KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice.

Benjamin S. Braun,Ophir D. Klein,K. Hirose ,Denny Schanze,Tannie Q. Huang,Pau Castel,Joaquím Grego-Bessa ,Carolina Morales ,Jasmine C. Wong,Amnon Sharir,Kevin Shannon,Pedro A. Pérez–Mancera ,Guo N. Huang,Martin Zenker,Susan Keefe ,David A. Tuveson,Dean Sheppard,Maria del pilar Alzamora,Scott C. Kogan,Jangkyung Kim

doi:10.1172/jci.insight.140495

Abstract

Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre–mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.

Highlights

The RASopathies are a heterogenous group of developmental disorders with overlapping phenotypic features that include craniofacial dysmorphism, short stature, developmental delay, learning disabilities, neurocutaneous abnormalities, cardiovascular defects, and a variable risk of tumorigenesis [1,2,3,4]
Blastocyst injections resulted in multiple coat color chimeras that were bred to WT C57BL/6 (B6) mice and selected for agouti offspring to achieve germline transmission of the KrasLSL-P34R and KrasLSL-T58I alleles (Supplemental Figure 1B and Supplemental Figure 2B)
Ras oncoproteins with glycine 12 (G12) substitutions show variable reductions in intrinsic GTPase activity and are insensitive to stimulation by neurofibromin and p120GAP, which are the predominant cellular GTPase-activating proteins (GAPs) [20, 36]

Summary

Introduction

The RASopathies are a heterogenous group of developmental disorders with overlapping phenotypic features that include craniofacial dysmorphism, short stature, developmental delay, learning disabilities, neurocutaneous abnormalities, cardiovascular defects, and a variable risk of tumorigenesis [1,2,3,4]. We reasoned that modeling additional germline KRAS mutations with different effects on intrinsic GTP hydrolysis and responses to GAPs would advance our current understanding of activated Ras signaling in development and tumorigenesis while generating mouse strains for biologic studies and preclinical drug testing. Based on the distinct clinical phenotypes of the respective patients, the potentially novel biochemical and functional properties of K-RasP34R and K-RasT58I, and the presence of recurrent somatic KRASP34R and KRAST58I mutations in cancer, we generated mice harboring conditional mutant P34R and T58I alleles and characterized their developmental, biochemical, and functional consequences. We show that heterozygous mutant mice model key RASopathy phenotypes and show that each mutation has distinct and context-dependent effects on signal transduction

Results

Discussion

Methods