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N. El Kadi
MA 11 - Emerging Diagnostic/Biomarkers in NSCLC (ID 668)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Advanced NSCLC
- Presentations: 1
MA 11.10 - EGFR TKI Treatment Induces Active Deamination of 5-Methylcytosine and Leads to Acquired T790M Resistant Mutation (ID 9056)
11:00 - 12:30 | Author(s): N. El Kadi
Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinoma patients of Caucasian ethnicity and in 50% of Asian descent. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This leads to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, a similar mechanism of C>T mutation is seen in imatinib treated CML and GIST tumors. Our data suggests that the C>T mutation is an acquired event secondary to 5-methylcytosine deamination with Activation Induced Cytosine Deamination enzyme (AICDA).
PC9 is a lung adenocarcinoma cell line known to have an EGFR del19 activation mutation and can acquire T790M mutation from T790M-negative clone. We utilized droplet digital PCR (ddPCR) to detect T790M mutations. In addition, qPCR was used to assess the expression of AICDA and NFκB pathway before and after TKI exposure.
Sub clones of PC9 cell line with no evidence of T790M mutation by 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. Furthermore, using a UDG assay, we show that AICDA recognizes a CAC motif and can deaminate cytosine at position 2369. By mass spectrometry we established that 2369 cytosine is methylated. Deamination of 5-methylcytosine leads to thymine directly rather than uracil, explaining the C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirmed that NFκB binds AICDA promoter and induces its expression. Similarly, using a mouse xenograft model, EGFR TKI increases the expression of NFκB and AICDA; this is abrogated by concurrent use of an IKKα inhibitor. Knocking down AICDA by shRNA, decreases the rate of T790M development in PC9 cell lines after TKI exposure. Assessing AICDA expression in patients at baseline (n=4) and upon T790M mutation progression (n=4), there was a significant 20-fold increase in its expression.
Our data suggest that upon exposure to EGFR TKI, AICDA is overexpressed through an NFκB dependent pathway, causing the deamination of 5-methylcytosine to thymine, manifesting as T790M mutation and leading to TKI resistance. This indicates that T790M is acquired and its development could be targeted.
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