Abstract 4365: Sensitivity of small cell lung cancer cells with defective Fanconi Anemia (FA) pathway to BCL2 inhibitors..
Authors: Gao Li , Duan Wenrui , Barnwell Brittany , Kalvala Arjun , Otterson Gregory A. , Villalona-Calero Miguel A. 
Published: August 2014

Bcl-2 is a central apoptotic inhibitor, and overexpression is associated with tumor progression and treatment resistance in cancers. Overexpression has been reported in up to 80% of small cell lung cancers (SCLC). ABT-263 (Nativoclax) is a potent and selective inhibitor of Bcl-2 and Bcl-xL, disrupting their interactions with pro-death proteins leading to the initiation of apoptosis within 2 hours post exposure. However a recent phase II study of single-agent ABT-263 showed low rate of response to single-agent treatment in advanced and recurrent SCLC. Thus, pre-selection of patients most likely to derive benefit from BCL-2 inhibitors will be needed for further development of these agents in SCLC. The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. The repair abnormalities resulting from deficiencies in FA pathway potentially select for the persistence of prosurvival pathways. We hypothesized that cancers with defective FA pathway would be more sensitive to not only DNA interstrand crosslinking based therapy, but also to treatments in which prosurvival pathways are targeted, like BCL-2 inhibition.

We utilized RNAi technology to create FANCD2 knockdown SCLC cancer cells. H719 cells were transduced with FANCD2-specific shRNA-expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles to create stably transduced cells. Successful FANCD2 knockdown was confirmed by Western blot by reduction in the FANCD2 protein. Cell viability was evaluated with MTT (Dimethylthiazolyl-2-5-diphenyltetrazolium bromide) analysis, and apoptosis was evaluated with Western immunoblot PARP cleavage assay.

The FA defective H719 small cell lung cancer cells and the control cells (transfected with empty vectors) were treated with ABT-263 at a dose of 2μM. The treated cells were then harvested at 6, 24 and 48 hours post treatment. MTT cell viability analysis showed that ABT-263 alone was cytotoxic to the FA deficient lung cancer cells with less viable cells comparing to controls 6-48 hours post treatment. In addition, Western immunoblot analysis with anti-PARP [poly (ADP-ribose) polymerase] antibody showed PARP cleavage was increased in the FA defective H719 cells as compared to control cells 6 hours post ABT-263.

Disruption of FA cascade has been reported in solid tumors. Recently we have developed a FA triple-staining immunofluorescence (FATSI) method to detect FANCD2 foci formation, which is capable of evaluating the functionality of the whole pathway using formalin fixed paraffin embedded (FFPE) tumor samples and have identified up to 15% of small cell lung cancer tumor samples to be functionally deficient. Based on our preliminary studies with the H719 cells, we propose that SCLCs with defective FA pathway would be more sensitive to BCL-2 inhibitors compared to those retaining an intact repair function.

Citation Format: Li Gao, Wenrui Duan, Brittany Barnwell, Arjun Kalvala, Gregory A. Otterson, Miguel A. Villalona-Calero. Sensitivity of small cell lung cancer cells with defective Fanconi Anemia (FA) pathway to BCL2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2013-4365