<p>Fusion genes and their sequences from TCGA (S1-2); Primers for fusion PCR (S3); BRAF mutants and mutagenesis primers (S4); (q)PCR primers for expression detection (S5); Fusion gene construction efficiency and success rate (S6)</p>
<p>Fusion expression detection by PCR (S1); MET fusion expression and signaling activation by immunoblot and N-terminal partner control assay (S2); NTRK2 fusion expression and signaling activation by immunoblot and N-terminal partner control assay (S3); Expression of BRAF fusion N-terminal partner controls and BRAF structure-function studies by immunoblot and qPCR (S4)</p>
<p>Fusion expression detection by PCR (S1); MET fusion expression and signaling activation by immunoblot and N-terminal partner control assay (S2); NTRK2 fusion expression and signaling activation by immunoblot and N-terminal partner control assay (S3); Expression of BRAF fusion N-terminal partner controls and BRAF structure-function studies by immunoblot and qPCR (S4)</p>
Bcl-2 family members are important regulators of apoptosis, and their altered expression is often involved in oncogenesis. Of particular importance are the levels of Bcl-2 family members in forming lymphomas. We studied two groups of murine thymic T cell lymphomas derived from either Bcl-2 or Bax overexpression in order to predict their sensitivity and resistance to treatments. While the growth rate and histological characteristics were similar for both lymphoma groups, Bax-derived lymphomas failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2-derived lymphomas often halted proliferation following radiation delivery and rarely had p53 mutations. Bax-derived lymphomas were uniformly sensitive to treatment with 2-deoxy-D-glucose (2DG) while all Bcl-2-derived lymphomas were resistant. This led us to hypothesize that the Bcl-2 family is involved in 2DG-induced cell death. Focusing on the mechanism of 2DG toxicity in Bax-derived lymphomas, our studies demonstrate the following: cell death involved the activation of proapoptotic Bax, was effectively blocked by anti-apoptotic Bcl-2, and was mediated, at least in part, by the BH3-only family member Bim. Based on these results, we explored whether a BH3 mimetic (ABT-737) could sensitize lymphomas to 2DG killing. Indeed, a combination of ABT-737 with 2DG enhanced killing in Bax-derived lymphomas and resensitized Bcl-2overexpressing lymphomas to 2DG. Since both 2DG and BH3 mimetics are currently in clinical trials, understanding their killing mechanisms and optimal combinations are of potential clinical significance. The work in this dissertation demonstrates a novel role of Bcl-2 family member proteins in regulating 2DG toxicity and may predict response to
Abstract Pancreatic cancer is often diagnosed at an advanced stage and has a poor prognosis. The overall survival rate is less than 4% at 5 years with most patients dying within one year. Therefore, more effective approaches to treat advanced pancreatic cancer and chemopreventive strategies are in urgent need. Recent reports of early dissemination in pancreatic cancer stress the need for early intervention. Activating mutations of the KRAS oncogene are possibly the single most common genetic abnormality in pancreatic cancer, present in ∼90%-95% of cases. In addition to its role in cancer progression, mutations of the KRAS gene are one of the earliest genetic abnormalities observed in pancreatic cancer, supporting its role as an initiating event for pancreatic cancer formation. Therefore, mutant KRAS gene or its gene product represents an obvious target for the prevention of pancreatic cancer. Unfortunately, direct inhibition of KRAS or its downstream effectors has been largely ineffective in treating pancreatic cancer in clinical trials. The ideas are to find an innovative approach to target KRAS. We recently proposed a new cancer chemoprevention approach SITEP (short-term intermittent therapy to eliminate premalignancy) to target the “drivers” of tumorigenesis to overcome the challenges in cancer chemoprevention. We demonstrated that a two-drug combination of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and a small molecule mimic of the mitochondrial protein Smac /DIABLO (Smac), through a synthetic lethal interaction, specifically targets mutant KRAS expressing premalignant cells for apoptosis without harming normal cells. We further showed that short-term, intermittent in vivo treatment with TRAIL and Smac mimic induced apoptosis in tumor cells and reduced tumor burden in a murine model of Kras-induced lung cancer. In this study, we examined whether targeting KRAS mutant cells for apoptosis in pancreatic precursor lesions (e.g., PanINs) by TRAIL and Smac mimic would be an effective approach for pancreatic cancer chemoprevention. We showed that constitutive expression of mutant KRAS led to the activation of downstream signaling events in the immortalized human pancreatic normal epithelial (HPNE) cells, including phosphorylation of ERK and AKT. TRAIL and Smac mimic treatment showed that control cells are resistant to either TRAIL, or Smac mimic, or TRAIL and Smac mimic combination, and expression of KRAS mutant sensitized cells to the combination of TRAIL and Smac mimic. Short term treatment of Pdx1-Kras mice showed that TRAIL and Smac mimic induced significant apoptosis in abnormal pancreatic ductal cells and reduced number of PanINs. We conclude that targeting oncogenic KRAS signaling pathways in precursor lesions by TRAIL and Smac mimic may prove useful in developing chemoprevention against pancreatic cancer. Citation Format: Oksana Zagorodna, Shaoyi Huang, Huamin Wang, Xiangwei Wu. Chemoprevention of pancreatic cancer by targeting Kras mutations for apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1076. doi:10.1158/1538-7445.AM2013-1076
