High-Throughput Identification, Modeling, and Analysis of Cancer Driver Genes In Vivo

Tang, Yuning J.; Shuldiner, Emily G.; Karmakar, Saswati; Winslow, Monte M.

doi:10.1101/cshperspect.a041382

Cited by 4 publications

(2 citation statements)

References 179 publications

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“…Alterations in tumor suppressor genes are ubiquitous in cancer and impact cellular processes that are critical to both cancer development and aging 24–27 . Integration of multiplexed CRISPR-based somatic genome engineering and tumor barcoding enables precise quantification of the growth of many genotypes of tumors in parallel 18,19,28 . To quantify the impact of aging on tumor suppressor gene function, we generated a pool of barcoded lentiviral vectors encoding Cre and targeting 25 known or putative tumor suppressor genes as well as “sg Inert ” non-targeting control vectors and a sgRNA targeting an essential gene ( Pcna ) (Lenti-sg RNA Aging /Cre , Fig.…”

Section: Resultsmentioning

confidence: 99%

Aging represses lung tumorigenesis and alters tumor suppression

Shuldiner,

Karmakar,

Tsai

et al. 2024

Preprint

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Most cancers are diagnosed in persons over the age of sixty, but little is known about how age impacts tumorigenesis. While aging is accompanied by mutation accumulation — widely understood to contribute to cancer risk — it is also associated with numerous other cellular and molecular changes likely to impact tumorigenesis. Moreover, cancer incidence decreases in the oldest part of the population, suggesting that very old age may reduce carcinogenesis. Here we show that aging represses tumor initiation and growth in genetically engineered mouse models of human lung cancer. Moreover, aging dampens the impact of inactivating many, but not all, tumor suppressor genes with the impact of inactivating PTEN, a negative regulator of the PI3K/AKT pathway, weakened to a disproportionate extent. Single-cell transcriptomic analysis revealed that neoplastic cells from tumors in old mice retain many age-related transcriptomic changes, showing that age has an enduring impact that persists through oncogenic transformation. Furthermore, the consequences of PTEN inactivation were strikingly age-dependent, with PTEN deficiency reducing signatures of aging in cancer cells and the tumor microenvironment. Our findings suggest that the relationship between age and lung cancer incidence may reflect an integration of the competing effects of driver mutation accumulation and tumor suppressive effects of aging.

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Section: Resultsmentioning

confidence: 99%

Aging represses lung tumorigenesis and alters tumor suppression

Shuldiner,

Karmakar,

Tsai

et al. 2024

Preprint

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“…However, the creation of new mouse alleles has remained a barrier to the study of more complex tumor genotypes, due to the expense and time required to generate a new allele and cross it with other alleles of interest. As a result, some common but complex genotypes have never been modeled 4 . Somatic genome editing with Cas9 has greatly increased the rate at which single genes can be studied, but the ability of Cas9 to model complex genotypes has been limited by the need for each guide to have its own promoter and tracrRNA, thereby requiring the laborious cloning of guides in sequence.…”

Section: Introductionmentioning

confidence: 99%

Efficient and multiplexed somatic genome editing with Cas12a mice

Hebert,

Xu,

Tang

et al. 2024

Preprint

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Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models have been restricted in their ability to create multiple targeted edits, which has limited investigations into complex genetic interactions that underlie development, homeostasis, and disease. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes. In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of oncogene-negative lung adenocarcinoma, small-cell lung cancer, and a canonical genotype of pancreatic ductal adenocarcinoma, all driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo.

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Combinatorialin vivogenome editing identifies widespread epistasis during lung tumorigenesis

Hebert,

Tang,

Andrejka

et al. 2024

Preprint

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Lung adenocarcinoma, the most common subtype of lung cancer, is genomically complex, with tumors containing tens to hundreds of non-synonymous mutations. However, little is understood about how genes interact with each other to enable tumorigenesis in vivo, largely due to a lack of methods for investigating genetic interactions in a high-throughput and multiplexed manner. Here, we employed a novel platform to generate tumors with all pairwise inactivation of ten tumor suppressor genes within an autochthonous mouse model of oncogenic KRAS-driven lung cancer. By quantifying the fitness of tumors with every single and double mutant genotype, we show that most tumor suppressor genetic interactions exhibited negative epistasis, with diminishing returns on tumor fitness. In contrast, Apc inactivation showed positive epistasis with the inactivation of several other genes, including dramatically synergistic effects on tumor fitness in combination with Lkb1 or Nf1 inactivation. This approach has the potential to expand the scope of genetic interactions that may be functionally characterized in vivo, which could lead to a better understanding of how complex tumor genotypes impact each step of carcinogenesis.

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High-Throughput Identification, Modeling, and Analysis of Cancer Driver Genes In Vivo

Cited by 4 publications

References 179 publications

Aging represses lung tumorigenesis and alters tumor suppression

Aging represses lung tumorigenesis and alters tumor suppression

Efficient and multiplexed somatic genome editing with Cas12a mice

Combinatorialin vivogenome editing identifies widespread epistasis during lung tumorigenesis

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