Abstract 1024: Expression pattern of ALDH1A1 and HLTF predicts sensitivity to lysosomal autophagy inhibitors in cancer cells

Abstract

Abstract Autophagy inhibition with hydroxychloroquine (HCQ)is cytotoxic to only a subset of cancer cell lines. Clinical trials involving HCQ in cancer patients are underway, but predictive biomarkers to identify patients most likely to respond are unavailable. To identify determinants of sensitivity to HCQ, an mRNA microarray analysis of HCQ-sensitive(S) compared to HCQ- resistant (R) colon and lung cancer cell lines identified differentially expressed genes in HCQ-S cells. Immunoblotting against the 2 most upregulated (Lysozyme (LYZ); aldehyde dehydrogenase 1 (ALDH1A1)) and 2 most down regulated proteins (helicase like transcription factor (HLTF); p-glycoprotein (ABCB1)) was performed in 35 human cancer cell lines. The IC50 and slope of the HCQ dose response curve (72 h MTT) were used to create a composite score to categorize cell lines as -S or -R, and correlated to protein expression of the 4 genes. Classification and regression tree (CART) analysis determined that high expression of the stem cell marker ALDH1A1 or low expression of both ALDH1A1 and HLTF identified 100% of sensitive cell lines. To understand how ALDH1A1 mechanistically interacts with HCQ, the aldefluor assay determined that a more potent dimeric CQ (Lys21) did not impair ALDH1A1 enzymatic function, however knockdown or chemical inhibition of ALDH1A1 impaired cellular uptake of fluorescently tagged Lys21 and conferred resistance to HCQ. Overexpression of ALDH1A1 conferred HCQ sensitivity in HCQ-R cells. To understand the interaction between HLTF and HCQ, we determined that HLTF promoter methylation correlated with silenced expression and sensitivity to HCQ. Forced expression of HLTF or treatment with a demethylating agent conferred resistance to HCQ-S cells. Knockdown of HLTF in HCQ-R cells conferred sensitivity. HCQ produced reactive oxygen species (ROS) irrespective of HLTF status. Cotreatment with a ROS scavenger mitigated HCQ cytotoxicity in HLTF silenced cells. DNA damage (p-H2AX) was observed at 100-fold lower HCQ concentrations in HLTF silenced compared to HLTF expressed cells. Overexpression of HLTF significantly reduced HCQ associated double strand breaks (Rad52 foci). Knockdown of DNA polymerase eta, a low fidelity DNA polymerase involved in translesion synthesis (TLS) completely abrogated HLTF-associated HCQ resistance. In vivo expression of HLTF mitigated the antitumor activity of the dimeric CQ Lys05 in a HCQ-S colon cancer xenograft. These results indicate ALDH1A1 acts as a sink for CQ derivatives to enter the cell, localize to the lysosome, and produce ROS-mediated DNA damage. The DNA damage can be counteracted by intact HLTF/Pol eta-dependent TLS. Analysis of the TCGA found that the ALDH1A1 high and HLTF low phenotype is prevalent across 10 human cancers. In conclusion this study identifies a 2 gene signature that can be translated into an IHC-based assay to identify patients most likely to respond to lysosomal autophagy inhibitors. Citation Format: Shengfu Piao, Arabinda Samanta, Xiaohong Ma, Quentin W. Mcafee, Vito W. Rebecca, Meghan Buckley, Eric J. Brown, Phyllis A. Gimotty, Ravi K. Amaravadi. Expression pattern of ALDH1A1 and HLTF predicts sensitivity to lysosomal autophagy inhibitors in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1024.