Multiplexed mosaic tumor models reveal natural phenotypic variations in drug response within and between populations [CRISPR screen]

Heterogeneity between patients poses a significant challenge in cancer drug development. Although success of a drug candidate in the clinic depends on efficacy across large numbers of diverse patients, only a few in vivo preclinical models are used for assaying a preclinical drug candidate due to the lack of scalability of traditional xenograft models. To address this limitation, we developed GENEVA, a scalable platform that enables the measurement of molecular and phenotypic responses to drug perturbations at single-cell resolution, both in 3D and in vivo. GENEVA models the genetic diversity of cancer by combining multiple patient-derived cell lines and cancer cell lines into pooled 3D cultures and xenograft models, allowing us to study drug responses across a wide range of genetic backgrounds within a single experiment. This platform integrates high-resolution transcriptomics with multiplexed single-cell profiling, providing a unique ability to uncover the interplay between cancer genotypes and drug responses. In this study, we apply GENEVA to investigate KRAS G12C inhibitors and demonstrate that mitochondrial activation is a key driver of cell death following KRAS inhibition. Additionally, we identify epithelial to mesenchymal transition (EMT) as a prominent resistance mechanism to KRAS G12C inhibition using in vivo GENEVA mice. These findings highlight the utility of GENEVA in identifying novel therapeutic targets and optimizing combination therapies, while underscoring its potential to bridge the gap between preclinical cancer models and patient outcomes by modeling the complexity of tumor heterogeneity in a scalable and robust assay. Overall design: Lung cancer cell lines (H358, H2122, H23, H441) transduced with dCas9-KRAB and pooled dual sgRNA libraries targeting approximately 100 genes were injected into the flanks of NSG mice to form mosaic xenografts. Two days post-implantation, mice were treated with Sotorasib ($100~mg/\text{kg}$) or vehicle. Dosing was performed by daily oral gavage for 13 days. Tumors were excised on day 14, and genomic DNA (gDNA) was extracted for subsequent guide RNA sequencing to quantify gene enrichment/depletion (Rho scores) and determine genetic dependencies.