An In Vivo Kras Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants

Abstract

KRAS is the most frequently mutated oncogene in cancer.Tumor sequencing has revealed a complex spectrum of KRAS mutations across different cancer types, yet there is little understanding how specific KRAS alterations impact tumor in initiation, progression, or therapy response. Using highfidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRAS G12C ), pancreas (KRAS G12R ) and colon (KRAS G13D ) cancers. Induction of each mutation in the developing mouse pancreas reveal striking quantitative and qualitative differences in the degree of ductal transformation and pre-malignant progression. Further, using organoid models we show that KRAS G13D mutants respond to EGFR inhibition, while the anti-proliferative effect of KRAS G12C -selective inhibitors can be overcome by upstream EGFR signaling. Together, these new mouse strains provide an ideal for investigating KRAS biology in vivo, and for developing pre-clinical precision oncology models of KRAS-mutant pancreas (G12R), colon (G13D), and lung (G12C) cancers.KRAS mutations in tumor development. However, these models alone do not recapitulate the spectrum of KRAS alterations in human cancer. Here we describe an efficient pipeline for engineering allelic series of conditional alleles that significantly expands repertoire of pre-clinical KRASdriven cancer models. Using high-fidelity CRISPR targeting in embryonic stem cell (ESC)-based mouse models (GEMM-ESCs) 11-14 , we engineered six new LSL-Kras mutant alleles (G12V, G12C, G13D, G12R, G12A, G12S) that represent the most frequent mutations at the G12/G13 hotspot, after G12D. Guided by clinical data, we generated conditional mice representing three tissue-selective alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C) and show that, even subtle mutational changes in the Kras oncogene, have a profound impact on tumor initiation in the pancreas.Conditional animal models, such as Lox-Stop-Lox (LSL)-Kras G12D and LSL-Kras G12Vgeo mice developed almost 20 years ago 9,10 , have been critical tools to dissect the role of . CC-BY-NC-ND 4.

Biological significance of KRAS mutant allele expression in ovarian endometriosis

“…Recently it has been shown that downstream expression and phenotypes of RAS differ depending on the KRAS mutated amino acids in mouse colon and pancreas 60 . Therefore, spatial analysis of genomic and transcriptomic data with consideration of mutant amino acids is necessary to clarify the biological function of cancer‐associated gene mutations in endometriosis.…”

The origins and genetic interactions of KRAS mutations are allele- and tissue-specific

“…For instance, a retrospective meta-analysis suggested that COAD tumors with a KRAS G13D allele were sensitive to anti-EGFR therapies, a treatment generally discouraged for KRAS -mutant tumors 21 . It has recently been proposed, via computational and experimental means, that differential interaction kinetics between KRAS G13D and the Ras GAP NF-1 explain this effect 22 – 24 . Another example is that the KRAS G12D allele is associated with worse overall survival in advanced PAAD, when compared to patients with WT KRAS , KRAS G12R, or KRAS G12V 25 .…”

The Role of Wild-Type RAS in Oncogenic RAS Transformation

“…In keeping with clinical observations, G12C and G12D mutations showed overall enhanced tumorigenesis in both the colon and pancreas compared to G12R or G13D mutations. Further, when assessing drug sensitivities in pancreatic organoids, Kras G13D -mutated organoids were much more sensitive to EGFR inhibition alone compared with other mutants, and Kras G12C -mutated organoids were sensitive to combining an EGFR inhibitor with covalent KRASG12C inhibition [ 154 ], paralleling the findings described above. Taken together, these data indicate that specific KRAS mutations may be more sensitive to inhibitors of WT RAS signaling, leading to organ-specific vulnerabilities based on mutation frequencies.…”

“…To investigate these findings in a controlled model, Zafra et al [ 154 ] recently generated an in vivo Kras allelic series where they directly compared tumorigenesis and drug sensitives of Kras G12C , Kras G12D , Kras G12R , and Kras G13D mutants. In keeping with clinical observations, G12C and G12D mutations showed overall enhanced tumorigenesis in both the colon and pancreas compared to G12R or G13D mutations.…”