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J.M. Amann
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-065 - RanBP9 is a Novel Prognostic and Predictive Biomarker for NSCLC and Affects Cellular Response to Cisplatin and PARP Inhibitors (ID 10002)
09:30 - 09:30 | Author(s): J.M. Amann
- Abstract
Background:
We have previously demonstrated the involvement of Ran Binding Protein 9 (RanBP9) in the DNA Damage Response (DDR) in Non Small Cell Lung Cancer (NSCLC) cells. Here, we investigate its role in response to DNA-damaging agents in vitro and as prognostic and predictive biomarker for NSCLC patients.
Method:
First, by IHC, we evaluated RanBP9 expression in tumor vs normal adjacent tissue (NAT). Then, we generated A549 RanBP9 WT and KO NSCLC cells using CRISPR/Cas9. We treated A549 RanBP9 WT and KO with cisplatin (CDDP) and PARP inhibitors. We assessed response to treatment by measuring cell toxicity, apoptosis and proliferation. Finally, we determined the expression of RanBP9 in cohort of NSCLC patients previously enrolled in the TAILOR trial.
Result:
In the present study, we report that significant overexpression of RanBP9 is a common event in lung cancer, as shown by an extensive immunohistochemical analysis of RanBP9 levels in 148 lung tumors of different histotypes and their normal adjacent tissue (p<0.02 - 0.001). RanBP9 expression was maintained/acquired in the nodal metastasis from 30 NSCLC patients, indicating its potential involvement in tumor aggressiveness. We also show that RanBP9 KO A549 NSCLC cell lines display a reduced DDR and higher levels of apoptosis upon cisplatin treatment both in vitro and in vivo. Accordingly, a retrospective analysis of 134 NSCLC patients revealed that higher levels of RanBP9 are associated with tumor stage (p<0.0001), and low response to platinum compounds as first-line treatment (PFS, HR~ (RanBP9 positive versus negative)~ 1.71, 95% CI 1.142 - 2.563, p = 0.0093; OS HR~ (RanBP9 + vs -) ~1.942, 95% CI 1.243-3.033, p=0.0036). Finally, we show that ablation of RanBP9 is associated with overactivation of Poly(ADP-ribose) Polymerase (PARP) and increases sensitivity to PARP inhibitors. Moreover, that use of PARP inhibitors enhances cisplatin anti-neoplastic efficacy in the absence of RanBP9.
Conclusion:
We identified RanBP9 as a novel predictive biomarker of response to genotoxic treatments in NSCLC patients. We also report that RanBP9 affects the response of NSCLC cells to PARP inhibitors in vitro. Our results open new avenues for the treatment of NSCLC patients based on their level of expression of RanBP9.
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P2.02-067 - LKB1 Loss Is Associated with DNA Hypomethylation in Human Lung Adenocarcinoma (ID 10164)
09:30 - 09:30 | Author(s): J.M. Amann
- 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.
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P3.03 - Chemotherapy/Targeted Therapy (ID 719)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Chemotherapy/Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.03-002 - Histone Deacetylase Inhibition Enhances the Antitumor Activity of a MEK Inhibitor in Lung Cancer Cells Harboring RAS Mutations (ID 7917)
09:30 - 09:30 | Author(s): J.M. Amann
- Abstract
Background:
Non-small-cell lung cancer (NSCLC) can be identified by precise molecular subsets based on genomic alterations that drive tumorigenesis and include mutations in EGFR, KRAS, and various ALK fusions. However, despite effective treatment for EGFR and ALK, promising therapeutics have not been developed for patients with KRAS mutations. Therefore, novel therapeutic strategies for KRAS mutated cancer based on molecular mechanisms are needed to improve their prognosis. It has been reported that one way the RAS-ERK pathway contributes to tumorigenesis is by affecting stability and localization of FOXO3a protein, an important regulator of cell death and the cell cycle.
Method:
We used NSCLC cells with RAS mutation to evaluate the effect of a MEK inhibitor in combination with a HDAC inhibitor through the expression and localization of FOXO proteins in vitro and in vivo. Protein expression was examined by Western blotting.
Result:
Combined treatment with a MEK inhibitor and a HDAC inhibitor showed synergistic effects on cell viability of RAS mutated lung cancer cells through activation of FOXOs, with a subsequent increase in BIM and cell cycle inhibitors. Moreover, in a mouse xenograft model, the combination of belinostat and trametinib significantly decreases tumor formation through FOXOs by increasing BIM and increase in cell cycle inhibitors p21 and p27.
Conclusion:
These findings demonstrate that FOXOs might be one of the critical pathways in RAS driven lung cancer cells, suggesting that the dual molecular targeted therapy for MEK and HDACs may be promising as novel therapeutic strategy in NSCLC with specific populations of RAS mutations.
