Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin
Jianing Xu ... Emily H. Cheng, James J. Hsieh
Jianing Xu ... Emily H. Cheng, James J. Hsieh
Published September 1, 2016
Citation Information: J Clin Invest. 2016;126(9):3526-3540. doi:10.1172/JCI86120.
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Categories: Research Article Oncology

Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin

Abstract

Genomic studies have linked mTORC1 pathway–activating mutations with exceptional response to treatment with allosteric inhibitors of mTORC1 called rapalogs. Rapalogs are approved for selected cancer types, including kidney and breast cancers. Here, we used sequencing data from 22 human kidney cancer cases to identify the activating mechanisms conferred by mTOR mutations observed in human cancers and advance precision therapeutics. mTOR mutations that clustered in focal adhesion kinase targeting domain (FAT) and kinase domains enhanced mTORC1 kinase activity, decreased nutrient reliance, and increased cell size. We identified 3 distinct mechanisms of hyperactivation, including reduced binding to DEP domain–containing MTOR-interacting protein (DEPTOR), resistance to regulatory associated protein of mTOR–mediated (RAPTOR-mediated) suppression, and altered kinase kinetics. Of the 28 mTOR double mutants, activating mutations could be divided into 6 complementation groups, resulting in synergistic Rag- and Ras homolog enriched in brain–independent (RHEB-independent) mTORC1 activation. mTOR mutants were resistant to DNA damage–inducible transcript 1–mediated (REDD1-mediated) inhibition, confirming that activating mutations can bypass the negative feedback pathway formed between HIF1 and mTORC1 in the absence of von Hippel–Lindau (VHL) tumor suppressor expression. Moreover, VHL-deficient cells that expressed activating mTOR mutants grew tumors that were sensitive to rapamycin treatment. These data may explain the high incidence of mTOR mutations observed in clear cell kidney cancer, where VHL loss and HIF activation is pathognomonic. Our study provides mechanistic and therapeutic insights concerning mTOR mutations in human diseases.

Authors

Jianing Xu, Can G. Pham, Steven K. Albanese, Yiyu Dong, Toshinao Oyama, Chung-Han Lee, Vanessa Rodrik-Outmezguine, Zhan Yao, Song Han, David Chen, Daniel L. Parton, John D. Chodera, Neal Rosen, Emily H. Cheng, James J. Hsieh

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Characterization of kidney cancer–derived mTOR missense mutations. (A) A...

Figure 1

Characterization of kidney cancer–derived mTOR missense mutations.

(A) Amino acid positions of mTOR missense mutations identified in ccRCC (TCGA_KIRC and RECORD-3). Numbers in parentheses indicate number of patients with mTOR missense mutations versus total number of patients sequenced (2 patients in the RECORD-3 study carry 2 different mTOR mutations). (B) Diagram shows the domain structure of mTOR, its regulatory interaction partners (negative regulators in pink, positive regulators in green, and a dual-role regulator in gray), and the substrates of mTORC1 complex. The positions within mTOR that are involved in the interaction with the regulatory partners are highlighted below the domain structure. The thickness of the horizontal bar of RAPTOR-mTOR interaction indicates the relative binding affinity. mTOR missense mutations derived from ccRCC are mapped and color coded to summarize their respective effects on mTORC1 signaling (activating mutations in red). KD N, kinase domain N lobe; KD C, kinase domain C lobe. (C) Most of the mTOR mutants in the FAT and kinase domains induce higher levels of S6K phosphorylation (T389) than WT mTOR. 293T cells were transfected with vectors expressing HA-tagged S6K and Flag-tagged mTOR. Forty-eight hours later, cells were lysed and whole cell lysates were subjected to immunoblot analysis using the indicated antibodies. Densitometry of phosphorylated S6K versus HA-S6K from 3 independent experiments is shown (mean ± SEM, n = 3 independent experiments). *P < 0.1; **P < 0.05 (t test). The arrowhead denotes a cross-reactive band. (D) All mTOR mutants tested are sensitive to rapamycin treatment. Tetracycline-inducible HeLa cells expressing WT or mutant mTOR were treated with the indicated doses of rapamycin for 1 hour prior to immunoblot analysis using the indicated antibodies.