Project description:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Familial predisposition to PDAC occurs in ~10% of cases, but causative genes have not been identified in most families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with multiple cases of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene never before directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.S36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Unbiased approaches implicate RABL3 in RAS pathway regulation: the transcriptome of juvenile rabl3 mutants reveals a KRAS upregulation signature, and affinity-purification mass spectrometry for proteins associated with RABL3 or RABL3_p.S36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, we find that RABL3_p.S36* accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Furthermore, rabl3 homozygous mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Finally, we identify additional germline mutations in RABL3 that impact RAS activity in vivo and have a significant burden in a cohort of patients with developmental disorders, suggesting a role in undiagnosed RASopathies. Moreover, RABL3 is upregulated in multiple human PDAC cell lines and knockdown abrogates proliferation, consistent with a broader role for RABL3 in PDAC. Our studies identify the RABL3 mutation as a new target for genetic testing in cancer families and uncover a novel mechanism for dysregulated RAS activity in development and cancer.

Project description:KRAS mutations are a major driver of pancreatic ductal adenocarcinoma (PDAC), promoting tumor initiation, progression, and metastasis. RASA2, a Ras GTPase-activating protein, modulates KRAS protein levels in wild-type contexts, yet its role in KRAS-mutant PDAC remains unclear. Here, we systematically investigated the biological function and molecular mechanisms of RASA2 in KRAS-mutant PDAC. Integrative analyses of multiple datasets and patient-derived samples demonstrated that RASA2 is consistently upregulated in KRAS-mutant PDAC and significantly associated with poor prognosis and metastatic progression. Gain- and loss-of-function studies revealed that RASA2 markedly enhances PDAC cell migration and invasion in both KRAS-mutant and KRAS-wild-type models, indicating that its pro-metastatic activity is largely independent of KRAS mutational status. Transcriptomic profiling and mechanistic interrogation uncovered that RASA2 activates GLI1 through a TGFB2-dependent, Sonic Hedgehog–independent non-canonical Hedgehog signaling pathway. Mechanistically, RASA2 directly interacts with the transcriptional regulator RTF1, facilitating H2BK120 ubiquitination at the TGFB2 promoter and thereby augmenting TGFB2 transcription. This epigenetic activation of TGFB2 subsequently drives non-canonical Hedgehog/GLI1 signaling to confer pro-metastatic traits. Pharmacologic inhibition of TGFB2 signaling or genetic silencing of GLI1 effectively abrogated RASA2-driven migratory, invasive, and metastatic phenotypes both in vitro and in vivo. Collectively, our findings identify RASA2 as a critical mediator of PDAC metastasis downstream of oncogenic KRAS and uncover a previously unrecognized RASA2–RTF1–H2BK120ub1–TGFB2–GLI1 signaling axis. KRAS mutation drives RASA2 upregulation, while the pro-metastatic activity of RASA2 is largely independent of KRAS mutational status. These findings reveal a previously unrecognized mechanism by which oncogenic KRAS promotes metastatic progression through RASA2-dependent epigenetic and transcriptional reprogramming and nominate the RASA2–TGFB2–GLI1 axis as a potential therapeutic target in PDAC

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Transcriptomic profiling of KRAS mutants in a panel of isogenic human pancreatic ductal adenocarcinoma (PDAC) cell lines

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