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Early Loss of Mitochondrial Complex I and Rewiring of Glutathione

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Early Loss of Mitochondrial Complex I and Rewiring of Glutathione Early loss of mitochondrial complex I and rewiring of PNAS PLUS glutathione metabolism in renal oncocytoma Raj K. Gopala,b,c,d, Sarah E. Calvoa,b,c, Angela R. Shihe, Frances L. Chavesf, Declan McGuonee, Eran Micka,b,c, Kerry A. Piercec, Yang Lia,g, Andrea Garofaloc,h, Eliezer M. Van Allenc,h, Clary B. Clishc, Esther Olivae, and Vamsi K. Moothaa,b,c,1 aHoward Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; bDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; cBroad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; dDepartment of Hematology/Oncology, Massachusetts General Hospital, Boston, MA 02114; eDepartment of Pathology, Massachusetts General Hospital, Boston, MA 02114; fMolecular Pathology Unit, Massachusetts General Hospital, Boston, MA 02114; gDepartment of Statistics, Harvard University, Cambridge, MA 02138; and hDepartment of Medical Oncology, Dana–Farber Cancer Institute, Boston, MA 02215 Contributed by Vamsi K. Mootha, April 17, 2018 (sent for review July 6, 2017; reviewed by Ralph J. DeBerardinis and Robert A. Weinberg) Renal oncocytomas are benign tumors characterized by a marked electron transport chain (10, 11). Sequencing of nuclear DNA in accumulation of mitochondria. We report a combined exome, RO has revealed a low somatic mutation rate without recurrent transcriptome, and metabolome analysis of these tumors. Joint nuclear gene mutations, although recurrent chromosome 1 loss analysis of the nuclear and mitochondrial (mtDNA) genomes and cyclin D1 rearrangement have been reported (12, 13). reveals loss-of-function mtDNA mutations occurring at high vari- In this study, we report the results of profiling DNA, RNA, ant allele fractions, consistent with positive selection, in genes and metabolites in RO to investigate the molecular and bio- encoding complex I as the most frequent genetic events. A subset chemical basis of these tumors. We performed whole exome of these tumors also exhibits chromosome 1 loss and/or cyclin sequencing (WES) on a cohort (termed ROMGH) of 19 histo- D1 overexpression, suggesting they follow complex I loss. Tran- pathologically proven RO samples with matched normal kidney scriptome data revealed that many pathways previously reported tissue. We then validated our findings by analyzing off-target to be altered in renal oncocytoma were simply differentially mtDNA reads from a WES study by Durinck et al. (12) on a expressed in the tumor’s cell of origin, the distal nephron, com- cohort of RO patients (termed ROGEN) in which mtDNA se- pared with other nephron segments. Using a heuristic approach to quence analysis was not performed. We reanalyzed RNA-seq account for cell-of-origin bias we uncovered strong expression al- terations in the gamma-glutamyl cycle, including glutathione syn- from this latter study taking cell of origin into consideration to thesis (increased GCLC) and glutathione degradation. Moreover, spotlight tumor-associated gene expression changes. Finally, we the most striking changes in metabolite profiling were elevations performed mass spectrometry-based metabolomics on 10 addi- in oxidized and reduced glutathione as well as γ-glutamyl-cysteine tional fresh frozen RO samples from our cancer center to and cysteinyl-glycine, dipeptide intermediates in glutathione bio- identify differentially abundant metabolites. Our integrative synthesis, and recycling, respectively. Biosynthesis of glutathione analysis reveals what appear to be the earliest genetic and bio- appears adaptive as blockade of GCLC impairs viability in cells chemical changes in this benign tumor. cultured with a complex I inhibitor. Our data suggest that loss- of-function mutations in complex I are a candidate driver event Significance in renal oncocytoma that is followed by frequent loss of chromo- some 1, cyclin D1 overexpression, and adaptive up-regulation of Renal oncocytomas are benign kidney tumors with numerous glutathione biosynthesis. mitochondria. Here, we analyze the mitochondrial (mtDNA) and nuclear genomes of these tumors. Our analysis finds oncocytoma | complex I | mtDNA | glutathione | γ-glutamyl cycle mtDNA mutations in complex I (the first step in mitochondrial respiration) to be early genetic events that likely contribute to enal oncocytoma (RO) is a rare and benign tumor, ac- tumor formation. Since mtDNA mutations can lead to severe Rcounting for ∼5% of kidney tumors (1). RO is distinct from degenerative disorders, the cellular responses allowing renal renal cell carcinomas (RCCs), which fall into three main histo- oncocytoma cells to grow are important to consider. To prop- logic subtypes: clear cell (ccRCC), papillary (pRCC), and chro- erly understand authentic gene expression changes in tumors, mophobe (chRCC) (2–4). Molecular and histopathological we found it important to consider the gene expression pattern observations suggest that ccRCC and pRCC arise from the of the tumor’s cell of origin, the distal nephron. By doing so, proximal tubules of the nephron while chRCC and RO are distal we uncover alterations in glutathione synthesis and turnover MEDICAL SCIENCES in origin (5, 6). Recognition of RO is important as it can be that likely represent an adaptive metabolic response in cured surgically. renal oncocytoma. ROs are composed of large polygonal cells with granular eo- sinophilic cytoplasm, prominent nucleoli, and occasional nuclear Author contributions: R.K.G., E.O., and V.K.M. designed research; R.K.G., S.E.C., A.R.S., atypia (7). The tumor cells are filled with abundant mito- F.L.C., D.M., K.A.P., A.G., E.M.V.A., C.B.C., and E.O. performed research; R.K.G., S.E.C., “ ” A.R.S., E.M., Y.L., A.G., E.M.V.A., C.B.C., and E.O. analyzed data; R.K.G., S.E.C., and chondria and are termed oncocytic. Oncocytic neoplasms V.K.M. wrote the paper; and V.K.M. supervised research. occur throughout the body but are most common in the thyroid Reviewers: R.J.D., University of Texas-Southwestern Medical Center; and R.A.W., Mas- and kidney (8). In electron micrographs, the numerous mito- sachusetts Institute of Technology, The David H. Koch Institute for Integrative chondria of these tumors show morphological abnormalities, Cancer Research. including increased size and centrally stacked cristae (8). In the The authors declare no conflict of interest. kidney, mitochondrial accumulation is not only seen in RO but This open access article is distributed under Creative Commons Attribution-NonCommercial- also in the eosinophilic variant of chRCC (9). NoDerivatives License 4.0 (CC BY-NC-ND). Genetic lesions both in the nuclear and mitochondrial ge- 1To whom correspondence should be addressed. Email: [email protected]. nomes have previously been reported in RO. It has long been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. appreciated that oncocytic tumors, including RO, bear disruptive 1073/pnas.1711888115/-/DCSupplemental. mutations in mtDNA genes encoding complex I subunits of the Published online June 18, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1711888115 PNAS | vol. 115 | no. 27 | E6283–E6290 Downloaded by guest on October 3, 2021 Nuclear Genomes in ROMGH Fig. S1A) and no recurrent focal copy number events. As pre- To search for somatic alterations in nuclear and mitochon- vious studies detected cyclin D1 rearrangements leading to cyclin drial genomes, we performed WES on 19 high purity formalin- D1 overexpression (13), we assessed cyclin D1 via immunohis- fixed paraffin-embedded (FFPE) RO samples and adjacent tochemistry (IHC) (14), finding strong expression in 3/19, mod- MGH erate expression in 4/19, and absent expression in 12/19 samples normal kidney (RO ). Mean nuclear DNA coverage was (SI Appendix, Fig. S1B and Dataset S1). Chromosome 1 was lost 171× in normals and 212× in tumors with a relatively low non- ∼ in 3/7 samples showing cyclin D1 overexpression by IHC (Fig. synonymous mutation rate of 15 mutations per sample (Data- 1A). Together, 63% of RO tumors show chromosome 1 loss and set S1). MutSig analysis did not reveal any significantly mutated 37% show cyclin D1 overexpression. nuclear genes. We then evaluated tumors for karyotypic alterations in the Mitochondrial Genomes in ROMGH nuclear DNA. ROs were largely diploid with hemizygous loss of We next analyzed off-target mtDNA reads from WES to identify chromosome 1 or 1p in 12/19 patients (Fig. 1A and SI Appendix, somatic mtDNA variants in ROMGH (Methods). We obtained A ROMGH N=19 ROGEN N=16 B Complex I Complex I ND1 1 3 O ND5 RNR1 RNR2 ND1 ND2 C CO2 ATP8/6CO ND3 ND4L/4 ND5 ND6 CYTB mtDNA ND4 AAAAA CI ND4L TTTTT ND3 CCCCC ND2 GGGGG ND6 mtDNA CYB CIII CO1 N=19 CO2 CIV CO3 MGH ATP6 RO CV ATP8 chr 1 cyclin D1 Somatic mtDNA variant Nuclear DNA Copy number N=16 missense loss Loss of function Cyclin D1 Immunohistochemistry (LoF) GEN absent RO Variant allele fraction mtDNA moderate <40 40 50 60 70 80 90 strong Mutation consequence (color): Loss of function NonSilent Silent/NonCoding Variant allele fraction (size): > 80% 40-79% < 40% C 84% 69% 62% 11% 17% 9% LoF complex I variant allele fraction 90−100% 80−90% 70−80% 60−70% 50−60% % tumors 40−50% 5-40% none 0.2 0.4 0.6 0.8 1.0 0.0 ROMGH ROGEN chRCC chRCC pRCC ccRCC N=19 N=16 eosin. classic N=12 N=104 N=8 N=19 Fig. 1. Analysis of mitochondrial and nuclear genomes in RO. (A) Somatic mutations in mtDNA for RO samples (columns) across two cohorts (ROMGH and ROGEN). Somatic protein-altering mutations in the 13 mtDNA genes are shown with color (red, LoF; black, missense) and shade to indicate VAF (see legend). OXPHOS complexes are indicated (CI/III/IV/V: complex I/III/IV/V, respectively). Chromosome 1 and cyclin D1 status are shown (see legend).
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