TFE3 Fusions Research Articles

Clear Cell Tumor With Melanocytic Differentiation and ACTIN-MITF Translocation: Report of 7 Cases of a Novel Entity.

Clear cell morphology is an uncommon finding in tumors. A subset of clear cell neoplasms also shows melanocytic differentiation, including clear cell sarcoma, PEComa, and some subtypes of renal cell carcinoma. A hallmark of these tumor types is the activation of a member of the MIT/TFE family of transcription factors, which includes MITF, TFE3, TFEB, and TFEC. Microphthalmia transcription factor (MITF is the master regulator of melanin synthesis, while TFEB plays a critical role in lysosome biogenesis. Cytogenetic translocations involving TFE3 and TFEB are now well described in multiple tumor types, but there has been little evidence to suggest similar regulation of MITF. Here we describe a series of 7 clear cell cutaneous neoplasms with melanocytic differentiation that are characterized by ACTIN-MITF gene fusions, either ACTB-MITF or ACTG1-MITF. The chromosomal breakpoints preserve MITF's dimerization and transcriptional activation domains, suggesting that these fusion proteins likely result in hyperactive MITF function, analogously to the previously reported TFE3 and TFEB fusions. Our findings indicate that MITF gene rearrangements may be key drivers of tumor pathogenesis and expand the spectrum of neoplasia associated with the MIT/TFE family.

Perivascular epithelioid cell tumors (PEComa) of the gynecologic tract.

PEComas of the female genital tract are rare mesenchymal neoplasms that are most common in the uterus, but also may occur in other gynecologic locations. As they morphologically and immunohistochemically resemble smooth muscle tumors, distinction between the two entities is often challenging, and may be aided by molecular analysis. Thus far, two distinct molecular groups-classic PEComas with TSC mutations and TFE3-translocation associated PEComas with TFE3 fusions have been described. Recognition of the first group is imperative as these patients may benefit from targeted therapy with mTOR inhibitors, if malignant. This review will focus on recognition of the morphologic and immunophenotypic features of PEComas, as well as the role of molecular testing in their diagnosis and treatment, analysis of the different algorithms to predict behavior, and differential diagnosis.

PRCC-TFE3 fusion-mediated PRKN/parkin-dependent mitophagy promotes cell survival and proliferation in PRCC-TFE3 translocation renal cell carcinoma

ABSTRACT TFE3 (transcription factor binding to IGHM enhancer 3) nuclear translocation and transcriptional activity has been implicated in PINK1-PRKN/parkin-dependent mitophagy. However, the transcriptional control governing the mitophagy in TFE3/Xp11.2 translocation renal cell carcinoma (TFE3 tRCC) is largely unknown. Here, we investigated the role and mechanisms of PRCC-TFE3 fusion protein, one of TFE3 fusion types in TFE3 tRCC, in governing mitophagy to promote development of PRCC-TFE3 tRCC. We observed and analyzed mitophagy, transcriptional control of PRCC-TFE3 on PINK1-PRKN-dependent mitophagy, PRCC-TFE3 fusions nuclear translocation, cancer cell survival and proliferation under mitochondrial oxidative damage in PRCC-TFE3 tRCC cell line. We found that nuclear-aggregated PRCC-TFE3 fusions constitutively activated expression of the target gene E3 ubiquitin ligase PRKN, leading to rapid PINK1-PRKN-dependent mitophagy that promoted cell survival under mitochondrial oxidative damage as well as cell proliferation through decreasing mitochondrial ROS formation. However, nuclear translocation of TFE3 fusions escaped from PINK1-PRKN-dependent mitophagy. Furthermore, we confirmed that PRCC-TFE3 fusion accelerated mitochondrial turnover by activating PPARGC1A/PGC1α-NRF1. In conclusion, our findings indicated a major role of PRCC-TFE3 fusion-mediated mitophagy and mitochondrial biogenesis in promoting proliferation of PRCC-TFE3 tRCC.

PRCC-TFE3 regulates migration and invasion of translocation renal cell carcinomas via activation of Drp1-dependent mitochondrial fission.

PRCC-TFE3 translocation renal cell carcinomas (tRCC) isa common subtype of TFE3 tRCCs in which TFE3 fusions areindicated as oncogenes to promote tumor development. PRCC-TFE3 fusions are often accumulated in the nucleus and related to poorer outcomes and higher stages (III/IV). In this study, we found that PRCC-TFE3 could positively regulate expression of both dynamin-related protein 1 (Drp1) and fission protein 1, and alter distribution of mitochondria, which could promote cell migration and invasion independent ofmatrix metalloproteinase-2 (MMP-2) and MMP-9. Together, our findings showed a new mechanism for PRCC-TFE3 tRCC cell migration and invasion by alteration of mitochondrial dynamics. Thus, targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing the progression of PRCC-TFE3 tRCC.

TFE3 Xp11.2 Translocation Renal Cell Carcinoma Mouse Model Reveals Novel Therapeutic Targets and Identifies GPNMB as a Diagnostic Marker for Human Disease.

Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. IMPLICATIONS: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Renal cell carcinoma (RCC) accounts for 2%–3% of all adultmalignancies, representing the seventh most common cancer inmen and the ninth most common cancer in women [1].Worldwide, there are ∼209000 new cases and 102000 deathsper year. The incidence of all stages of RCC has increased overthe past several years, contributing to a steadily increasing mor-tality rate per unit population. Active and passive cigarettesmoking is an established risk factor for RCC as well as hyper-tension. However, anti-hypertensive medications such as diure-tics are not independently associated with RCC development.RCC also appears to be more common in patients with obesity,end-stage renal failure, acquired renal cystic disease and tuber-ous sclerosis.Approximately 2%–3% of RCC are hereditary and severalautosomal dominant syndromes are described, each with a dis-tinct genetic basis and phenotype, the most common one beingVon Hippel Lindau disease.In recent years, many new genes associated with RCC havebeen reported (such as PBRM1, SETD2, BAP1). Their roles inpathogenesis and as prognostic biomarkers are currently underinvestigation.

TFE3 fusions escape from controlling of mTOR signaling pathway and accumulate in the nucleus promoting genes expression in Xp11.2 translocation renal cell carcinomas

BackgroundXp11.2 translocation renal cell carcinoma (tRCC) is mainly caused by translocation of the TFE3 gene located on chromosome Xp11.2 and is characterized by overexpression of the TFE3 fusion gene. Patients are diagnosed with tRCC usually before 45 years of age with poor prognosis. We investigated this disease using two tRCC cell lines, UOK109 and UOK120, in this study.MethodsThe purpose of this study was to investigate the pathogenic mechanism of TFE3 fusions in tRCC based on its subcellular localization, nuclear translocation and transcriptional activity. The expression of TFE3 fusions and other related genes were analyzed by quantitative reverse transcription PCR (qRT-PCR) and Western blot. The subcellular localization of TFE3 was determined using immunofluorescence. The transcriptional activity of TFE3 fusions was measured using a luciferase reporter assay and ChIP analysis. In some experiments, TFE3 fusions were depleted by RNAi or gene knockdown. The TFE3 fusion segments were cloned into a plasmid expression system for expression in cells.ResultsOur results demonstrated that TFE3 fusions were overexpressed in tRCC with a strong nuclear retention irrespective of treatment with an mTORC1 inhibitor or not. TFE3 fusions lost its co-localization with lysosomal proteins and decreased its interaction with the chaperone 14–3-3 proteins in UOK109 and UOK120 cells. However, the fusion segments of TFE3 could not translocate to the nucleus and inhibition of Gsk3β could increase the cytoplasmic retention of TFE3 fusions. Both the luciferase reporter assay and ChIP analysis demonstrated that TFE3 fusions could bind to the promoters of the target genes as a wild-type TFE3 protein. Knockdown of TFE3 results in decreased expression of those genes responsible for lysosomal biogenesis and other target genes. The ChIP-seq data further verified that, in addition to lysosomal genes, TFE3 fusions could regulate genes involved in cellular responses to hypoxic stress and transcription.ConclusionsOur results indicated that the overexpressed TFE3 fusions were capable of escaping from the control by the mTOR signaling pathway and were accumulated in the nucleus in UOK109 and UOK120 cells. The nuclear retention of TFE3 fusions promoted the expression of lysosomal genes and other target genes, facilitating cancer cell resistance against an extreme environment.

Abstract 3109: Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P<0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P<0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner. Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3109.

Commentary on: “Comprehensive molecular characterization of papillary renal-cell carcinoma.” Cancer Genome Atlas Research Network.: N Engl J Med. 2016 Jan 14;374(2):135–45.

BackgroundPapillary renal-cell carcinoma, which accounts for 15%–20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation, and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. MethodsWe performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. ResultsTypes 1 and 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into 3 individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase. ConclusionsTypes 1 and 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CpG island methylator phenotype in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least 3 subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.)

Abstract 4475: Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathway resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we sought to study the biology underlying oncogenesis in tRCC involving TFE3 fusion with genes associated with pre-mRNA splicing factor machinery; NONO, SFPQ and PRCC. Methods: To investigate the biology of different TFE3 fusion proteins, oncogenic phenotypes and oncogenic signaling were compared among cells with distinct endogenous expression of TFE3 fusions such as SFPQ-TFE3, NONO-TFE3, PRCC-TFE3. Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. TFE3 fusion transcripts were identified with RT-PCR. TFE3 fusion preferential DNA binding was assessed with ChIP-sequencing. Oncogenic phenotype assessment was done in 2D culture and two different 3D models; 1) Solid tumor model, in which cancer cell interaction with extracellular matrix and stromal cells, were represented in multiculture-spheroid system, 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid. Results: Dissimilar oncogenic phenotypes were seen among RP-R07, UOK-109 and UOK-146 in 2D and 3D culture. The presence of nuclear E-Cadherin was observed in all three cells model, with increasing level of nuclear expression in fusion partner dependent manner (P<0.05). Distinctive profiles of global phosphotyrosine and global phosphoserine were observed in cells model harboring different TFE3 fusions, despite common nuclear signal of TFE3. Nuclear expression of selected kinases and phosphokinases such as AKT, FAK, mTOR, VGEFR-2 were identified among TFE3 fusion cells with different level of expression (P<0.05). These results suggest distinct alteration of kinases nucleocytoplasmic shuttling regulation in tRCC cells (P<0.05), as compared to non TFE3 fusion cells. TFE3 ChIP-seq data in RP-R07 indicate novel DNA target involving metabolism and TGF-β signaling related genes. Conclusions: Our results indicate a distinct biology underlying tRCC oncogenesis in different TFE3 fusion models involving kinase reprograming and nucleocytoplasmic shuttling regulation. These differences also suggest the possibility of generating personalized treatments targeting specific TFE3 fusion genes associated with the spliceosome machinery. Citation Format: Nur P. Damayanti, Sreenivasulu Chintala, Ashley Orillion, Remi Adelaiye-Ogala, May F. Elbanna, Pete Hollenhorst, Roberto Pili. Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2017-4475

Comprehensive genomic profiling (CGP) of advanced papillary renal cell carcinoma (PRCC) to reveal distinctions from TCGA dataset.

4517 Background: Biologic characterization of PRCC by TCGA reveals alterations in MET in type 1 disease, and CDKN2Asilencing, SETD2 mutations and TFE3 fusions in type 2 disease. A limitation of TCGA data is that of 161 pts analyzed, 73% and 3% had M0 and M1 disease, respectively. We present the gene alterations (GA) identified in pts with advanced PRCC.Methods: FFPE tissue was obtained from 169 patients with PRCC. Diagnosis and PRCC subtyping was centrally confirmed by 2 board-certified pathologists. DNA was extracted and comprehensive genomic profiling (CGP) was performed (median coverage, 648X) in a CLIA-certified central laboratory. Results: Of 169 pts with advanced PRCC, 39 (23%) were type 1, 108 (64%) were type 2 and 22 (13%) were unclassified. 66 samples (39%) were from a metastatic site biopsy, and 103 were from the kidney (61%). An average of 2.4 GA/tumor were detected. In type 1 pts, commonly altered genes were MET (33%: 8 activating mutations, 5 amplifications), TERT (30%), CDKN2A/B (13%), and EGFR (8%). In type 2 pts, commonly altered genes were CDKN2A/B (18%), TERT (18%), NF2 (13%) and FH (13%); METGAs (5 mutations, 3 amplifications) were observed in 7.4% of type 2 pts. MET GA was significantly associated with type 1 PRCC (p = 0.0002) and NF2 GA and alterations in SWI/SNF complex genes were both significantly associated with type2 PRCC (p = 0.02 and p = 0.007, respectively). Overall, frequent alterations were found in genes involved in SWI/SNF complexes (25%), chromatin modification (23%) and cell cycle regulation (22%). RAS/RAF pathway (9%) and PI3K/mTOR pathway (8%). Notable differences with TCGA include higher frequencies of MET and NF2 GAs, association of GAs in the SWI/SNF with type 2 PRCC, and frequent CDKN2A/B alteration in both type 1 and type 2 disease. MET alteration was lower in metastases vs primary (type 1: 15% vs 38%; type 2: 4% vs 10%). Conclusions: In a cohort of PRCC patients slightly larger than the TCGA experience, key differences in CRGA frequency were observed. These likely underscore the marked difference in stage distribution between datasets. Results of this study may inform trials of relevant targeted therapy in metastatic PRCC.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma

BackgroundPapillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.MethodsWe performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.ResultsType 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2–antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).ConclusionsType 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.)

Identification of a novel PARP14-TFE3 gene fusion from 10-year-old FFPE tissue by RNA-seq.

Xp11 (TFE3) translocation renal cell carcinoma (RCC) is officially recognized as a distinct subtype of RCC in the 2004 WHO classification. This neoplasm is characterized by several chromosomal translocations between the TFE3-involving Xp11.2 breakpoint and various fusion partners. To date, five partner genes have been identified, that is, PRCC in 1q21, PSF in 1q34, ASPL in 17q25, CLTC in 17q23, and NONO in Xq12; and three additional translocations have been reported with no partner gene being defined: t(X;3)(p11;q23), t(X;10)(p11;q23), and t(X;19)(p11;q13). Here, we report the identification of a novel TFE3 fusion partner, PARP14 in chromosome band3q21. We used RNA-seq on a 10-year-old FFPE (formalin-fixed, paraffin-embedded) tissue sample, which carried t(X;3)(p11;q23) as detected in the original cytogenetic study. The fusion transcript connected the 5'-end of the first two exons of PARP14 to the 3'-end of five exons of TFE3, which was verified by reverse transcription PCR (RT-PCR) and Sanger sequencing. Similar to other TFE3 fusions previously reported, the predicted PARP14-TFE3 product retains the nuclear localization and DNA-binding domains of TFE3. This finding expands the list of TFE3 translocation partner genes and re-emphasizes the essential oncogenic role of TFE3 fusion proteins in this tumor. Our result also clearly demonstrated the feasibility of identifying chromosomal translocation by RNA-seq in clinical FFPE, which are easily accessible and associated with valuable clinical information. © 2015 Wiley Periodicals, Inc.

Combining integrated genomics and functional genomics to dissect the biology of a cancer‐associated, aberrant transcription factor, the ASPSCR1–TFE3 fusion oncoprotein

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.

TFE3 Fusions Activate MET Signaling by Transcriptional Up-regulation, Defining Another Class of Tumors as Candidates for Therapeutic MET Inhibition

Specific chromosomal translocations encoding chimeric transcription factors are considered to play crucial oncogenic roles in a variety of human cancers but the fusion proteins themselves seldom represent suitable therapeutic targets. Oncogenic TFE3 fusion proteins define a subset of pediatric renal adenocarcinomas and one fusion (ASPL-TFE3) is also characteristic of alveolar soft part sarcoma (ASPS). By expression profiling, we identified the MET receptor tyrosine kinase gene as significantly overexpressed in ASPS relative to four other types of primitive sarcomas. We therefore examined MET as a direct transcriptional target of ASPL-TFE3. ASPL-TFE3 binds to the MET promoter and strongly activates it. Likewise, PSF-TFE3 and NONO-TFE3 also bind this promoter. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and activation of downstream signaling in the presence of hepatocyte growth factor (HGF). In cancer cell lines containing endogenous TFE3 fusion proteins, inhibiting MET by RNA interference or by the inhibitor PHA665752 abolishes HGF-dependent MET activation, causing decreased cell growth and loss of HGF-dependent phenotypes. MET is thus a potential therapeutic target in these cancers. Aberrant transcriptional up-regulation of MET by oncogenic TFE3 fusion proteins represents another mechanism by which certain cancers become dependent on MET signaling. The identification of kinase signaling pathways transcriptionally up-regulated by oncogenic fusion proteins may reveal more accessible therapeutic targets in this class of human cancers.

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

Cloning of an Alpha-TFEB fusion in renal tumors harboring the t(6;11)(p21;q13) chromosome translocation.

MITF, TFE3, TFEB, and TFEC comprise a transcription factor family (MiT) that regulates key developmental pathways in several cell lineages. Like MYC, MiT members are basic helix-loop-helix-leucine zipper transcription factors. MiT members share virtually perfect homology in their DNA binding domains and bind a common DNA motif. Translocations of TFE3 occur in specific subsets of human renal cell carcinomas and in alveolar soft part sarcomas. Although multiple translocation partners are fused to TFE3, each translocation product retains TFE3's basic helix-loop-helix leucine zipper. We have identified the genes fused by the chromosomal translocation t(6;11)(p21.1;q13), characteristic of another subset of renal neoplasms. In two primary tumors we found that Alpha, an intronless gene, rearranges with the first intron of TFEB, just upstream of TFEB's initiation ATG, preserving the entire TFEB coding sequence. Fluorescence in situ hybridization confirmed the involvement of both TFEB and Alpha in this translocation. Although the Alpha promoter drives expression of this fusion gene, the Alpha gene does not contribute to the ORF. Whereas TFE3 is typically fused to partner proteins in subsets of renal tumors, we found that wild-type, unfused TFE3 stimulates clonogenic growth in a cell-based assay, suggesting that dysregulated expression, rather than altered function of TFEB or TFE3 fusions, may confer neoplastic properties, a mechanism reminiscent of MYC activation by promoter substitution in Burkitt's lymphoma. Alpha-TFEB is thus identified as a fusion gene in a subset of pediatric renal neoplasms.

PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors.

In papillary renal cell carcinomas the TFE3 transcription factor becomes fused to the PSF and NonO pre-mRNA splicing factors and most commonly to a protein of unknown function designated PRCC. In this study we have examined the ability of the resulting PRCC-TFE3 and NonO-TFE3 fusions to activate transcription from the plasminogen activator inhibitor-1 (PAI-1) promoter. The results show that only fusion to PRCC enhanced transcriptional activation, indicating that the ability to enhance the level of transcription from endogenous TFE3 promoters is not a consistent feature of TFE3 fusions. In investigations of the normal function of PRCC we observed that PRCC expressed as a green fluorescent fusion protein colocalizes within the nucleus with Sm pre-mRNA splicing factors. It was also found that endogenous PRCC is coimmunoprecipitated by antibodies that recognize a variety of pre-mRNA splicing factors including SC35, PRL1 and CDC5. Association with the cellular splicing machinery is therefore, a common feature of the proteins that become fused to TFE3 in papillary renal cell carcinomas.