Author of

• 18972

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  P2.02 - Biology/Pathology (ID 616)

  • Event: WCLC 2017
  • Type: Poster Session with Presenters Present
  • Track: Biology/Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)

  • 23265

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    P2.02-067 - LKB1 Loss Is Associated with DNA Hypomethylation in Human Lung Adenocarcinoma (ID 10164)

    09:30 - 16:00  |  Presenting Author(s): Michael John Koenig
    • Abstract

    Background:
    Lung cancer is the leading cause of cancer-associated mortality in the United States. LKB1 loss, for which there are no targeted therapies, occurs in 30% of lung adenocarcinomas. Our group has developed an RNA-based genetic signature for LKB1 loss and applied it to samples in the Cancer Genome Atlas (TCGA). Our studies have identified a novel role for LKB1 in regulation of DNA methylation and histone acetylation/methylation. While global demethylation has been noted in many cancers, LKB1-deficient lung tumors display a greater degree of demethylation compared to tumors that express functional LKB1.
    
    Method:
    RNA-Seq results the TCGA lung adenocarcinoma provisional dataset were analyzed using a genetic classifier for LKB1 loss; samples were separated into wildtype and loss cohorts. Approximately 420 of the samples classified were also characterized using Illumina 450k methylation arrays. We used this data to analyze differentially methylated CpGs. Motif analysis of common transcription factor binding sites near hypomethylated CpGs was accomplished using HOMER. To assess transcription factor localization and binding in vitro, we used a retroviral gene expression system to restore LKB1 function in previously deficient lung cancer cell lines and a CRISPR/Cas9 approach to knock out LKB1 in wildtype cell lines.
    
    Result:
    We observed that LKB1 loss is associated with widespread demethylation of CpG islands throughout the genomes of TCGA samples. Of approximately 138,000 differentially methylated probes, 131,000 are significantly hypomethylated in LKB1 loss (adj. p-value cutoff = .01). We observed that DNMT1, which maintains methylated CpG sites, is downregulated following LKB1 loss. HOMER motif analysis of common transcription factor binding sites in the top 5000 hypomethylated sites implicated several transcription factors that are associated with hypomethylated CpGs—most notably FOXA1/2/3, Nur77, CEBPB, and KLF5; the FOXA family and KLF family have been described as pioneering transcription factors in genomic demethylation. Fractionation and Western blot of A549 cells show that inducing LKB1 expression attenuates FOXA1 and FOXA3 chromatin binding as well as overall expression of FOXA1 and FOXA2.
    
    Conclusion:
    These results have broad implications for gene regulation in LKB1-loss lung tumors and a full understanding of these changes might uncover drug targets specific for these tumors. LKB1’s association with global demethylation suggests that therapeutics targeting methylation such as decitabine may have different effects on LKB1 loss and LKB1 WT tumors, a hypothesis which we are currently testing. We are also continuing to study FOXA1/2/3 and KLF5 to determine the mechanism by which LKB1 regulates its demethylation program.