James Brugarolas

mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways.

Loss of PTEN function leads to activation of phosphoinositide 3-kinase (PI3K) signaling and Akt. Clinical trials are now testing whether mammalian target of rapamycin (mTOR) inhibition is useful in treating PTEN-null cancers. Here, we report that mTOR inhibition induced apoptosis of epithelial cells and the complete reversal of a neoplastic phenotype in the prostate of mice expressing human AKT1 in the ventral prostate. Induction of cell death required the mitochondrial pathway, as prostate-specific coexpression of BCL2 blocked apoptosis. Thus, there is an mTOR-dependent survival signal required downstream of Akt. Bcl2 expression, however, only partially restored intraluminal cell growth in the setting of mTOR inhibition. Expression profiling showed that Hif-1α targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition. These data suggest that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1α, and that clinical resistance to mTOR inhibitors may emerge through BCL2 expression and/or upregulation of HIF-1α activity.

TSC2 regulates VEGF through mTOR-dependent and -independent pathways

Inactivation of the TSC2 tumor suppressor protein causes tuberous sclerosis complex (TSC), a disease characterized by highly vascular tumors. TSC2 has multiple functions including inhibition of mTOR (mammalian target of Rapamycin). We found that TSC2 regulates VEGF through mTOR-dependent and -independent pathways. TSC2 loss results in the accumulation of HIF-1alpha and increased expression of HIF-responsive genes including VEGF. Wild-type TSC2, but not a disease-associated TSC2 mutant, downregulates HIF. Rapamycin normalizes HIF levels in TSC2(-/-) cells, indicating that TSC2 regulates HIF by inhibiting mTOR. In contrast, Rapamycin only partially downregulates VEGF in this setting, implying an mTOR-independent link between TSC2 loss and VEGF. This pathway may involve chromatin remodeling since the HDAC inhibitor Trichostatin A downregulates VEGF in TSC2(-/-) cells.

BAP1 loss defines a new class of renal cell carcinoma

The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes, including BAP1. The BAP1 protein, a nuclear deubiquitinase, is inactivated in 15% of clear cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation but not deubiquitination of monoubiquitinated histone 2A lysine 119 (H2AK119ub1). BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Notably, mutations in BAP1 and PBRM1 anticorrelate in tumors (P = 3 × 10−5), and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q = 0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q = 0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

Regulation of TFEB and V-ATPases by mTORC1

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V‐ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V‐ATPases. mTORC1 regulates V‐ATPase expression both in cells and in mice. V‐ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V‐ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis.

The p53 tumor suppressor limits cellular proliferation by inducing either G1 arrest or apoptosis, depending on the cellular context. To determine if these pathways are mechanistically distinct, we have examined the effects of different p53 mutants in p53 null primary mouse embryo fibroblasts. We chose this system as it is highly physiological and ensures that the interpretation of the results will not be confounded by the presence of endogenous p53 or oncoproteins which target p53. Using single cell microinjection assays for both G1 arrest and apoptosis, with loss‐of‐function and chimeric gain‐of‐function mutants, we have demonstrated that transcriptional activation is critical for both processes. Replacement of the p53 activation domain with that of VP16, or replacement of the p53 oligomerization domain with that of GCN4, reconstituted both G1 arrest and apoptosis activities. However, despite the importance of transcriptional activation in both processes, the target gene requirements are different. The p21 cyclin‐dependent kinase inhibitor, which has been shown to be a direct target of p53 and a component of the radiation‐induced G1 arrest response, is dispensable for oncogene‐induced apoptosis, suggesting that these two p53‐dependent transcriptional pathways are distinct.

Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation

BACKGROUND Clear-cell renal-cell carcinomas display divergent clinical behaviours. However, the molecular genetic events driving these behaviours are unknown. We discovered that BAP1 is mutated in about 15% of clear-cell renal-cell carcinoma, and that BAP1 and PBRM1 mutations are largely mutually exclusive. The aim of this study was to investigate the clinicopathological significance of these molecular subtypes and to determine whether patients with BAP1-mutant and PBRM1-mutant tumours had different overall survival. METHODS In this retrospective analysis, we assessed 145 patients with primary clear-cell renal-cell carcinoma and defined PBRM1 and BAP1 mutation status from the University of Texas Southwestern Medical Center (UTSW), TX, USA, between 1998 and 2011. We classified patients into those with BAP1-mutant tumours and those with tumours exclusively mutated for PBRM1 (PBRM1-mutant). We used a second independent cohort (n=327) from The Cancer Genome Atlas (TCGA) for validation. In both cohorts, more than 80% of patients had localised or locoregional disease at presentation. Overall both cohorts were similar, although the TCGA had more patients with metastatic and higher-grade disease, and more TCGA patients presented before molecularly targeted therapies became available. FINDINGS The median overall survival in the UTSW cohort was significantly shorter for patients with BAP1-mutant tumours (4·6 years; 95% CI 2·1-7·2), than for patients with PBRM1-mutant tumours (10·6 years; 9·8-11·5), corresponding to a HR of 2·7 (95% CI 0·99-7·6, p=0·044). Median overall survival in the TCGA cohort was 1·9 years (95% CI 0·6-3·3) for patients with BAP1-mutant tumours and 5·4 years (4·0-6·8) for those with PBRM1-mutant tumours. A HR similar to the UTSW cohort was noted in the TCGA cohort (2·8; 95% CI 1·4-5·9; p=0·004). Patients with mutations in both BAP1 and PBRM1, although a minority (three in UTSW cohort and four in TCGA cohort), had the worst overall survival (median 2·1 years, 95% CI 0·3-3·8, for the UTSW cohort, and 0·2 years, 0·0-1·2, for the TCGA cohort). INTERPRETATION Our findings identify mutation-defined subtypes of clear-cell renal-cell carcinoma with distinct clinical outcomes, a high-risk BAP1-mutant group and a favourable PBRM1-mutant group. These data establish the basis for a molecular genetic classification of clear-cell renal-cell carcinoma that could influence treatment decisions in the future. The existence of different molecular subtypes with disparate outcomes should be considered in the design and assessment of clinical studies. FUNDING Cancer Prevention and Research Institution of Texas and National Cancer Institute.

Targeting renal cell carcinoma with a HIF-2 antagonist

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α–HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Spectrum of diverse genomic alterations define non–clear cell renal carcinoma subtypes

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non–clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.

Molecular Genetics of Clear-Cell Renal Cell Carcinoma

Renal cell carcinoma of clear-cell type (ccRCC) is an enigmatic tumor type, characterized by frequent inactivation of the VHL gene (infrequently mutated in other tumor types), responsiveness to angiogenesis inhibitors, and resistance to both chemotherapy and conventional radiation therapy. ccRCC tumors exhibit substantial mutation heterogeneity. Recent studies using massively parallel sequencing technologies have implicated several novel driver genes. In VHL wild-type tumors, mutations were discovered in TCEB1, which encodes Elongin C, a protein that binds to VHL and is required for its function. Several additional tumor suppressor genes have been identified near the VHL gene, within a region that is frequently deleted in ccRCC on chromosome 3p: SETD2, BAP1, and PBRM1. Mutations in BAP1 and PBRM1 are largely mutually exclusive and are associated with different tumor biology and patient outcomes. In addition, the mTORC1 pathway is deregulated by mutations in MTOR, TSC1, PIK3CA, and PTEN in approximately 20% of ccRCCs. Mutations in TSC1, and possibly other genes, may predict for sensitivity to mTORC1 inhibitors. These discoveries provide insight into ccRCC development and set the foundation for the first molecular genetic classification of the disease, paving the way for subtype-specific therapies.

State of the science: An update on renal cell carcinoma

Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication. Mol Cancer Res; 10(7); 859–80. ©2012 AACR.