PRC-2 Epigenetic Chromatin Reprogramming in ALK-positive (ALK+) Lung Cancer Initial Emergence of Precision Drug Resistance.

12020 Background: Despite remarkable upfront response to ALK tyrosine kinase inhibitors (TKIs) in ALK+ lung cancer, acquired resistance eventually develops. Molecular mechanisms of the initial emergence of drug resistance are poorly understood. Methods: ALK+ H3122, H2228 and a patient-derived Ma-ALK001.S NSCLC cell lines were used in our study. In vitro MTS viability and murine in vivo xenograft assays were performed. siRNA knockdown and CRISPR/cas9 gene knockout were used for mechanistic studies. ChIP-qPCR was conducted to analyze HOXB3 epigenetic marks. RNA-seq was also performed for transcriptome analysis. Tumor microarray (TMA) biomarker study was adopted for outcome analysis of the polycomb repressive complex-2 (PRC-2) in NSCLC. Results: Our study revealed a rapid-onset emergence of adaptive drug-resistant ALK+ lung cancer cells within the first 14 days upon TKI treatment initiation. The expression of stem cell transcription factors, most notably HOXB3, was induced adaptively within the tumor cells undergoing drug escape. Similarly, tumoral TGFβ2 autocrine expression both at mRNA and protein levels were significantly elevated, and it regulated the downstream HOXB3 expression and mitochondrial prosurvival BCL-2/BCL-xL signaling, cancer stemness and EMT markers. RNA-seq revealed a rapid-onset adaptive global transcriptome reprogramming in the drug-escaping persister tumor cells. ChIP-qPCR showed resistance emerged via epigenetic regulation of the untreated bivalent HOXB3 promoter closed chromatin state transforming to stem-like open state during drug-escape, based on H3K4me3/H3K27me3 methylation mark balance. Findings were validated via CRISPR/cas9-EZH2 knockout studies. AQUA-TMA biomarker and survival outcome analysis confirmed the clinical relevance of EZH2/UTX of PRC-2. Finally, direct EZH2 inhibition or knock-out promoted ALK-TKI drug resistance, while UTX inhibition enhanced drug sensitivity. Conclusions: ALK+ lung cancer achieves initial rapid-onset emergence of adaptive drug escape through PRC-2 epigenetic chromatin reprogramming to regulate cancer plasticity-EMT/stemness interplay via the TGFβ2-EZH2/UTX-HOXB3-BCL-2/BCL-xL cascade.