsic tumor suppressor mechanism restraining tumor progression. In addition to this durable proliferative block, senescent cells adopt altered morphologies, transcriptional profiles, and metabolism, while often possessing unusual heterochromatin formation termed senescence-associated heterochromatic foci. To uncover genes that are required to permit proliferation in the face of sustained oncogene signaling, we conducted an shRNA-based genetic screen in primary cells expressing inducible BRAF. Here we show that depletion of a known glycolysis regulator, islet amylin polypeptide (IAPP also known as amylin), prevents RAS and BRAF oncogene-induced senescence (OIS) in human cells. Importantly, depletion of IAPP resulted in changes of the cells' metabolome and this metabolic reprogramming was associated with widespread alterations in chromatin modifications compared with senescent cells. Conversely, exogenous treatment of IAPP-depleted cells with amylin restored OIS. Together, our results demonstrate that the metabolic regulator IAPP is important regulator of OIS. Moreover, they suggest that IAPP analog treatment or activation of IAPP signaling in RAS/BRAF mutant tumors may have therapeutic potential through senescence induction. IMPLICATIONS: These findings demonstrate that IAPP is a novel metabolic regulator of oncogene-induced senescence and use of IAPP analogs may be therapeutically effective to restore growth arrest to BRAF and/or RAS mutant cancers.

Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.

Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.

A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.