Abstract 2119: DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness

Abstract

Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance. Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay. Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism. Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC. Citation Format: Courtney Houchen, Janani Panneerselvam, Priyanga Mohandoss, Nathaniel Weygant, Randal May, Dongfeng Qu, Naushad Ali, Timothy Wang, Chinthalapally Rao, Michael Bronze, Parthasarathy Chandrakesan. DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2119.