P53-deficient Non-small Cell Lung Research Articles

Abstract 1429: DNA damage response to dianhydrogalactitol (VAL-083) in p53-deficient non-small cell lung cancer cells

Abstract Non-small cell lung cancer (NSCLC) treatment usually involves surgery and chemotherapy with tyrosine kinase inhibitors (TKI) in patients with EGFR mutations (10-15% in Western population, 40% in Asian populations) or with platinum-based regimens. Response to TKI treatment is short lived, and tumors recur with new mutations, primarily T790M. Recurrent NSCLC with T790M is sensitive to third generation TKIs, but resistance usually emerges through new mutations, including KRAS. Resistance to cisplatin and carboplatin, partly due to p53 mutation, is also a major clinical limitation and long-term prognosis in NSCLC is poor. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent with demonstrated clinical activity against NSCLC in historical NCI-sponsored trials and VAL-083 is approved for lung cancer treatment in China. However, the mechanism-of-action of how DNA damage signals are propagated and their effects on NSCLC cells are not fully understood. Therefore, we examined VAL-083 in a panel of 11 human NSCLC cell lines harboring wild-type p53 (H460, A549, H226), mutant p53 (H1975, SkLU1, H2122, H157, H1792, H23) or null p53 (H838, H1299). Importantly, as determined by the 5-day MTT assay, VAL-083 was cytotoxic against all 11 cell lines at low μM concentrations and cytotoxicity was independent of p53 status. We chose 3 TKI-resistant cell lines with different mutation profiles for cell cycle kinetics studies: i) H1975 (p53 mut, EGFR-T790M mut, KRAS wt) with IC50 0.9μM, ii) A549 (p53 wt, EGFR wt, KRAS mut) with IC50 1.8μM, and iii) H157 (p53 mut, EGFR wt, KRAS mut) with IC50 4.5μM. In all 3 cell lines, early response at 18 hr showed dose-dependent increase in cells in S-phase, with continued slow cell cycle progression resulting in accumulation of cells in G2/M phase by 36 hr, suggesting persistent cell cycle arrest. DNA damage signaling was examined by immunoblot analysis in A549 and H1975 cells. In p53-wt A549, VAL-083 induced the phospho-Ser15 form of p53, total p53 and total p21, thus indicating that VAL-083 treatment activated p53 function. On the other hand, in p53-mutant H1975, VAL-083 treatment did not result in consistent p53 or p21 increases, but did readily induce phospho-Ser15 p53. This is consistent with a lack of p53 function, as anticipated for mutant p53 cells. Interestingly, H1975 was 2-fold more sensitive to VAL-083 than A549, suggesting, that in p53-mutant cells, VAL-083 acts through a p53-independent mechanism. Examination of ATR, ATM, Chk1 and Chk2 indicated that DNA damage by VAL-083 prompted phosphorylation of these kinases. Notably, the total anti-apoptotic Chk1 was more prominently reduced in H1975 than A549, which may partly explain the stronger cytotoxicity of VAL-083 in p53-mutant H1975. These preclinical data strongly support VAL-083 as a potential treatment of mutant p53 and TKI-resistant NSCLC, and indicate DNA damage signaling is mediated via ATM, ATR, Chk1 and Chk2. Citation Format: Anne Steino, Guangan He, Jeffrey A. Bacha, Dennis M. Brown, Zahid Siddik. DNA damage response to dianhydrogalactitol (VAL-083) in p53-deficient non-small cell lung cancer cells [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 1429. doi:10.1158/1538-7445.AM2017-1429

Abstract 460: Influence of low oxygen conditions on the in vitro radiosensitizing effect of gemcitabine, in p53 wild type and p53 deficient non-small cell lung carcinoma cells

Abstract Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. As the antimetabolite gemcitabine is a potent radiosensitizer, often used in the clinic, the influence of oxygen deficiency on the radiosensitizing effect of gemcitabine was investigated. Materials & methods: The human tumor cell lines included were A549 (lung carcinoma cell line, wt p53), A549-E6 (transduced with the HPV type 16 E6 gene, abrogating the p53 function) and A549-LXSN (vector transfected control, functional p53). Anoxic conditions (<0.1% O2,) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0-15 nM gemcitabine for 24h directly before radiation (0-8 Gy). Immediately following radiation, anoxic cells were reoxygenated and all cells were washed with drug-free medium. Cell cycle analysis and determination of apoptotic cell death was performed using flow cytometry. P53 expression was studied by Western blot. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxia and anoxia (dose enhancement factor 1.27-1.70 and 1.28-2.06 respectively). Combination index (CI) analysis showed an additive to strongly synergistic interaction between gemcitabine and radiation under both normal and reduced oxygen conditions (CI 0.672-1.083 and 0.587-1.090 respectively). Two-way ANOVA revealed that cell survival was significantly influenced by the concentration of gemcitabine, radiation dose, oxygen tension and cell line used. Post hoc analysis indicated a significant difference between A549-LXSN and A549-E6 cells, suggesting a potential role for p53 in radiosensitization by gemcitabine. Independent of the p53 functionality, gemcitabine was able to overcome the anoxia-induced G0/1 phase block and established a significant S-phase block in both normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased significantly with the combination of gemcitabine and radiation, and was significantly influenced by the oxygen concentration. Interestingly, a significant difference between A549-LXSN and A549-E6 was observed. Conclusion: This study demonstrated, for the first time, that gemcitabine retains its radiosensitizing potential under low oxygen conditions. The p53 protein might be involved in this process, not by affecting cell cycle progression, but rather by acting on the induction of apoptotic cell death. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 460.