In:
Science, American Association for the Advancement of Science (AAAS), Vol. 376, No. 6592 ( 2022-04-29)
Abstract:
Cancer develops from cells that become malignant because of mutations in multiple genes—often accumulated over long time periods—that produce a phenotypic diversity across patient tumors. Specific genetic alterations in particular cancer types have been linked to prognosis, to response or resistance to therapies (especially targeted therapeutics), and to a tumor’s propensity to acquire further mutations, among other phenotypes. However, genotype-phenotype connections are challenging to infer in patients, as any two tumors differ genetically in too many ways to isolate the effect of one or several mutations. The ability to systematically connect cancer-associated mutations or combinations thereof with their phenotypic consequences would advance our understanding of the mechanisms of cancer pathogenesis and genetically linked disease features. RATIONALE We reasoned that genome editing and the fitness advantage of cancer-associated mutations could be leveraged to generate human cellular models of tumor development. Such genome-edited models would replicate the precise genetics, lineage relationships, and stepwise progression of cancer and allow us to establish genotype-to-phenotype links in a controlled experimental design. While similar models have been realized for tumors originating from self-renewing stem cells as cells of origin, specifically in colorectal cancer, no comparable models exist for tumor types that arise from nonstem differentiated cells. We present an approach that starts from the nonstem cell of origin of melanoma, the healthy human melanocyte; this approach then generates a series of cells with precise genome editing of mutations in key cancer genes, thus expanding the horizon of possible cellular models of cancer development. RESULTS We generated a progressive series of genome-edited human models of melanoma. We started from healthy human melanocytes and introduced, in a stepwise fashion, mutations in up to five genes spanning six pathways commonly dysregulated in melanoma: CDKN2A (part of the RB pathway), BRAF (MAPK), TERT (telomerase), PTEN (PI3K/AKT), TP53 (p53), and APC (Wnt), for a total of nine genetically distinct cellular models. We characterized these models during growth in vitro and after intradermal injection through mouse xenografts, using physiological assessment, histopathology, and single-cell RNA sequencing (scRNA-Seq), leveraging computational methods and machine learning algorithms. Through these models, we connected melanocyte genotypes to phenotypes such as gene expression programs, replicative immortality, malignancy, rapid tumor growth, tumor pigmentation, metastasis, and histopathological features. In vitro, consecutive mutations produced an ordered progression through expression space. In vivo, mutations in malignant cells also affected the cell-type composition and expression states of tumor-infiltrating microenvironment cells. Our melanoma models shared genotype-associated expression programs with patient melanomas and partially recapitulated patient melanoma genotype-associated histopathological features. CONCLUSION The genotype-phenotype connections we identify highlight how the impact of mutations often depends on genetic context. Such genetic epistasis makes understanding the phenotypic consequences of the mutational landscape of human cancers a combinatorial problem whose study requires modeling strategies that can scale to multiple mutations, such as the one presented in this study. Genome-edited human models of cancer enable the identification of causal relationships between defined sets of genetic alterations and disease-relevant phenotypes, furthering understanding of how cancer mutations help give rise to the diverse and varied phenotypes of human malignancy. Genome-engineered human cell models connect melanoma genotypes to phenotypes. Sequential, precise gene editing of human melanocytes produced a series of melanoma models. The editing approach we developed—without selection markers or single-cell cloning—is applicable to other cell types, expanding the toolkit of available cancer models. Through in-depth phenotypic characterization of in vitro cells and in vivo tumors from xenografts in immunodeficient mice, multimutant melanoma genotypes were causally linked to specific phenotypes.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abi8175
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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