A Landscape Of Deubiquinating Enzymes (Dub) In Lung Cancer Using Activity Based Protein Profiling (Abpp)

Dysregulation of ubiquitin signaling has been linked to many diseases, including cancer. Numerous DUBs are implicated in key pro-cancer pathways, including regulation of HIF1α, control of RAS signaling, signaling through both canonical and non-canonical NFκB pathways, and regulation of p53 activity. Overexpression of USP14, USP17 and USP28 is linked to promoting non-small cell lung cancer (NSCLC). OTUB1 has shown to trigger lung cancer progress by inhibiting RAS monoubiquitination in KRAS mutant NSCLC. Notably, BAP1 (tumor suppressor) expression decreases in lung cancer tissues although its mRNA levels did not reflect it. Based on these findings, we hypothesized that profiling DUB proteins and their activity in lung cancer cells and tissues could uncover new therapeutic targets. Since proteogenomics studies have revealed that mRNA expression is not a reliable predictor of the protein expression, we have used ABPP assays to profile DUB. ABPP, which uses chemical probes directed against the active sites of enzymes to interrogate the functional state of enzymes in biological samples, can serve as an important tool to nominate DUB targets. HA-tagged active site-directed ubiquitin probe, HA-Ub-VME was used to evaluate the DUBs profile of KRAS and EGFR mutant cell lines. Cell lysate was labeled in presence or absence of DUB inhibitors (DUBi) and the modified enzymes were visualized by anti-HA immunoblotting. Labeled proteome was enriched and analyzed using mass spectrometry and data was analyzed using APOSTL. In addition to DUB profiling, we screened lung cancer cell lines to DUBi, including small cell lung cancer, KRAS mutant, EGFR mutant, and models of EGFR TKI resistance. Immunoblotting in various KRAS and EGFR mutant cell lines displayed diverse profiles of DUBs. Enrichment of DUBs in A549 cells showed 23 DUBs and competition with bAP15, a well-known DUBi for USP14 resulted in reduced probe binding to USP14. We optimized the approach to capture maximum DUBs by examining parameters such as protein/probe ratio, probe incubation time and probing different lung cancer cells that may harvest a larger or complementary repertoire of DUBs. A preliminary screen of DUBi across 20 SCLC and NSCLC cell lines lead to a panel of DUBi (IC50=100nM-5µM), with broad or selective activity in particular cell lines. These results are being used for competitive DUB profiling using ABPP to comprehend target engagement and resulting changes in cell viability induced by DUBi. These results enable future studies to profile the DUB landscape in lung cancer cell lines, PDX tumor cohorts, and tumor tissues from patients. DUB ABPP can identify important DUBs in lung cancer cells to both identify new drug targets, as well as discern mechanism of action of drugs identified from compound screening campaigns. Citation Format: Shikha Mahajan, Bin Fang, Fumi Kinose, Eric B. Haura. A landscape of deubiquinating enzymes (DUB) in lung cancer using activity based protein profiling (ABPP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5222. doi:10.1158/1538-7445.AM2017-5222