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The HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR mutated and wildtype NSCLC cells

ABSTRACT: Background: The receptor tyrosine kinase (RTK) EGFR is overexpressed and mutated in NSCLC. These mutations can be targeted by RTK inhibitors (TKIs) such as erlotinib. Chromatin-modifying agents may offer a novel therapeutic approach by sensitizing tumor cells to TKIs. Methods: The NSCLC cell lines HCC827 (EGFR mutant, adenocarcinoma), A549 (EGFR wt, adenocarcinoma) andNCI-H460 (EGFR wt, large cell carcinoma) were analyzed by SNP6.0 array. Changes in proliferation after panobinostat (LBH-589, PS) and erlotinib treatment were quantified by WST-1 assay and apoptosis by Annexin V/7-AAD flow cytometry. Abundance of target proteins and histone marks (acH3, H3K4me1/2/3) was determined by immunoblotting. Results: As expected, the EGFR wt cell lines A549 and NCI-H460 were quite insensitive to the growth-inhibitory effect of single-agent erlotinib (IC50 70-100 μM), compared to HCC827 (IC50 < 0.02 μM). PS treatment diminished growth to <50 % in both EGFR wt cells, and <30 % in HCC827. The combination of both drugs reduced proliferation by >95 % in HCC827, ≥70 % in A549, but not further in NCI-H460. PS alone induced differentiation and expression of p21WAF1/CIP1 and p53 and decreased CHK1 in all three cell lines, with almost no further effect when combined with erlotinib. In contrast, combination treatment additively decreased pEGFR, pERK, pAKT in A549, and synergistically induced acH3 in both adenocarcinoma lines. Surprisingly, we saw an induction of H3K4 methylation marks after erlotinib treatment in HCC827 (and to a lesser extent in A549) that was even further enhanced by combination with PS. Conclusion: We were able to show that PS synergistically sensitized lung adenocarcinoma cells to the antiproliferative effects of erlotinib. Since single-agent erlotinib has only modest clinical effects in lung adenocarcinoma EGFR wt patients, its combination with an HDACi might offer a promising therapy approach.

ORGANISM(S): Homo sapiens

PROVIDER: GSE63784 | GEO | 2014/12/03

SECONDARY ACCESSION(S): PRJNA269073

REPOSITORIES: GEO

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Project description:Erlotinib is a tyrosine kinase inhibitor (TKI) that is approved as a second-line monotherapy in patients with advanced non-small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest since many tumors express wild-type EGF receptor (wtEGFR) lacking a TKI-sensitizing mutation, develop a second-site EGFR mutation, e.g., EGFR-L858R/T790M, or activate an alternate receptor tyrosine kinase, e.g., through MET amplification. To test potential drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for nuclear factor-M-NM-:B (NF-M-NM-:B) transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M) and H1993 (MET amplification) NSCLC cells, the combination of erlotinib and quinacrine was highly synergistic, as quantified by Chou-Talalay combination indices. The combination inhibited colony formation, induced cell cycle arrest and apoptosis, and slowed xenograft tumor growth. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-M-NM-:B-dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Transcriptomic profiling showed that quinacrine, alone or in combination with erlotinib, significantly affected cell cycle-related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug synergy, we identified genes that were more strongly suppressed by the combination than by either single treatment, and whose increased expression predicted poorer survival in lung adenocarcinoma patients. Combination of quinacrine and erlotinib is synergistic in NSCLC through suppression of FACT, resulting in inhibition of cell cycle progression. RNA was isolated from cells treated with 1 M-NM-<M erlotinib, 3 M-NM-<M or 5 M-NM-<M quinacrine or a combination of both for 6, 12, 24, or 48 h using the RNeasy Mini Kit (QIAGEN) according to the manufacturerM-bM-^@M-^Ys instructions. Microarray analysis was performed using the Affymetrix Human Gene 2.1 ST Array at the Gene Expression & Genotyping Core Facility at Case Comprehensive Cancer Center. Raw CEL files were pre-processed using the Affymetrix Expression Console Software 1.30 with Robust Multi-array Average (RMA) normalization (background correction, quantile normalization and log2 transformation). Probes were annotated using the HuGene 2.1 st hg19 probeset annotation files downloaded from the Affymetrix website.

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The HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR mutated and wildtype NSCLC cells

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