MA17.02 Identify Vulnerable Pathways and Improve Treatment Outcomes in LKB1-Deficient Lung Tumors

Abstract

The LKB1 tumor suppressor is inactivated in about 20% of non-small cell lung cancers (NSCLC) by mutations. Cancer cells with LKB1 deficiency exert complex effects on signal transduction and transcriptional regulation, which may cause these cells more susceptible to certain therapies comparing to cells with intact LKB1 function. Phenformin, an antidiabetic medicine from the biguanides class, has shown activities against NSCLC. Phenformin as a single agent has been shown to reduce tumor burden and prolonged survival in Kras;Lkb1 compound mutant mice but not Kras;p53 mice, suggesting specific activities in tumor with LKB1 deficiency. Currently patients with unresectable locally advanced NSCLC are treated standardly with concurrent chemoradiotherapy followed by checkpoint inhibitor, durvalumab. In this project, we test treatment sensitivity to radiotherapy and/or phenformin in lung cancer cells with intact or deficient LKB1. Human lung cancer cell lines described below were used in (1) clonogenic survival assays as well as (2) generating tumor xenograft on nude mice for tumor growth delay experiments. A549, HCC15 and Calu-1 cell lines obtained from ATCC were cultured in RPMI1640 containing 5% FBS, without antibiotics. A549 cells (LKB1 deficient, TP53 WT and KRAS mutated) or HCC15 (LKB1 deficient, TP53 mutated and KRAS WT) were transfected with empty vector or WT LKB1 or LKB1-K78I plasmids; Calu-1 (LKB1 WT, KRAS mutated and p53 deleted) transfected with empty vector or LKB1 CRIPR KO were generated as described previously. A549 cells with transfected WT-LKB1 were significantly more resistant to ionizing radiation (IR) induced cell kill (8.7% survival at 8 Gy) comparing to cells transfected with empty vector (3.7%) or kinase-dead LKB1 genes (4.2%). Similarly, HCC15 cells with transfected WT-LKB1 are significantly more resistant to IR induced cell kill (7.5%) comparing to cells transfected with empty vector (4.1%) or kinase-dead LKB1 genes (3.4%). Calu-1 cells harbor WT LKB1, and it is significantly more resistant to IR induced cell kill (8.3%) comparing to their counterpart with LKB1 KO (Calu-1 transfected with LKB1 CRIPR KO) (4.1%). When A549 cells were pretreated with 30 μM phenformin prior to, during and after IR, there was no change in survival in cells transfected with WT LKB1; however there was significant further reduction in survival in cells transfected with empty vector (LKB1 deficient). Xenograft tumors were generated in nude mice with A549 cells with the above genetic alterations. There was significant further tumor growth delay in those with A549 with deficient LKB1 comparing to those with A549 with WT LKB1 gene add-back. This tumor growth delay was further enhanced when these mice were treated with oral phenformin prior to, during and after IR treatment, confirming the in vitro experimental results. Human lung cancer with deficient LKB1 are more sensitive to ionizing radiation in vitro and in vivo. This was regardless of the TP53 or KRAS mutation status. A549 cells with deficient LKB1 were also more sensitive to phenformin treatment. Phenformin treatment further sensitized LKB1 deficient lung cancer cells to IR. This suggested that LKB1 can serve as a predictive biomarker to triage patient treatments.