Abstract 1429: DNA damage response to dianhydrogalactitol (VAL-083) in p53-deficient non-small cell lung cancer cells
Authors: Steino Anne , He Guangan , Bacha Jeffrey A. , Brown Dennis M. , Siddik Zahid 
Published: July 2017

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