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A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

ABSTRACT: The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin β5) as the essential integrin α/β pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the β-propeller domain of ITGAV for integrin αVβ5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the β-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVβ5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

SUBMITTER: Mattson NM

PROVIDER: S-EPMC10948361 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

Mattson Nicole M NM Chan Anthony K N AKN Miyashita Kazuya K Mukhaleva Elizaveta E Chang Wen-Han WH Yang Lu L Ma Ning N Wang Yingyu Y Pokharel Sheela Pangeni SP Li Mingli M Liu Qiao Q Xu Xiaobao X Chen Renee R Singh Priyanka P Zhang Leisi L Elsayed Zeinab Z Chen Bryan B Keen Denise D Pirrotte Patrick P Rosen Steven T ST Chen Jianjun J LaBarge Mark A MA Shively John E JE Vaidehi Nagarajan N Rockne Russell C RC Feng Mingye M Chen Chun-Wei CW

Nature structural & molecular biology 20240205 3

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin β5) as the essential integrin α/β pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the inte ...[more]

PMID: 38316881

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Project description:BackgroundRNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool.ResultsWe synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach.ConclusionsIn summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.

Dataset Information

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

Publications

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

Similar Datasets

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