Abstract PR05: The kinome of chemotherapy-resistant TNBC and identification of targetable kinases

Abstract

Abstract Triple-negative breast cancer (TNBC) accounts for around 15% of all breast cancer cases, with over 35,000 newly diagnosed women per year in the US. TNBC patients are at highest risk for recurrence, and neoadjuvant standard chemotherapy gives pathologic complete response in about 30% of these patients. Currently, no targeted therapy has been conclusively established to improve the outcome of TNBC patients, though successful phase II studies have been completed (1, 2). Therefore, identification of mechanisms that would be targetable is of great importance to improve the management of TNBC significantly. The aim of this study is to identify a unique set of hyperactivated kinases in chemotherapy-resistant TNBC cell lines that can potentially be targeted to achieve therapeutic response using a newly developed high-throughput kinase activity-mapping (HT-KAM) assay. The HT-KAM assay is our new screening technology to assess the catalytic activity of many kinases in parallel, which relies on collections of peptide probes that are used as combinatorial sensors to measure the phosphor-catalytic activity of kinases in large-scale high-throughput ATP-consumption assays (3). The HT-KAM system provides access to a new, untapped, and large resource of biologically meaningful measurements, both as a means to map the entire cancer kinome, and as a means to convert global phospho-signatures into functional patterns of kinase activity signatures. Kinome maps represent how kinase-signaling networks are rewired by drugs/targeted therapies in the context of different cellular backgrounds and exogenously mutated proteins/pathways, and provide insight into potentially targetable kinases. We previously established PhosphoAtlas, a heavily curated, comprehensive catalog database of 1,733 functionally interconnected proteins comprising the human phospho-reactome, including 4,748 unique edges that connect kinases to a target (776 kinase genes, 1,276 substrate protein genes, 2,492 heptameric peptide sequences [HPS]) (4). Here, we made a selection of 640 peptide sensors, capturing the functionality of >110 kinases over >900 kinase-substrate nodes directly relevant to tumor biology that represent the majority of the curated interconnected proteins, from our PhosphoAtlas (4). Earlier, the HT-KAM assay on 225 peptides was applied to explore mechanisms driving the unresponsiveness of colorectal and melanoma cancers to anti-BRAFV600E therapy in cell culture and patient-derived xenografts (PDX) (3). This successfully produced the predictive oncogenic kinome of melanoma tissues from patients suffering from fatal metastatic disease and, more importantly, identified new kinases/nodes that could be targeted to overcome drug resistance (3). In this study, we characterized the phospho-catalytic signatures of 10 TNBC cell lines (BT-549, HCC1143, HCC1395, HCC1937, HCC38, HCC70, HS578T, MDA-MB-231, MDA-MB-436, MDA-MB-468) across 640 peptides, either untreated or treated for 5 weeks with a chemotherapeutic drug (carboplatin, doxorubicin, 5-FU) at IC50 concentrations. For validation purposes of the anticipated phospho-signatures, we also assessed the catalytic activity of selected purified recombinant kinases. Based on preliminary data, we anticipate showing a comprehensive map of the oncogenic kinome of TNBC cell lines, enabling us to distinguish TNBC cell lines based on their origin and treatment.