Dysregulation of the p16(INK4a)-CyclinD-CDK4/6-Rb pathways in patients with NSCLC (Non-Small Cell Lung Cancer) is a rational therapeutic target. The current study investigated the radiosensitizing potential of a novel CDK4/6 inhibitor, LY2835219 (LY, Abemaciclib) in NSCLC cell lines with varied genomic context to identify genomic and metabolic biomarkers that are predictive of a response over conventional EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. NSCLC cell lines were exposed to LY (0-10uM) for 24 hr immediately after 0-10 Gy radiation. Cell survival was assessed by clonogenic assay and cell cycle distribution was quantified by flow cytometry. Dose modifying factors (DMF) were calculated at 10% survival from radiation survival curves. Altered DNA repair pathways and metabolic profiling of cells post LY treatment was assessed by immunoblot and LC/MS mass spectrometry analysis. LY treatment enhanced radiosensitivity of EGFR-TKI sensitive (HCC827, PC9) and EGFR-TKI resistant (H820 and H1975) cell lines with DMF of 1.3 (±0.06), 1.4 (±0.30), 1.5 (±0.51) and 1.3 (±0.02), respectively. Values in the parenthesis indicates standard deviation. Wild type EGFR expressing cells (A549 and H460) also showed enhanced radiosensitivity by LY with DMF of 1.6 (±0.09) and 1.75(±0.15), respectively. Interestingly, no radiation enhancement by LY was observed for cells deficient in functional PTEN (H1650), Rb (H82) and p53 (H460 DNp53 and H1299) protein. Radiosensitization was also observed for cells made resistant to third generation EGFR-TKI, AZD9291. Flow cytometry analysis of majority of cell types exposed to LY exhibited 55% to 94% G1 arrest (depending on cell type). Mechanistically, the combinatorial treatment in radiosensitive cells showed elevated phosphorylated-γH2Ax. Combination treatment also reduced expression of ATR, ATM, DNA-PK, Rad51 and Chk-2 suggestive of reduced DNA repair compared to radiation alone. LY treatment brought major changes in the glycolysis/TCA/ total amino acids. LY increased significantly Acetyl-CoA, fumarate and malate, indicating enhanced oxidative phosphorylation. LY significantly elevated Uric acid levels suggestive of oxidative stress and elevated nucleotide degradation. Finally, administration of 100mg/kg LY2835219 for five days in combination with fractionated dose of radiation (3 Gy) significantly delayed tumor regrowth in H460 xenograft (p< 0.014). Collectively, our pre-clinical data indicates altered Rb, p53 and PTEN status are distinct predictive biomarkers of response for LY mediated radiosensitivity and provides an alternative therapeutic option in overcoming EGFR-TKI resistance in NSCLC.
Note: This abstract was not presented at the meeting.
Citation Format: Sarwat Naz, William DeGraff, Anastasia Sowers, Rajani Choudhuri, Maria Wissler, Janet Gamson, John Cook, James B. Mitchell. Enhanced radiosenistivity of EGFR-TKI sensitive and resistant NSCLC cells by abemaciclib is mediated by altered DNA repair and metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5861. doi:10.1158/1538-7445.AM2017-5861