In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy

Abstract

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

Random clonal expansion as a limiting factor in transplantable in vivo CRISPR/Cas9 screens

“…The copyright holder for this this version posted November 29, 2021. ; https://doi.org/10.1101/2021.11.28.469740 doi: bioRxiv preprint ~65,000 gRNA was used with only three gRNA on average against each gene(S. Chen et al 2015;Song et al 2017;Rushworth et al 2020;Yau et al 2017;Dai et al 2021;Kodama et al 2017). Interestingly, the two of these studies that reported the highest detection of gRNA in tumours inoculated 3 × 10 7 cells into mice (Yau et al 2017;Dai et al 2021), a 3-fold increase on our inoculum. This extends to a ~6-fold increase in cells/gRNA (~450 vs ~83 in our study) when the half-library is factored in and may have contributed to greater averaging across clones in tumours and a reduction in count variance compared to our study.…”

“…Transplantable models, conversely, are more amenable to larger gRNA libraries. Cancer cell lines transduced at scale with whole-genome gRNA libraries can be injected into mice at large cell numbers to ensure high representation of each gRNA at inoculation (S. Chen et al 2015;Song et al 2017;Yau et al 2017;Rushworth et al 2020;Kodama et al 2017;Dai et al 2021). However, the majority of whole-genome transplantable in vivo CRISPR screens report a dramatic loss of clonal diversity during tumour growth, due to the selection and bottleneck of cellular evolution imposed by the transition from in vitro to in vivo preprint (which was not certified by peer review) is the author/funder.…”

CRISPR based therapeutics: a new paradigm in cancer precision medicine

“…Single cell CRISPR screening can also be combined with transcriptome (RNA-seq) or open chromatin sequencing (ATAC-seq) to decipher perturbation associated changes in the RNA pool and chromatin landscape respectively [ 47 – 50 ]. CRISPR screens have been utilized by many in the recent years for identification of genes that contribute to several hallmarks of cancer progression including primary tumor growth, drug resistance, epithelial-to-mesenchymal transition, cancer stemness, metabolic adaptations and metastasis [ 51 – 60 ]. Genome-wide CRISPR screens have also been employed to screen for regulators of genetic dependencies or synthetic lethal gene interactions/drug targets in different cancers [ 61 – 66 ].…”

Common computational tools for analyzing CRISPR screens

“…CRISPR editing in in vivo model in conducted by creating the mutant cell population of interest in a dish and then implanting those into a mouse, often subcutaneously or intravenously (Figure 1D). In the last few years, CRISPR technology has been used in living model organisms for studying various cancers and cancer specific processes [29][30][31][32][33][34][35], though the complexity of the approach limits these screens to targeted gene panels.…”