Author of

• 16549

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  P1.02 - Poster Session with Presenters Present (ID 454)

  • Event: WCLC 2016
  • Type: Poster Presenters Present
  • Track: Biology/Pathology
  • Presentations: 2
  • Moderators:
  • Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)

  • 16604

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    P1.02-055 - Synthetic Lethality Dictates the Mutual Exclusivity of Oncogenic Mutations in Lung Adenocarcinoma (ID 6190)

    14:30 - 14:30  |  Author(s): W. Lockwood
    • Abstract

    Background:
    EGFR mutations are present in ~15% and KRAS mutations in ~30% of lung adenocarcinomas (LACs). Yet, as several observers have noted, no individual LAC appears to carry mutations in both, even in the setting of resistance to EGFR kinase inhibitors. The typical explanation given is that the two genes are entirely functionally redundant, thereby eliminating any selective advantage to mutation of both. In this study we tested an alternative hypothesis for this mutual exclusivity: that co-occurrence of mutations in both KRAS and EGFR is deleterious to the evolving cancer cell. Furthermore, we aimed to characterize the specific cellular effects and signalling pathways induced by mutant KRAS and EGFR co-induction and determine whether this information could be used to design new strategies to inhibit LACs.
    
    Methods:
    Next-generation sequence data for over 600 human LACs were acquired from public sources and analyzed for co-incidence of KRAS and EGFR mutation and the relationship to smoking status. Transgenic mice that express both mutant oncogenes specifically in the lung epithelium were generated to test the effects on LAC development. LAC cell lines with endogenous mutations in either KRAS or EGFR were engineered with lentiviral vectors to conditionally express the second oncogene and assessed for cell viability, gene expression and protein phosphorylation changes. Temporal phosphoproteome assessment of cells co-expressing both oncogenes is currently ongoing to determine the specific signalling pathways affected.
    
    Results:
    We confirmed the mutual exclusivity of KRAS and EGFR mutations and demonstrated that this relationship is not due to limited power to detect concurrent mutations in either smokers or non-smokers. While no effect on tumor latency was observed in transgenic mice that express both mutant oncogenes, the resulting tumors preferentially expressed only one of the two transgenic oncogenes, indicating negative selection against co-expression. Introduction of the second oncogene into LAC cells expressing either mutant KRAS or EGFR stimulated the loss of cell viability. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK was observed in cells expressing both mutants suggesting that an overly active MAPK signaling pathway may mediate this synthetic lethal phenotype. Lastly, this effect is dependent on functionally active EGFR, since inhibition of the EGFR tyrosine kinase with erlotinib rescued cells from the detrimental effects of co-expression.
    
    Conclusion:
    Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.
    
    

  • 16622

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    P1.02-073 - Characterizing the Genomes of Lung Adenocarcinomas from Never Smokers Reveals SHPRH as a Novel Candidate Tumour Suppressor Gene (ID 4772)

    14:30 - 14:30  |  Author(s): W. Lockwood
    • Abstract

    Background:
    Approximately 15 to 25% of lung adenocarcinomas (LAC) arise in never smokers. They develop through mechanisms distinct from those that affect smokers and are associated with unique histological and molecular characteristics. A significant fraction of LACs in never smokers do not have mutations in known oncogenic driver genes such as EGFR/ALK/KRAS. Furthermore, mutations in oncogenic driver genes appear to be insufficient for tumorigenesis, suggesting that additional alterations are required.
    
    Methods:
    To address these issues, we used whole-exome sequencing to comprehensively study 15 LACs from never smokers - seven “triple negative” tumors (with normal EGFR/ALK/KRAS) and eight EGFR-mutant tumors - with the goal of identifying novel mutant genes in these subsets. To identify mutated genes that confer a selective advantage a multistep approach was used to filter variants based on gene expression level, background mutation rate and gene size. Targeted sequencing of 180 genes in the original 15 and an extended panel of 85 tumor/normal pairs validated these alterations and indicated their prevalence in LAC. Sequence data was integrated with copy number and gene expression levels to determine mechanisms, and consequences, of gene disruption. Animal and cell models were used to functionally validate identified genes of interest and explore their role in LAC biology.
    
    Results:
    32 unique genes demonstrated significant evidence of conferring a selective advantage including known oncogenes (EGFR/ERBB2/MET) and tumor suppressor genes (p53/RB1/ATM). In addition, RNA-seq revealed fusions involving RET or ROS1 in one tumour each. The variations in MET consisted of truncating and splice-site mutations that we are currently investigating in transgenic mouse models. Pathway analysis indicated frequent mutation in genes implicated in PI3-kinase signaling, RNA splicing and histone modification. Importantly, we identified the hemizygous and homozygous loss of multiple genes from chromosome arm 6q - a genetic locus associated with familial lung cancer susceptibility - including a novel candidate tumor suppressor gene, SHPRH, based on its high frequency of biallelic disruption. SHPRH is an evolutionarily conserved E3-ligase that mediates crucial processes related to DNA repair. We found that SHPRH silencing increased transformation of normal lung cells, increased DNA damage and induced cell cycle changes while SHPRH inhibition sensitized LAC cells to topoisomerase II and PARP inhibitors.
    
    Conclusion:
    SHPRH inactivation may induce genetic alterations that cooperate with mutations in driver oncogenes to promote LAC development. Together, this work will expand our understanding of LAC initiation and progression in never smokers and may offer new biomarkers for response to therapy.