LKB1 Loss Upregulates ALKBH5 and Contributes to Aggressive Phenotypes of KRAS Mutated Lung Cancer

Abstract

Background: Oncogenic KRAS mutations combined with loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the function, regulation and mechanism of m6A in this aggressive phenotypes remain largely unclear.Methods: The clinic-pathological role of m6A was evaluated in a cohort of lung cancer tissues and further validated by public databases and integrating bioinformatics analyses. We examined the upstream and downstream regulation of ALKBH5 (AlkB family member 5, an m6A demethylase) using quantitative real-time PCR, western blot, bisulfite genome sequencing, luciferase reporter assay, methylated DNA immunoprecipitation, m6A-RNA Immunoprecipitation (m6A-RIP) and m6A-seq data analysis. Results: We found that LKB1 loss decreased m6A levels and correlated with disease progression and poor survival for KRAS mutant lung cancer patients. The effect on m6A levels and the disease progression and survival was mediated by increasing levels of ALKBH5. LKB1 inactivation increased ALKBH5 transcription in multiple KRAS mutated tumor types, including colon, pancreas, and lung. Conversely, LKB1 overexpression decreased DNA methylation of the CTCF-binding motif on the ALKBH5 promoter, which enhanced CTCF binding and inhibited histone modifications, including H3K4me3, H3K9ac, and H3K27ac. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. Conclusions: Loss of LKB1 in KRAS mutated cancers promoted ALKBH5 transcription, decreased m6A levels, and increased the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS mutated lung cancer.