Data from Germline <i>USP36</i> Mutation Confers Resistance to EGFR-TKIs by Upregulating MLLT3 Expression in Patients with Non–Small Cell Lung Cancer

<div>AbstractPurpose:<p>Although somatic mutations were explored in depth, limited biomarkers were found to predict the resistance of EGFR tyrosine kinase inhibitors (EGFR-TKI). Previous studies reported N6-methyladenosine (m6A) levels regulated response of EGFR-TKIs; whether the germline variants located in m6A sites affected resistance of EGFR-TKIs is still unknown.</p>Experimental Design:<p>Patients with non–small cell lung cancer (NSCLC) with EGFR-activating mutation were enrolled to investigate predictors for response of EGFR-TKIs using a genome-wide-variant-m6A analysis. Bioinformatics analysis and series of molecular biology assays were used to uncover the underlying mechanism.</p>Results:<p>We identified the germline mutation <i>USP36</i> rs3744797 (C > A, K814N) was associated with survival of patients with NSCLC treated with gefitinib [median progression-free survival (PFS): CC vs. CA, 16.30 vs. 10.50 months, <i>P</i> < 0.0001, HR = 2.45] and erlotinib (median PFS: CC vs. CA, 14.13 vs. 9.47 months, <i>P</i> = 0.041, HR = 2.63). Functionally, the C > A change significantly upregulated USP36 expression by reducing its m6A level. Meanwhile, rs3744797_A (<i>USP36</i> MUT) was found to facilitate proliferation, migration, and resistance to EGFR-TKIs via upregulating MLLT3 expression <i>in vitro</i> and <i>in vivo</i>. More importantly, MLLT3 and USP36 levels are tightly correlated in patients with NSCLC, which were associated with prognosis of patients. Mechanistically, <i>USP36</i> MUT stabilized MLLT3 by deubiquitinating MLLT3 in nucleoli and consequently activating its downstream signaling (HIF1α and Snai). Furthermore, inhibition of MLLT3 alleviated <i>USP36</i> variant–induced EGFR-TKIs resistance in EGFR-mutant NSCLC.</p>Conclusions:<p>These findings characterized rs3744797 as an oncogenic variant in mediating EGFR-TKI resistance and tumor aggressiveness through deubiquitinating MLLT3, highlighting the variant as a predictive biomarker for EGFR-TKI response in NSCLC.</p></div>