Abstract Introduction Inactivating somatic mutations in the NF1 tumor suppressor gene, encoding a prototypical RASGAP, drive the development of 11% of LUACs. Extensive genetic and biochemical evidence supports a central role of aberrant RAS signaling in the pathogenesis of NF1-deficient tumors. However, variable dependence on different RAS effector pathways and inconsistent responses to MEK and PI3K/MTOR inhibitors have been reported in experimental models of Nf1-related carcinogenesis. We hypothesized that significantly co-occurring genetic alterations may underpin signaling diversification downstream of mutant NF1. Methods Our dataset includes 519 LUACs from TCGA and a cohort reported by Imielinski et al. Assessment of co-occurring genetic events was based on Fisher's exact test with a statistical cutoff of p≤0.05 (≤0.1 for the validation datasets). Supervised analysis of RNASeq and reverse phase protein array (RPPA) data and computation of MAPK and PI3K proteomic scores was conducted for each co-mutated gene as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results We mapped dominant expression signatures in NF1-mutant LUAC using GSEA. Signatures relating to poor prognosis, EMT, E2F3-mediated gene expression and immune engagement were enriched in NF1-mutant tumors. RPPA confirmed lower expression of E-cadherin (P = 0.015) and higher expression of fibronectin (P = 0.018) in NF1-mutant compared to KRAS-mutant LUAC. NF1-mutant tumors showed significant heterogeneity in MAPK and PI3K/AKT/MTOR pathway activation. Compared to KRAS-mutant LUAC, NF1-mutant tumors had significantly lower MAPK score (P = 0.005) but comparable PI3K score (P = 0.407). Analysis of co-occurring genetic events identified 1201 genes as significantly differentially mutated between NF1-mutant and NF1-wild-type LUACs in the TCGA cohort. LANCL2 (P = 6.7×10−06) and TP53 (1.63×10−05) were the top hits in this dataset. 135 genes validated in a second LUAC dataset. Strikingly, a core set of genes were significantly co-mutated with NF1 across a range of human tumors. Somatic mutations in KIAA1549L were associated with highly significant increase in PI3K score (P<0.001). Higher PI3K score was also noted in tumors bearing co-mutations in SORCS3 (P = 0.049), and PAPP-A2 (P = 0.027), a matrix metalloproteinase with a reported role in proteolytic cleavage of IGFBP5. Functional assays to confirm the impact of these and other selected co-mutations and their effect on responsiveness to MEK and PI3K/MTOR inhibitors are underway. Conclusions Co-occurring genetic alterations impact both the amplitude and directionality of signaling downstream of NF1 inactivation in LUAC and may therefore account for heterogeneity in both biological behavior and clinical response to targeted therapies. Citation Format: Ferdinandos Skoulidis, Pan Tong, Jing Wang, John V. Heymach. Impact of co-occurring genetic events on RAS signaling diversification in NF1-mutant lung adenocarcinoma (LUAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2150. doi:10.1158/1538-7445.AM2015-2150