Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based.
Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression.
Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance.
Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer [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 931.