Ras-like in most tissues 1 (RIT1) encodes a ubiquitously expressed RAS-family small GTPase. Recently, somatic RIT1 mutations were identified in lung adenocarcinoma and myeloid malignancies, and germline RIT1 mutations were found in. RIT1 is also implicated hematopoietic malignancies and in Noonan syndrome, a “Rasopathy” affecting craniofacial development and associated with congenital heart disease. Of a variety of RIT1 mutations discovered, the most recurrent mutation observed was M90I. In this study, murine models were used to determine whether expression of RIT1M90I mutation is sufficient for tumor initiation or developmental phenotypes observed in Noonan patients.

Mice were genetically engineered at the Rosa26 locus to harbor RIT1M90I under the control of a lox-Cre system (LSL-RIT1M90I). The Cre recombinase protein was introduced via intratracheal delivery of adenovirus to the lungs. At one-year post treatment, 7 of the 10 LSL-RIT1M90I mice examined exhibited the expected genetic recombination. Tumors were observed in 3 of these mice, one of which was wild-type for p53. No visible tumors were seen in mice lacking the RIT1M90I gene or in cases where recombination was not observed, regardless of p53 status.

Mouse embryonic fibroblasts (MEFs) were infected by adenovirus to recapitulate the in vivo studies and evaluate transformation. In vitro experiments conducted in RIT1M90I MEFs demonstrated faithful recombination of the transgene upon treatment with Cre. Protein analysis of these same cells reflected higher levels of RIT1 protein in transgenic cells treated with Cre, while in all other conditions, RIT1 was undetectable. Preliminary data from an immortalization assay suggested enhanced proliferation of MEFs expressing the RIT1M90I gene compared to wild-type counterparts.

Cre recombinase was expressed body-wide in tamoxifen (4OHT) inducible mice (UBC-Cre-ERT2) LSL-RIT1M90I animals crossed to the inducible UBC-Cre-ERT2 system exhibited faithful recombination of the transgene upon treatment with 4OHT.

To determine the consequences of constitutive expression of RIT1M90I, RIT1M90I sires were crossed to dams harboring constitutively active Cre recombinase or to a constitutively active Cre SRY-box promoter (Sox2-Cre). Of the 100 pups observed, none contained both RIT1M90I and Cre recombinase (p < 0.0001), indicating embryonic lethality induced by RIT1 activation.

These data demonstrate the powerful effect of RIT1 activation on murine embryonic development and will enable future efforts on the characterization of the mechanism of RIT1-induced Noonan Syndrome and lung cancer.

Citation Format: Maria McSharry. In vivo modeling of RIT1 function in development and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1765.