Abstract 1696: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer
Authors: Wu Bogang , Sun Sheng , Thimmiah Nayana , Nagayama Aiko , Coates James , Thant Win , Li David , Doench John , Bardia Aditya , Ellisen Leif 
Published: June 2023

Abstract
Antibody drug conjugates (ADC) are emerging as paradigm-changing precision therapeutics. Sacituzumab Govitecan (SG), which combines a Topoisomerase I (TOP1) inhibitor payload (SN38) with hRS7, an antibody targeting the tumor-selective antigen TROP2, is proving to be a successful cancer therapeutic for some highly refractory cancers including triple negative breast cancer (TNBC). However, mechanisms and biomarkers of sensitivity and resistance to SG remain poorly understood. Prior clinical trials have identified positive correlations between TROP2 expression and better outcome for metastatic TNBC patients receiving SG. Additionally, patient-derived specimen analyses by our group identified mutations in both TROP2 and TOP1 in post-progression metastatic lesions, suggesting multifaceted resistance mechanisms.
To systematically interrogate ADC sensitivity mechanisms, we utilized genome-wide CRISPR-Cas9 knockout screening to identify novel regulators mediating SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, and endosome trafficking/sorting. The top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Conversely, the very top SG resistance-inducing hit in the genome was PARG, encoding the glycohydrolase that opposes PARP enzymatic activity. Furthermore, the synergistic lethality between TOP1 and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. In addition, to identify SG/ADC specific genes and pathways, we performed a secondary round of CRISPR screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG.
In conclusion, through a systematic approach using CRISPR screening, we identified several novel resistance and sensitizing pathways that are implicated in ADC delivery and target protein turnover. We are currently prioritizing the druggable genes and pathways in preparation for proof-of-concept studies combining ADCs with select targeted therapeutics to achieve greater tumor killing efficacy.
Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James Coates, Win Thant, David Li, John Doench, Aditya Bardia, Leif Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [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 1696.