The molecular mechanisms by which oncoproteins transform normal cells remains a fundamental question in cancer biology. Recent DNA sequencing studies have demonstrated that most normal tissues harbor a large number of cancer-associated mutations; however, despite the presence of such oncogenic events, most tissues remain untransformed histologically. These findings are in agreement with observations made in exceptional individuals carrying germline or mosaic cancer-associated mutations, which despite exhibiting distinctive and severe syndromes, do not often develop tumors. I have recently defined the term ‘oncoprotein duality' as the ability of a specific oncogene to drive not only a specific cancer, but also a congenital disorder (Castel et al, 2020 Nat Rev Cancer). Such an outcome depends on whether the mutations occur somatically or in the germline and where and when the mutations arise during embryonic development. In this context, the RAS family of guanosine triphosphatases (GTPases) is of particular interest because mutations in numerous members of this family are found both in cancer and in a group of congenital disorders termed RASopathies, which include Costello, Cardio-facio-cutaneous, and Noonan syndromes among others. I hypothesize that studying the molecular function of RAS oncoproteins in this setting will help elucidate their oncogenic role in cancer. To better understand oncoprotein duality, I have analyzed all RAS driver mutations in tumors and congenital disorders and found that NRAS, HRAS, KRAS, and RIT1 are frequently found mutated in both conditions. Interestingly, mutations in NRAS, HRAS, and KRAS exhibited differential allelic preference when comparing cancer and germline disorders. In cancer, gain-of-function mutations in NRAS, HRAS, and KRAS tend to occur in the classic hotspots Gly 12, Gly 13, and Gln 61; however, mutations in these sites are infrequent in germline disorders, probably due to embryonic lethality. In contrast, RIT1 mutations occur at the same region in both sporadic cancers and germline disorders, clustering in a highly conserved domain termed switch II, which is important for protein-protein interactions. RIT1 mutations show increased MAPK signaling in response to growth factor stimulation when expressed in both normal and cancer cells, but not to the same extent as other RAS oncoproteins. To further study the effect of RIT1 activating mutations at the organismal and somatic level, I have generated a conditional knock-in mouse model in which the pathogenic variant Rit1M90I can be induced in a Cre recombinase dependent manner. This mutant allele occurs at 2-5% frequency in lung adenocarcinoma and in myeloid leukemias and it is thought to be an oncogenic driver. Moreover, this mutation, when expressed in the germline, causes the neurodevelopmental disorder Noonan syndrome, which is caused by autosomal dominant mutations in Ras/MAPK genes. Heterozygous mice with germline Rit1M90I mutation recapitulated the classic features of Noonan syndrome, including decreased body size, craniofacial dysmorphia, and hypertrophic cardiomyopathy. Primary mouse cell lines containing the conditional allele confirmed the dysregulated growth factor response responsible for the developmental phenotype. To establish the role of this pathogenic variant in the formation of lung adenocarcinoma, I somatically expressed the Rit1M90I oncoprotein in the lung of conditional mice by using intranasal instillation of adenovirus expressing Cre recombinase. Rit1M90I was found to be a poor oncogene: very few lung adenocarcinomas were observed in these mice after one year. In contrast, mice carrying the KrasG12D allele, a bona fide oncogenic event in lung cancer, developed tumors a few months after adenoviral infection. Similarly, expression of the RIT1M90I oncoprotein in myeloid cells using the tissue-specific strain Mx1-Cre led to a myeloproliferative disorder after several months upon Cre recombination. These results suggest that Rit1M90I requires additional hits to promote malignant transformation in the lung and in myeloid cells and we have initiated in vivo CRISPR/Cas9 screening to identify putative cooperators, such as tumor suppressors. Next, to elucidate the mechanism by which RIT1 mutations promote dysregulation of the MAPK pathway, we undertook a mass spectrometry approach to identify putative regulators of RIT1. By using affinity purification of wild type RIT1, we identified LZTR1 as a novel interactor of this RAS GTPase. LZTR1 is a Kelch/BTB/BACK domain-containing protein that acts as an adaptor for protein degradation through binding to the Cullin-3 ubiquitin ligase. Despite this information, mostly based on LZTR1 domain architecture and previous reports, a substrate had not yet been identified. I have demonstrated that RIT1, but not other RAS GTPases, is a physiological substrate of LZTR1, as evidenced by the ability of LZTR1 to promote RIT1 proteasomal degradation through Lysine 48-linked ubiquitination at specific residues. Importantly, RIT1 mutants found in cancer and Noonan syndrome fail to interact with LZTR1 and exhibit decreased ubiquitination and proteolysis. Emerging evidence suggests that LZTR1 is mutated in a large number of tumors, including glioblastoma and liver cancer, but also in genetic disorders such as schwannomatosis and Noonan syndrome. In the case of Noonan syndrome, LZTR1 mutations are loss-of-function with autosomal recessive segregation, and are mutually exclusive with RIT1 mutations, revealing not only a biochemical, but also a genetic interaction between these two causative genes. To better understand the biochemical role of such mutations, we have performed a comprehensive assessment of the functional role of each reported mutation on LZTR1, creating a genotype-phenotype association based on the properties of each mutant found in cancer and congenital disorders. Consistent with our proposed mechanism, LZTR1 pathogenic mutations result in improper RIT1 degradation and accumulation, leading to the hyperactivation of the MAPK pathway. LZTR1 knockout, which mimics loss-of-function mutations, is embryonically lethal in mice due to a severe form of cardiomyopathy. However, primary cell lines derived from LZTR1 knockout mice reveal increased RIT1 protein levels and, as a result, dysregulated growth factor signaling responses. These observations were confirmed in cancer cell lines in which LZTR1 was knocked out using CRISPR/Cas9. My ongoing research proposes a biochemical and genetic connection between the RIT1 oncogene and the tumor suppressor LZTR1 and provides mechanistic insight into the pathogenesis of different human cancers, including lung adenocarcinoma and myeloid leukemias. Moreover, while previous reports have suggested that RAS proteins can be regulated by protein degradation, we provide the first evidence that dysregulation of this mechanism is pathogenic in both human cancers and developmental disorders. Further work is ongoing in the lab to understand the tissue-specific factors that are required to promote RIT1 oncoprotein-driven cancers and to design the most appropriate therapeutic interventions for each condition. Citation Format: Pau Castel. Oncoprotein duality in cancer and developmental syndromes: Lessons learned from RAS GTPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG08.