Abstract 3839: Discovery of GSTZ1 as a novel target for drug refractory non-small cell lung cancer by using fragment-based chemical proteomics
Authors: Liao Yi , Chan Sean Chin , Welsh Eric A. , Fang Bin , Sun Luxin , Schönbrunn Ernst , Koomen John M. , Duckett Derek R. , Haura Eric B. , Monastyrskyi Andrii , Rix Uwe 
Published: April 2023

Abstract
A subset of non-small cell lung cancer (NSCLC) patients respond poorly to clinical drugs targeting defined oncogenic driver mutations. To improve the efficacy of these drugs in drug refractory NSCLC, the identification of supporting therapeutic targets and discovery of the binding molecules are urgently needed. Photoreactive fragment-like probes can discover target proteins that constitute novel cellular vulnerabilities and provide the chemical basis for drug discovery. However, the rational design of the fragment probe library, target prioritization, and sample throughput are still challenging. We applied a structurally diverse panel of BioCore fragments fully functionalized with orthogonal diazirine and alkyne moieties. These probes were used for protein crosslinking in live cells and subsequent target identification through label-free quantitative LC-MS/MS analysis. High-confidence targets were queried against a pharmacogenomic database (DepMap) and prioritized through multiple cross-comparison with other probes. The top-ranked probe-binding target was validated using a competitive affinity assay, an enzymatic activity assay, and RNA interference. Proteome-wide tyrosine reactivity was profiled using sulfur-triazole exchange chemistry (SuTEx). The tyrosine phosphorylation of proteins was investigated using a pY-100 antibody and western blotting. Using sotorasib-refractory KRAS G12C H1792 lung cancer cells, we identified 932 probe-enriched proteins from panel-wide cross-comparisons suggesting the high potential of exploring the ligandable proteome. We also performed intensive cross-comparison analysis and identified 31 unique high-confidence targets, with glutathione S-transferase zeta 1 (GSTZ1) identified as a unique target of probe 17. We found that high expression of GSTZ1 was significantly associated with poorer NSCLC patient survival. Probe 17 was validated to physically bind to GSTZ1 and inhibit the enzymatic activity of GSTZ1. In addition, GSTZ1 gene knockdown sensitized drug-refractory NSCLC cells with KRAS G12C, FGFR1 amplification, and DDR2 mutation to clinical targeted drugs and induced more cell apoptosis in combination with these targeted drugs. SuTEx proteomics suggests modulation of drug resistance pathways leading to the identification of altered KRAS and FGFR1 tyrosine phosphorylation by GSTZ1, which provides a functional insight into the mechanism of drug sensitization. We developed a chemical biology workflow for the simultaneous discovery of high-confidence targets and their binding probe molecules, such as probe 17 and GSTZ1. GSTZ1 was found to cooperate with oncogenic alterations in supporting refractory NSCLC cell survival signaling, which may form the biological basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs.
Citation Format: Yi Liao, Sean Chin Chan, Eric A. Welsh, Bin Fang, Luxin Sun, Ernst Schönbrunn, John M. Koomen, Derek R. Duckett, Eric B. Haura, Andrii Monastyrskyi, Uwe Rix. Discovery of GSTZ1 as a novel target for drug refractory non-small cell lung cancer by using fragment-based chemical proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3839.