BackgroundMutations in MTOR and TSC1/2 can only explain part of variability in mTOR inhibitor response. Here, we performed a comprehensive molecular characterization of tumors with high sensitivity to these drugs to uncover novel mechanisms of response.MethodsChromophobe renal cell carcinoma (chRCC) tumors were analyzed by whole-exome sequencing. Rapamycin sensitivity, expression of mTOR pathway effectors, ubiquitylome analyses to identify USP9X substrates, p62 immunoprecipitation and autophagy assessment through immunofluorescence assays for p62 and LC3 were performed in USP9X depleted HeLa and 786-O cells for mechanistic investigation. ResultsWhole-exome sequencing of chRCC patients with high mTOR inhibitor sensitivity, uncovered USP9X deubiquitinase as the only mutated gene shared by these tumors. The clonal characteristics of the mutations, revealed by studying multiple primary and metastatic samples, together with the low USP9X mutation rate in unselected chRCC series, implied a causal link between USP9X and mTOR inhibitor sensitivity. The high sensitivity was recapitulated in vitro, and while no direct effect on mTORC1 was detected, an unbiased ubiquitylome analysis revealed p62 as a direct USP9X target. Bortezomib treatment, which also led to p62 ubiquitination, increased rapamycin effect. Finally, immunofluorescence assays for p62 and LC3 confirmed dysregulated autophagy in USP9X depleted cells, supporting a synthetic lethal interaction between rapamycin-induced autophagy via mTOR-axis and USP9X loss.ConclusionsOur study uncovers USP9X as a potential novel marker of sensitivity to mTOR inhibitors and suggests the inhibition of this gene as a clinically exploitable strategy able to increase sensitivity to these drugs through a novel p62 regulatory mechanism.