Search

Purpose of the study Oesophageal cancer is the seventh most common cause of cancer related death worldwide. Disease relapse is frequent and treatment options are limited. With the exception of HER-2 and EGFR amplification, there are currently no validated biomarkers to predict response to biological therapies in this disease. The purpose of this study was to identify new biomarker defined therapeutic approaches for patients with oesophageal cancer. Experimental approach We have undertaken a functional screening approach focused on the druggable kinome to generate a first map of the genetic dependencies found in oesophageal cancer. A panel of 19 oesophageal and 130 non-oesophageal cancer cell lines were screened with siRNA targeting 720 kinases to generate functional profiles. By integrating this dataset with exome sequencing and copy number data, we identified key genetic dependencies (KGDs) associated with oesophageal histology (adenocarcinoma or squamous cell carcinoma) or key driver genotypes including amplifications in c-MYC, CCND1, EGFR and ERBB2, as well as loss of functional mutations in SMAD4 and ARID1A. Results Interrogation of the oesophageal functional genomics dataset established oncogene addiction effects including c-MYC, which is amplified in 30% of oesophageal carcinomas. We confirmed that a novel isoform of Bruton agammaglobulinaemia tyrosine kinase gene (BTK-C) was expressed in oesophageal cancer models and that silencing of BTK resulted in selective lethality in c-MYC amplified oesophageal cell lines. To further assess the impact of BTK inhibition in oesophageal cancer, we treated a panel of oesophageal cell lines with increasing doses of ibrutinib, a small molecule tyrosine kinase inhibitor of BTK. Analysis of ibrutinib SF50 revealed that there was preferential sensitivity to ibrutinib in c-MYC amplified oesophageal cell lines. Furthermore, we observed a significant association between mutation of the chromatin remodelling factor gene SMARCA4 and dependency upon the bromodomain protein BRD4. We also identified SMAD4 kinase genetic dependencies including AKT1 and a number of its substrates (FGR, MAP3K3, CHEK1 and WEE1). We noted a densely connected group of kinases that regulate the mitotic cell cycle in the SMAD4 dependency network, and found that SMAD4 mutant cell lines exhibited an increased sensitivity to drugs, including paclitaxel, that target the mitotic checkpoint. Conclusions Our results support the potential of BTK small molecule inhibition as a novel therapeutic strategy in c-MYC amplified oesophageal carcinoma. Loss of function SMAD4 mutations represents a candidate biomarker of response to drugs that target the mitotic checkpoint in oesophageal cancer. Citation Format: Irene Chong, lauren Aronson, David Cunningham, James Campbell, Colm Ryan, Michael Davidson, Ian Chau, Alan Ashworth, Christopher J. Lord. Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 69.

Background: Breast cancer is the most frequently diagnosed malignancy (besides non-melanoma skin cancer) and is the second leading cause for cancer-related death among US women. Most breast cancers overexpress the estrogen receptor (ER) or, to a lesser extent, the HER2 receptor and thus are responsive to hormonal therapies. However nearly a quarter of patients do not express ER, HER2, or progesterone receptor (“triple-negative breast cancer”) and are clinically managed as a single disease with nonspecific chemotherapies. Novel targeted therapies are urgently needed to treat these aggressive cancers. Recent large-scale studies integrating multiple dimensions of genomic data have revealed that alterations to the MAP kinase pathways are critical for the development and progression of triple-negative cancers (TNBCs). In particular, mutations in MAP3K genes have been associated with receptor-negative tumors. In this study, we hypothesized that TNBCs depend on unique MAP3K proteins distinct from those required by non-TNBCs to transduce growth signals. Methods: We performed an unbiased screen of the MAP3K family to identify genes critical for growth and tumorigenicity in six TNBC and six ER-positive cell lines. Pools of siRNA were deposited into optical microplates and cell lines were liposomally reverse-transfected. Depending on the growth rate, cells were stained with DAPI at 4 or 6 days post-transfection and nuclei were detected by high-throughput microscopy. To test for inhibition of anchorage-independent growth, cells were transfected with siRNA and the next day seeded in soft agar in 6-well dishes. Colonies were counted by machine at 1-3 weeks and compared to control siRNA-treated cells. For death assays, siRNA-treated cells were grown for 2 days and collected for western blot detection of annexin V and cleaved caspase 3. Results: We determined that MAP3K3 (MEKK3) and TAO2 (MAP3K17) are essential for anchorage-dependent growth of TNBC cells, but are not required for the proliferation of ER-positive cell lines. Among the TNBC cells, there were variable responses to knockdown of these kinases. While the growth of MDA-MB-231, HCC70, and HCC1937 cells were greatly inhibited after MAP3K3 knockdown, the growth of HCC1937 cells was unaffected by MAP3K3 knockdown but was instead suppressed by TAO2 inhibition. Knockdown of MAP3K3 and TAO2 also resulted in a dramatic reduction of anchorage-independent colony formation in these TNBC cells but not in ER-positive lines. We demonstrated that this reduction in growth is due to suppression of proliferation, and not by induction of apoptosis via annexin V apoptotic assay and detection of cleaved caspases. Conclusion: These data suggest that TNBCs evolve a dependence on specific MAP kinase pathways, and that targeting MAP3K3 and TAO2 may be an effective strategy to treat the subset of TNBCs that rely on these molecules to potentiate growth. Citation Format: Graham M. Poage, Abhijit Mazumdar, Ivan P. Uray, Powel H. Brown. Identification of mitogen-activated protein kinases which are required for the growth of triple-negative breast cancers. [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 557. doi:10.1158/1538-7445.AM2013-557

Gene amplification in the 17q chromosomal region is observed frequently in breast cancers. An integrative bioinformatics analysis nominated MAP3K3 gene, located in 17q23, as a potential therapeutic target in breast cancer. This gene encodes the mitogen-activated protein kinase kinase kinase 3 (MEKK3), but has not yet been associated with cancer-causal genetic aberrations. We found that MAP3K3 was amplified in approximately 8–20% of breast carcinomas, and that its over-expression was an independent prognostic marker for poor outcome with respect to relapse-free and overall survival, especially among the estrogen receptor-positive breast cancer patients. shRNA-mediated knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation of MAP3K3-amplified breast cancer cell lines MCF7 and MDA-MB361, and promoted breast cancer cell apoptosis induced by TNFα, TRAIL, or a doxorubicin. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized mammary epithelial cells (MCF-10A). Together, these results suggest that MAP3K3 is a potential biomarker indicating poor prognosis, contributes to resistance to therapy, and is an oncogene in breast carcinogenesis. Therefore, therapeutic targeting of MAP3K3 may be attractive in breast cancer patients with MAP3K3-amplified breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-03.