Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity

Abstract

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.

Maintaining protein stability of ∆Np63 via USP 28 is required by squamous cancer cells

“…To do so, we made use of a genetically modified mouse model (GEMM) harbouring conditional alleles for Tp53 ( Tp53 flox / flox ) and a Cre‐activatable mutant KRas ( lsl‐KRasG12D ). We intratracheally administered AAV encoding Cre recombinase, packaged with the capsid 2/8, which facilitates a tropism towards tracheal and alveolar cells (Fig EV3H; Winters et al , ). Twelve weeks post‐infection, mice were sacrificed and showed clear signs of tumour formation (Fig EV3I).…”

Capturing the primordial Kras mutation initiating urethane carcinogenesis

“…This bias of urethane for (C)A>T/G substitutions similarly argues against mutations arising at an appreciable level in codons 12 (G 34 GT) or 13 (G 37 GC) in exon 1, as neither fit the CAN pattern in either strand orientation. Related to this, despite the fact that oncogenic mutations at G 12 , and to a lesser extent G 13 , occur frequently in human cancers 19 and when introduced into the lungs of mice are tumorigenic to varying degrees 20 , they are rarely recovered from urethane-induced tumors 3 . We therefore sequenced the transcribed strand of exon 1 of Kras by MDS from genomic DNA isolated from the lungs of mice 1, 2, 3, and 4 weeks after exposure to urethane or PBS.…”

RAS Mutations Are Not Created Equal

“…However, whether other effectors that contribute to KRAS-dependent cancer growth (e.g., PI3K) have disrupted binding was not determined. A recent mouse model-based analysis addressed whether oncogenic potency may influence tissue-specific KRAS mutation profiles, but only focused on mutations at G12 and G13 (8). To specifically address a basis for A146 mutations in different cancer types, Poulin and colleagues generated new mouse strains harboring the A146T mutation in the endogenous Kras locus, identical otherwise to previously wellcharacterized KRAS G12D -driven mouse models of lung and pancreatic cancers (9,10).…”