Abstract 1679A: HLTF gene silencing predicts sensitivity to lysosomal autophagy inhibitors in cancer cells.
Authors: Piao Shengfu , Samanta Arabinda , Ma Xiao-hong , Mcafee Quentin , Wilkins Ben , Christensen James , O'Dwyer Peter , Amaravadi Ravi K. 
Published: August 2014

Autophagy inhibition with hydroxychloroquine (HCQ), either alone or when combined with other anticancer agents, is cytotoxic to only in a subset of human cancer cell lines. Clinical trials involving HCQ in cancer patients are underway, but predictive biomarkers to guide the development of autophagy inhibitors are not available. To identify determinants of sensitivity to HCQ, an mRNA microarray analysis of HCQ-sensitive compared to HCQ- resistant colon cancer and lung cancer cell lines identified helicase-like transcription factor(HLTF) as the number one most down regulated gene in HCQ-sensitive cell lines. In a larger panel of cancer cell lines HLTF promoter methylation correlated with silenced expression and sensitivity to HCQ. Forced expression of HLTF by transfection or treatment with a demethylating agent conferred resistance to HCQ-sensitive cell lines. Knockdown of HLTF in HCQ-resistant cell lines conferred sensitivity. To determine the mechanism that links HLTF to lysosomal autophagy inhibition, we posed the hypothesis that HCQ-related ROS produced DNA damage that was repaired by HLTF. Lysosomal autophagy inhibition with HCQ produced reactive oxygen species (ROS) irrespective of HLTF status. Cotreatment with an ROS scavenger but not cathepsin inhibitors mitigated HCQ associated cytotoxicity in HLTF silenced cells. DNA damage was observed at 100-fold lower HCQ concentrations in HLTF silenced cell lines than in HLTF expressed cells. Knockdown of pol eta, a low fidelity DNA polymerase involved in translesion synthesis, completely abrogated HLTF-associated resistance to HCQ. HLTF expression and caspase inhibition but not either alone completely abrogated all DNA damage and cytotoxicity associated with HCQ in HLTF silenced cells. These results outline a potentially new mechanistic pathway that links lysosomal autophagy inhibition with ROS-mediated DNA damage and secondary caspase-dependent apoptosis, which can be prevented by intact HLTF-pol eta translesion synthesis activity. Next, combinations of HCQ with PI3K/mTOR, FAK, or IGFR1 inhibitors currently under clinical investigation were tested in cells with silenced or expressed HLTF. Additive cytotoxicity with HCQ was observed only in cell lines with silenced HLTF irrespective of the inhibitor combination tested. Finally low expression of HLTF as measured by immunohistochemistry predicted responders in a preliminary cohort of colon cancer patients treated on a phase I clinical trial of FOLFOX/bevacizumab and HCQ. Given that previous clinical studies have determined that HLTF gene silencing is prevalent in 20-40% of many epithelial malignancies, these results provide rationale for development of HLTF gene silencing as a first predictive biomarker for autophagy inhibitors that could be applied to a broad subset of human cancers.

Citation Format: Shengfu Piao, Arabinda Samanta, Xiao-hong Ma, Quentin Mcafee, Ben Wilkins, James Christensen, Peter O'Dwyer, Ravi K. Amaravadi. HLTF gene silencing predicts sensitivity to lysosomal autophagy inhibitors in cancer cells. [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 1679A. doi:10.1158/1538-7445.AM2013-1679A