Cancer genomics studies have nominated thousands of putative cancer driver genes
1
; a major challenge is to develop high-throughput and accurate models to define their functions. Here we devised a scalable cancer spheroid model and performed genome-wide CRISPR screens in 2D-monolayers and 3D lung cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulate those of
in vivo
tumors, and genes with differential sensitivities between 2D and 3D are strongly enriched for significant mutations in lung cancers. These analyses also revealed novel drivers essential for cancer growth in 3D and
in vivo
, but not in 2D. Notably, we discovered that CPD (Carboxypeptidase D) is responsible for removal of a c-terminal RKRR motif
2
of IGF1R α-chain, critical for receptor activity. CPD expression correlates with patient outcomes in lung cancer, and loss of CPD reduced tumor growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy to perform CRISPR screens in spheroids to uncover cancer vulnerabilities.
Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer.However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53 -/human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation.ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin.Convergently, high-throughput compound screening indicated selective vulnerability of ARID1Adeficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wntinhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.Research.
Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood.Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR/Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding-protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse Kras G12D -driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and key component of p53-mediated tumor suppression.We thank Laurakay Bruhn, Steven Altschuler, Ben Borgo, Peter Sheffield and Carsten Carstens of Agilent Inc. for oligonucleotide synthesis and helpful discussions. We thank Lin He for the Eμ-Myc lymphoma cells, Andreas Strasser and Ana Janic for the Zmat3 null MEFs, and Julien Sage and Aaron Gitler for critical reading of the manuscript.
Loss of the von Hippel–Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.
Conflict of Interest: C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He also previously served on the Board of Directors of Merck and Charles River Laboratories. J.D.R. is a co-founder of Raze Therapeutics, advisor to the Barer Institute and L.E.A.F. Pharmaceuticals, and inventor of Princeton University patents regarding 1C metabolism inhibitors. J.M. is scientific advisor and owns stock of Scholar Rock.
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