Abstract 6234: Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression
Authors: Mattson Nicole , Miyashita Kazuya , Chan Anthony , Yang Lu , Pokharel Sheela Pangeni , Liu Wei , li Ming , Liu Qiao , Xu Xiaobao , Singh Priyanka , Zhang Leisi , Feng Mingye , Chen Chun-Wei 
Published: April 2023

Abstract
Integrins have long been sought as therapeutic targets in cancer, but there are no FDA approved drugs to target this gene family. Since integrins require the dimerization between an alpha and beta subunit to function, we developed a CRISPR library targeting all 26 alpha and beta subunits to identify which pair is the most important. We found a strong correlation between ITGAV and ITGB5 (Integrin Beta 5), which form a known heterodimer, in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. As the more essential partner, we designed a CRISPR tiling scan targeting the entire coding region of ITGAV and determine the essential nature of each amino acid. We found a sub-domain within the beta propeller to be important for ITGAV function, the CRISPR identified pocket (CIP). Genetic targeting of this pocket disrupted the ability to detect the heterodimer signal of ITGAV-ITGB5 by a specific antibody. Furthermore, we confirmed that essential amino acids within the CIP responsible for the heterodimer stability by the NanoBRET energy transfer system. Knowing the role of the CIP in heterodimer stability we searched for a small molecule to bind in the CIP to phenocopy our genetic findings. A combined in-silico and in-vitro high throughput drug screen started tested compounds for cell killing and disruption of the ITGAV-ITGB5 heterodimer. “Compound 2” (cpd_2) showed both strong cell killing and dissociation of the heterodimer. Disruption was measured by detection by flow cytometry and through the NanoBRET system. Cpd_2’s binding was confirmed with thermal shift studies with purified ITGAV beta propeller, validating that this compound is behaving and binding as we expect at the CIP. Next, we sought to understand how the ITGAV-ITGB5 heterodimer controls cancer cell survival. From the Dependency Map Public database (DepMap) we found that Rac1 dependency is significantly correlated only with ITGAV and ITGB5. We hypothesized that this meant that ITGAV-ITGB5 regulated Rac1 activity. Loss of Rac1 phenocopies knock out of ITGAV or ITGB5 and loss of either Rac1 or ITGAV led to dysregulation of the actin cytoskeleton, a known downstream feature of Rac1. A constitutively Rac1 (CA_Rac1) rescues the effects of targeting of ITGAV or treatment with cpd_2. Altogether this data suggests that ITGAV-ITGB5 signals to Rac1 and that attenuation of their function can be reversed by an active Rac1 In summary, our study highlights a novel strategy of targeting the integrin heterodimer ITGAV-ITGB5 with a small molecule binding to a novel subdomain, CIP, of ITGAV. The CIP is responsible for the heterodimer stability of ITGAV and ITGB5. Disruption of this heterodimer led to the dysregulation of Rac1 and destabilization of the actin cytoskeleton. This mechanism highlights a novel strategy for targeting integrins as a therapeutic target in cancer.
Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Liu, Ming li, Qiao Liu, Xiaobao Xu, Priyanka Singh, Leisi Zhang, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [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 6234.