Evidence for p15 cleavage site in myc-specific proteins of avian MC29 and OK10 viruses

Myc-related proteins were precipitated from MC29 virus-transformed cells (PR-2) and from OK10 virus-transformed cells (9C) by anti-gag and anti-myc sera. Immunoprecipitates were cleaved with the avian retroviral protease p15 and the cleavage products analyzed in SDS-PAGE. Cleavage fragments of p110gag-myc (product of MC29 virus) and p58myc (product of OK10 virus) showed the presence of a p15 cleavage site within the myc-specific region. The site is missing in deletion mutants of MC29 virus.     

Knockdown of CkrL by shRNA deteriorates hypoxia/reoxygenation‐induced H9C2 cardiomyocyte apoptosis and survival inhibition Via Bax and downregulation of P‐Erk1/2

Integrin β1 subunit and its downstream molecule integrin‐linked kinase and focal adhesion kinase have been confirmed to be essential to cell survival and inhibition of apoptosis and hypoxia/reoxygenation (H/R)‐induced injuries in cardiomyocytes. However, it is still unclear whether CrkL [v‐crk avian sarcoma virus CT‐10 oncogene homolog (Crk)‐like], which acts also as a component of the integrin pathway, could also affect H/R‐induced injuries in the cardiomyocytes. The rat‐derived H9C2 cardiomyocytes were infected with a CrkL small hairpin RNA interference recombinant lentivirus, which knockdowns the endogenous CrkL expression in the cardiomyocytes. Apoptosis, cell proliferation and survival were examined in the H9C2 cardiomyocytes treated with either H/R or not. Results showed that knockdown of CrkL could significantly increase apoptosis and inhibition of the cell proliferation and survival and deteriorate the previously mentioned injuries induced by H/R. In contrast, overexpression of human CrkL could relieve the exacerbation of the previously mentioned injuries induced by CrkL knockdown in the H9C2 cardiomyocytes via regulation of Bax and extracellular signal‐regulated kinase1/2 (p‐ERK1/2). In conclusion, these results confirmed that knockdown of CrkL could deteriorate H/R‐induced apoptosis and cell survival inhibition in rat‐derived H9C2 cardiomyocytes via Bax and downregulation of p‐ERK1/2. It implies that CrkL could mitigate H/R‐induced injuries in the cardiomyocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Zebrafish model of KRAS-initiated pancreatic endocrine tumor

Pancreatic cancer constitutes a genetic disease in which somatic mutations in the KRAS proto-oncogene are detected in 95% of cases. Activation of the KRAS proto-oncogene represents an initiating event in pancreatic tumorigenesis. Here, we established a zebrafish pancreatic neoplasia model that recapitulates human pancreatic tumors. Toward this end, we generated a stable CRE/Lox-based zebrafish model system to express oncogenic KRAS^G12D  in the elastase3I domain of the zebrafish pancreas. Lineage tracing experiments showed that early KRAS^G12D -responsive pancreatic progenitors contribute to endocrine in addition to exocrine cells. In this system, 10% and 40% of zebrafish developed pancreatic tumors by 6 and 12 months, respectively. The histological profiles of these experimental tumors bore a striking resemblance to those of pancreatic endocrine tumors. Immunohistochemical analysis including the endocrine cell-specific marker confirmed the pancreatic tumor region as a characteristic endocrine tumor. Taken together, our zebrafish model data revealed that pancreatic endocrine tumors originate from early KRAS^G12D -responsive pancreatic progenitor cells. These findings demonstrated that this zebrafish model may be suitable as an experimental and preclinical system to evaluate different strategies for targeting pancreatic endocrine tumors and ultimately improve the outcome for patients with pancreatic endocrine tumors.     

Therapeutic Targeting the Allosteric Cysteinome of RAS and Kinase Families

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRAS^G12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.     

SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth.However,for a significant portion of human cancer,how Hippo signaling is perturbed remains unknown.To answer this question,we performed a genome-wide screening for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome.In our screen,Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth.Interestingly,a mammalian homolog of Prosap,SHANK2,is the most frequently amplified gene on 11 q13,a major tumor amplicon in human cancer.Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification.More importantly,across the human cancer genome,SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon.Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 activator,and as a potential oncogene,SHANK2 promotes cellular transformation and tumor formation in vivo.In cancer cell lines with deregulated Hippo pathway,depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation.Taken together,these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator,commonly amplified in human cancer and potently promotes cancer.Our study for the first time illustrated oncogenic function of SHANK2,one of the most frequently amplified gene in human cancer.Furthermore,given that in normal adult tissues,SHANK2 s expression is largely restricted to the nervous system,SHANK2 may represent an interesting target for anticancer therapy.