Translocation Renal Cell Carcinoma Research Articles

Exploring G-quadruplex structure in PRCC-TFE3 fusion Oncogene: Plausible use as anti cancer therapy for translocation Renal cell carcinoma (tRCC)

The TFE3 fusion gene, byproduct of Xp11.2 translocation, is the diagnostic marker for translocation renal cell carcinoma (tRCC). Absence of any clinically recognized therapy for tRCC, pressing a need to create novel and efficient therapeutic approaches. Previous studies shown that stabilization of the G-quadruplex structure in oncogenes suppresses their expression machinery. To combat the oncogenesis caused by fusion genes, our objective is to locate and stabilize the G-quadruplex structure within the PRCC-TFE3 fusion gene. Using the Quadruplex-forming G Rich Sequences (QGRS) mapper and the Non-B DNA motif search tool (nBMST) online server, we found putative G-quadruplex forming sequences (PQS) in the PRCC-TFE3 fusion gene. Circular dichroism demonstrating a parallel G-quadruplex in the targeted sequence. Fluorescence and UV-vis spectroscopy results suggest that pyridostatin binds to this newly discovered G-quadruplex. The PCR stop assay, as well as transcriptional or translational inhibition using real time PCR and Dual luciferase assay, revealed that stable G-quadruplex formation affects biological processes. Confocal microscopy of HEK293T cells transfected with the fusion transcript confirmed G-quadruplexes formation in cell. This investigation may shed light on G-quadruplex’s functions in fusion genes and may help in the development of therapies specifically targeted against fusion oncogenes, which would enhance the capability of current tRCC therapy approach.

Clinical Characteristics and Outcomes of TFE3-Rearranged/TFEB-Altered Renal Cell Carcinoma with Systemic Therapies, Including Tyrosine Kinase Inhibitors or Immune Checkpoint Inhibitors: An Observational Study

Purpose: <i>TFE3</i>-rearranged/<i>TFEB</i>-altered renal cell carcinoma (RCC) is a rare subtype of RCC. Due to its rarity, there is an unmet medical need for effective therapies in advanced settings. The study aims to investigate the clinical and histopathological characteristics of patients with microphthalmia transcription factor family/ transcription factor E (MiTF/TFE) translocation RCC and the clinical outcomes of systemic therapies, including tyrosine kinase inhibitors (TKIs) or immune checkpoint inhibitors (ICIs).Materials and Methods: This was a single-center, retrospective study. We identified 32 eligible patients among a total of 37 patients diagnosed with MiTF/TFE translocation RCC between January 2004 and September 2021, and the study included 9 patients who were treated with systemic therapies. We collected data on clinical characteristics, targeted sequencing, and clinical outcomes.Results: The median age of the 32 patients was 45.5 years. Histologically, 26 patients (81.3%) had <i>TFE3</i>-rearranged RCC, and only 1 patient (3.1%) had <i>TFEB</i>-altered RCC. Curative or cytoreductive nephrectomy was performed in all 27 patients (84.4%), and 4 patients (12.6%) were diagnosed with metastatic disease at the time of the initial diagnosis. Nine patients (28.1%) were treated with systemic therapy with TKIs, 2 (6.3%) of whom received simultaneous TKI and ICI treatment. The response to systemic therapy (TKI or ICI) and duration of response ranged from complete response to progressive disease. Excluding 1 patient who was treated with a TKI in the adjuvant setting, the overall response rate in 8 metastatic patients was 50% and the complete response rate was 37.5%. The median follow-up period was 29 months. The median progressionfree survival was 21 months, median overall survival was not achieved, and 2 deaths occurred.Conclusions: Our findings suggest that TKI for treatment for metastatic <i>TFE3</i>-rearranged RCC is efficacious, with an overall response rate of 50% and a median progression-free survival of 21 months.

Abstract 7580: The antihistamine terfenadine inhibits TFE3 dimerization, has antitumor activity and synergizes with tyrosine kinase inhibitors in translocation renal cell carcinoma

Abstract Translocation renal cell carcinoma is a subtype of kidney cancer characterized by gene fusions involving MiT transcription factor family, TFE3 and TFEB, with various gene partners. In general, these tumors do not respond to conventional therapies, which stresses the importance of developing new therapeutical approaches. To date, several TFE3 fusion gene partners have been identified. However, the mechanism driving TFE3 cooperation with various gene partners remains to be elucidated. We have previously shown the vulnerability of dimerization domain of multiple TFE3-fusion proteins, and we have screened the FDA approved drugs library LOPAC and a small molecule library (Microsource) to identify compounds that inhibit TFE3 dimerization. The antihistamine terfenadine was found to inhibit TFE3 dimerization and had antiproliferative activity in different models of tRCC, both in vitro and in vivo. Interestingly, our more recent data show that terfenadine reduced the amount of TFE3 in the nuclei. We have also observed enhanced antiproliferative activity of terfenadine in HEK293 cells overexpressing PRCC-TFE3, SPFQ-TFE3 or NONO-TFE3 compared to TFE3 alone or vector control, suggesting a targeted effect of terfenadine on cells driven by these TFE3-SF fusions. In addition, knock-down of SFPQ-TFE3 in HEK293 partially reversed this enhanced sensitivity to terfenadine. Cell proliferation and combination index analysis were performed in cell lines endogenously expressing the most common TFE3-fusion proteins cells to determinate the combination with different tyrosine kinase inhibitors. Immunofluorescence and confocal microscopy were performed to assess the impact on TFE3 fusion protein levels. The combination of terfenadine with sunitinib showed a synergistic effect in cells endogenously expressing PRCC-TFE3 (IC50=1.5µM; Fraction Affected > 0.05) and NONO-TFE3 (IC50=1.8µM; Fraction Affected<0.7) fusions. The combination of terfenadine with crizotinib showed a synergistic effect in cells expressing SFPQ-TFE3 fusion (IC50=0.7µM; Fraction Affected>0.7). The combination of terfenadine with dovitinib showed a synergistic effect in cells expressing PRCC-TFE3 fusion (IC50=1µM; Fraction Affected>0.4). These preliminary data suggest a different response to the combination with different TKIs. This observation underlines the importance of identifying the TFE3 partner gene for targeted therapy. Overall, our results suggest that targeting the dimerization domain along with different oncogenic pathways activated in tRCC cells may represent a new therapeutic approach for this disease. Citation Format: Ilaria Delle Fontane, Ricardo Cordova, Christopher Rupert, Sabrina Orsi, Abbas Jawadwala, David E. Heppener, Roberto Pili. The antihistamine terfenadine inhibits TFE3 dimerization, has antitumor activity and synergizes with tyrosine kinase inhibitors in translocation renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7580.

Abstract 1786: Metabolic rewiring induced by protein or calorie restriction provides unique therapeutic vulnerabilities in translocation and select clear cell renal cell carcinomas

Abstract While the approval of several combinations with immune checkpoint inhibitors have significantly improved the outcome of kidney cancer patients, advanced clear cell and non-clear cell renal cell carcinoma (RCC) remain a clinical challenge. There has been a longstanding interest in using dysregulated metabolism as a therapeutic target due to its association with the pathogenesis and progression of several cancer types. As one of these highly metabolic tumors, RCC has the potential to greatly benefit from strategies targeting tumor metabolism. Dietary interventions are an innovative adjunct therapy that has the potential to induce a wide range of anti-cancer effects by disrupting metabolism due to the role diet plays in providing tumors with macromolecules for energy metabolism. Unfortunately, tumor cell plasticity, intratumor heterogeneity, and a lack of understanding regarding metabolic vulnerabilities in tumors limit the efficacy of implementing these strategies and a fundamental gap in the field remains as to how basic metabolic requirements of the tumor can be targeted with specific dietary interventions. In this study, the inherent metabolic phenotypes of translocation RCC (tRCC) and clear cell RCC (ccRCC) were characterized by integrating in vivo metabolomics and transcriptomics data, assessing mitochondrial and glycolytic activity in vitro via Seahorse, and performing immunohistochemical analyses of patient samples. Interestingly, the respective mitochondrial and glycolytic dependencies in tRCC and ccRCC patient-derived xenograft (PDX) models appear to influence the response to nutrient deprivation as seen by in vitro assays conducted after amino acid or glucose restriction. Importantly, these results indicate that dietary interventions may be able to selectively target metabolic vulnerabilities in tRCC and ccRCC. Ongoing combination studies with either protein or calorie restriction and targeted therapies, such as standard of care receptor tyrosine-kinase inhibitors, suggest the benefit of using dietary interventions as an adjunct therapy in RCC. Overall, these studies reveal that selective dietary interventions alter the metabolic landscape of RCC and expose vulnerabilities that can be exploited with targeted therapy. As dietary interventions are a safe, affordable, and potentially well tolerated adjunct therapy, we anticipate that the results of these studies will provide the biologic and mechanistic support for translating this strategy to advanced RCC patients. Citation Format: Sabrina A. Orsi, Christopher Rupert, Sean Colligan, Musea Chang, Abbas Jawadwala, Ilaria Delle Fontane, Filomena de Nigris, Roberto Pili. Metabolic rewiring induced by protein or calorie restriction provides unique therapeutic vulnerabilities in translocation and select clear cell renal cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1786.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Oncogenic fusions in renal cell carcinoma.

475 Background: Detecting actionable or diagnostic fusions in solid tumors may significantly alter clinical decisions, including therapy selection. Some fusions have matched drugs approved for use in the patient’s cancer, or in another cancer. Others have published evidence of benefit with particular drugs but are not included in guidelines, and some may represent an inclusion criterion for clinical trials. While diagnostic fusions may not have associated therapies, they are cancer-defining and can lead to re-classification of tumors. Here we describe actionable and diagnostic fusions we have observed in renal cell carcinoma (RCC). Methods: Tumor samples from patients diagnosed with RCC were analyzed using the OncoExTra assay, which identifies somatic mutations and gene fusions through tumor-normal, whole-exome DNA and whole-transcriptome RNA sequencing. All actionable fusions, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer, are detectable using this assay. We examined samples from patients with RCC who had the OncoExtra assay performed between May 2018 and Mar 2023 and identified both actionable and diagnostic fusions. Results: Among 389 tumor samples from patients diagnosed with RCC, there were 11 (2.8%) actionable and 1 (0.3%) diagnostic fusion identified. The actionable fusions included 6 (1.5%) TFE3, 1 (0.3%) ALK, 1 (0.3%) EGFR, 1 (0.3%) ERBB4, 1 (0.3%) YAP and 1 (0.3%) FGFR2, and the diagnostic fusion was NAB2/STAT6. The 6 TFE3 oncogene fusions involved 4 different partner genes ( PRCC was the partner in 3 fusions). ALK, EGFR, ERBB and FGFR fusions are found across a variety of cancers, and all have matched FDA-approved drug therapies in at least one cancer type. For example, ALK fusions, including the observed EML4/ALK fusion, which is common in non-small cell lung cancer, have several matched ALK-inhibitor therapies. TFE3 (transcription factor binding to IGHM enhancer 3) gene fusions are a diagnostic genomic alteration in microphthalmia-associated transcription factor (MiT) translocation RCC, which is a rare but aggressive subtype of RCC. While TFE3 fusions do not have FDA-approved matched therapies in any cancer, they are known to result in PIK3K/AKT/mTOR and MET activation, suggesting inhibitors of these pathways may be considered in these patients. The NAB2/STAT6 diagnostic fusion is characteristic of solitary fibrous tumors, suggesting that the patient likely had this kind of tumor. Conclusions: Whole-transcriptome sequencing allowed the identification of actionable fusions with the potential to affect clinical decisions regarding therapy in 2.8% of RCC patient tumors. In addition, one patient was found to have a fusion that is characteristic of a solitary fibrous tumor, demonstrating the power of genomic profiling to aid diagnosis.

The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs

Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.

TFE3-Splicing Factor Fusions Represent Functional Drivers and Druggable Targets in Translocation Renal Cell Carcinoma.

TFE3-splicing factor fusions possess both transcription and splicing factor functions that remodel the transcriptome and spliceosome and can be targeted with dimerization inhibitors to suppress the growth of translocation renal cell carcinoma.

The Mutant Menage- Mit Family Translocation Renal Cell Carcinoma

Microphthalmia associated transcription factor (MiT) family translocation renal cell carcinoma is neoplasm which enunciates genetic fusions within members of MiT family of transcription factors, especially TFE3 gene and TFEB gene. Neoplasm frequently incriminates the adult population ≤ 35 years of age with an intense female predilection. Tumefaction exhibits a papillary, solid or alveolar pattern of tumour configuration and is composed of clear cells or non-cohesive, pseudo stratified, eosinophilic epithelial cells. Tumour cells are pervaded with voluminous cytoplasm and pleomorphic nuclei of advanced grade. Neoplastic cells are intensely immune reactive to TFE3 or TFEB, PAX8, cathepsin K, CD10, alpha methyacyl CoA racemase (AMACR), vimentin or E-cadherin. Tumour cells appear immune non-reactive to cytokeratin, epithelial membrane antigen (EMA), carbonic anhydrase IX(CAIX), CD45, calretinin and smooth muscle actin(SMA). MiT family translocation renal cell carcinoma requires segregation from neoplasms such as clear cell papillary renal cell carcinoma, clear cell renal cell carcinoma, epithelioid angiomyolipoma, chromophobe renal cell carcinoma or papillary renal cell carcinoma. Neoplasm may be appropriately alleviated by cogent manoeuvers as surgical extermination of the neoplasm.

Potential role of 68Ga-PSMA PET/CT in metastatic renal cell cancer: A prospective study

PurposeProstate-specific membrane antigen (PSMA), in addition to its utility in prostate cancer, is also an angiogenic imaging marker for hypervascular tumors like renal cell carcinoma (RCC). Our study aims to assess the potential role of 68Ga-PSMA-11 positron emission tomography (PET)/CT in metastatic RCC and compare it with contrast-enhanced computed tomography (CECT). MethodsBiopsy-proven RCC patients with known or suspected distant metastases who underwent 68Ga-PSMA-11 PET/CT for staging/restaging were prospectively recruited. Those patients who had undergone 18F-FDG PET/CT within six weeks of 68Ga-PSMA PET/CT were also included retrospectively for comparative analysis. A patient-based and lesion-based analysis was done to compare the lesion detection rates of CECT, 68Ga-PSMA-11 PET and 18F-FDG PET. PET-based quantitative parameters were also compared between both the PET modalities. Impact of baseline parameters on survival was assessed using Cox regression analysis. A p-value of < 0.05 was considered significant. ResultsThirty-seven patients with median age 60 years ± 13 years (range = 26–76 years) were included in the study. Twenty-seven patients had clear cell (cc) RCC, six had papillary RCC (pRCC), and one each had an eosinophilic variant of ccRCC, collecting duct RCC, translocation RCC and poorly differentiated RCC. 68Ga-PSMA-11 PET performed better in detecting marrow and equivocal bone lesions and worse in detecting liver lesions compared to CECT. 68Ga-PSMA-11 PET-based angiogenic tumor burden estimation using Total Lesion-PSMA (TL-PSMA) and PSMA-Total volume (PSMA-TV) had a prognostic impact on the survival of patients. 68Ga-PSMA-11 PET also detected more lesions and showed significantly higher SUVmax than 18F-FDG PET. Conclusion68Ga-PSMA-11 PET/CT performs better than CECT and 18F-FDG PET/CT in metastatic evaluation and has prognostic value in the management of clear cell RCC.

Renal cell carcinoma in the contralateral kidney with TFE3 gene translocation following chemotherapy for childhood nephroblastoma: A case report and literature review

Key Clinical MessageRenal cell carcinoma as a secondary malignant neoplasm is relatively rare; however, the possibility of secondary renal cell carcinoma following chemoradiotherapy for childhood nephroblastoma should be considered.AbstractThe occurrence of secondary renal cell carcinoma (RCC) following chemoradiotherapy for nephroblastoma is relatively rare, especially in microphthalmia transcription factor family translocation renal cell carcinoma. A 13‐year‐old Japanese male was referred to our department for treatment of a right kidney mass. The patient had undergone open left nephrectomy and adjuvant chemotherapy for nephroblastoma, 12 years before. Diagnostic imaging revealed a tumor in the right kidney and a lesion suspected to be metastasis in the left eighth rib. Chromophobe RCC or translocation RCC was suspected from the imaging pattern. TNM classification was cT1aN0M1, and the clinical stage was IV. Partial nephrectomy by robot‐assisted surgery for the right renal tumor and resection of the left eighth rib were performed. Pathologically, the renal tumor was diagnosed as translocation RCC, and the rib lesion demonstrated no evidence of malignancy. We are currently undergoing imaging follow‐up and the patient has been recurrence‐free for 15 months. In this study, we present a rare case of secondary translocation RCC after successful treatment of nephroblastoma.

Positive GPNMB Immunostaining Differentiates Renal Cell Carcinoma With Fibromyomatous Stroma Associated With TSC1/2/MTOR Alterations From Others.

Renal cell carcinoma with fibromyomatous stroma (RCCFMS) include ELOC/TCEB1 -mutated renal cell carcinoma (RCC) and those with TSC1/2 / MTOR alterations. Besides morphologic similarity, most of these tumors is known to be diffusely positive for carbonic anhydrase IX and cytokeratin 7 by immunohistochemistry. We previously showed strong and diffuse expression of GPNMB (glycoprotein nonmetastatic B) in translocation RCC and eosinophilic renal neoplasms with known TSC1/2/MTOR alterations. We retrospectively identified molecularly confirmed cases of TCEB1/ELOC -mutated RCC (7 tumors from 7 patients), and RCCFMS with alterations in TSC1/2/MTOR (6 tumors from 5 patients, 1 patient with tuberous sclerosis syndrome). In addition, we included 7 clear cell papillary renal cell tumors (CCPRCTs) and 8 clear cell RCC, as they can also present morphologic overlap with RCCFMS. Morphologically, RCCs with TSC1/2/MTOR alterations and those with TCEB1/ELOC mutations were indistinguishable and characterized by papillary, nested, or tubular architecture, with tumor cells with clear cytoplasm and low nuclear grade. By immunohistochemistry, cytokeratin 7 was positive in 5/7 (71%) of TCEB1/ELOC -mutated RCCs, 6/6 (100%) of RCCs with TSC1/2/mTOR alterations, and 7/7 (100%) of CCPRCTs ( P =not significant). Carbonic anhydrase IX was positive in 7/7 TCEB1/ELOC -mutated RCCs, 6/6 (100%) of RCCs with TSC1/2/MTOR alterations, and 7/7 (100%) of CCPRCTs ( P =NS). GPNMB was strongly and diffusely positive in all tumors with TSC1/2/MTOR alterations (6/6), while negative in all TCEB1/ELOC -mutated RCCs (0/6), or CCPRCTs (0/7) ( P =0.002). Two of 8 clear cell RCC showed focal weak staining, while 6/8 were negative. In conclusion, the results support the use of GPNMB to distinguish RCCFMS with TSC1/2/MTOR alterations from others with similar morphology.

Ultrasound diagnosis of renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion in children and adolescents.

Many studies have reported Xp 11.2 translocation renal cancer in radioimaging,but there is little literature on the evaluation of Xp11.2 translocation renal cell carcinoma by ultrasound. To investigate the ultrasonographic features and diagnostic value of renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion in children and adolescents. The clinical and ultrasonographic data of 10 patients with renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion confirmed by pathology in our hospital were analyzed retrospectively. The age ranged from 3 to 18 years old, including 7 males and 3 females. The tumor location, size, boundary, echo, hemorrhage, cystic change, calcification, blood flow, lymph node status and metastasis were mainly observed, and the results were compared with the pathological results. There were 10 masses in 10 cases of renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion, including 4 in the right kidney and 6 in the left kidney; the maximum diameter line is 5-23cm; 9 cases had clear mass boundary (90%); 9 masses (90%) showed mixed cystic and solid masses with high echo of solid components, and 1 mass (10%) showed huge multilocular cystic mass with multiple septations; necrosis and cystic changes were seen in all 10 masses (100%); calcification in 5 masses (50%); blood flow signals were seen in the solid components of the mass (100%). Renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion in children and adolescents are mostly large cystic and solid mixed echo masses, with high echo of solid components, and often accompanied by cystic changes and calcification. Its ultrasonic manifestations have certain characteristics. Color Doppler ultrasound has a certain diagnostic value for this disease.

Dissection of tumor-intrinsic and tumor-extrinsic features of MiT/TFE translocation renal cell carcinoma via single-cell RNA sequencing

Abstract Background Translocation renal cell carcinoma (tRCC) is a rare and aggressive subtype of renal cell carcinoma driven by oncogenic gene fusions involving MiT/TFE family transcription factors, most commonly TFE3. Currently, there are no molecularly tailored treatments for tRCC, and standard-of-care therapies utilized for other RCC subtypes are typically less effective in tRCC. Emerging data suggest that tRCC is molecularly distinct from more common RCC subtypes. However, an incomplete understanding of both tumor-intrinsic drivers and the tumor microenvironment (TME) features of tRCC presents barriers to developing effective therapeutics for this cancer. Methods We used single-nucleus RNA sequencing (snRNA) to profile ten samples of tRCC tumors and one adjacent normal tissue. Five of the samples also underwent multiome profiling with snATAC-seq while nine of the tumor samples underwent whole genome sequencing and RNA-sequencing. We preprocessed and obtained the raw fasta files using cellranger and extracted RNA counts and chromatin accessibility peaks using the Seurat/Signac algorithm. Scrublet was used to exclude data from droplets containing more than one cell by performing doublet detection and removal on gene-barcode matrices. Cells with fewer than 200 genes detected or more than 5% of counts attributed to mitochondrially-encoded transcripts were removed before across-sample integration. We also excluded genes detected in fewer than three cells across all samples. The combined cohort was analyzed using the MergeData function in Seurat, and cancer cells were selected based on known tRCC gene markers and inferred transcriptional copy number variations estimated via the inferCNV package and verified from WGS data. Non-tumor cell types were determined through manual annotation via known marker genes. We analyzed patterns of gene expression at the single-cell level using the Seurat V4 package and module score functions. Differential expression analysis comparing cells from different clusters or treatment exposure groups was performed using a two-sided Wilcoxon rank-sum test with Bonferroni FDR correction. Results Following quality control and integration, 71,124 single-cells were used for analysis (67,762 from tumor samples) – representing 43,214 tumor cells, 8,352 monocytes, 4,692 T cells, 4,614 endothelial/stromal cells, and normal kidney cells. The normal-adjacent sample represented multiple cell types from the normal kidney, including cells from distal connecting tubule, connecting duct, proximal tubule, thick ascending limb, podocytes, and endothelial cells. Analysis of chromatin accessibility profiles from snATAC-seq and snRNA-seq data of tumor cells was employed to identify unique cell states and a putative cell of origin for tRCCs. Tumor cell analysis also demonstrated intra- and inter-tumoral transcriptional variability in genetically similar tumor subclusters, each with distinct regulon activity. Immune subpopulation analyses revealed higher proportions of resting T cells in treatment-naive tRCC samples and higher proportions of progenitor-exhausted and terminal-exhausted populations in an immune checkpoint inhibitor (ICI) treated sample, indicating immune reprogramming in response to ICI in this mutationally quiet tumor. To further understand the role of, and factors permissive to, anti-tumor T cell responses in tRCC (which is characterized by a low tumor mutational burden) we explored the hypothesis that MiT/TFE fusions may constitute tumor neoantigens. We computationally identified fusion-associated neoantigens corresponding to these tRCC samples, as well as across a larger aggregate dataset of tRCC samples profiled by bulk RNA-Seq. We predicted binding affinities between fusion-associated neoantigens and HLA types and identified multiple peptide candidates with high HLA-binding affinity, which will be validated for their immunogenicity in vitro. Conclusions Overall, our results highlight tumor-intrinsic and tumor-extrinsic determinants of immunogenicity in tRCC and may guide the development of immunotherapeutic strategies with strong mechanistic rationale in tRCC. CDMRP DOD Funding: yes

Racial Disparities and Molecular Insights in Translocation Renal Cell Carcinoma: Advancing Understanding and Treatment Approaches.

This commentary remarks on the study conducted by Lu et al, which contributes to the existing knowledge on translocation renal cell carcinoma (tRCC) by examining how race may influence susceptibility to tRCC and how distinct transcriptomic profiles among different races may explain variations in tRCC prognosis.

Abstract PR013: An integrative genomics approach converges on a critical OXPHOS program driven by MiT/TFE fusions in translocation renal cell carcinoma

Abstract Translocation renal cell carcinoma (tRCC) is a rare and aggressive subtype of non-clear cell renal cell carcinoma (RCC) that represents 1-5% of kidney cancer in adults. Biologically, tRCC is driven by rearrangements that involve a member of the MiT/TFE transcription factor gene family, most commonly TFE3. There are currently no molecularly-targeted therapies specific to tRCC, owing in part to an incomplete mechanistic understanding of precisely how MiT/TFE gene fusions exert their oncogenic function. In prior work, we leveraged “histologic overlap” between tRCC and other RCC subtypes to identify tRCC cases from across multiple genomic, clinical trial, and retrospective RCC datasets, with the goal of defining the molecular landscape of this disease. Here, by analyzing pathways selectively activated in tRCC relative to other RCC subtypes in this aggregate dataset, we observed that tRCCs display strong enrichment for genes involved in oxidative phosphorylation (OXPHOS), reactive oxidative species (ROS) sensing, and the response to oxidative stress mediated by the transcription factor NRF2 (nuclear factor erythroid-derived2-like 2 [NFE2L2]). We performed chromatin immunoprecipitation and sequencing (ChIP-Seq) on a panel of tRCC and clear-cell RCC (ccRCC) cell lines, using antibodies against TFE3 as well as the active enhancer mark H3K27ac. We found that tRCC cell lines display a distinctive epigenomic profile characterized by activation of a host of genes involved in driving OXPHOS as well as NRF2 signaling. We further found that TFE3 fusions and NRF2 physically interact and genomically co-localize at multiple genes critical for mediating the response to antioxidant stress, suggesting that this metabolic phenotype is transcriptionally driven by TFE3 fusions. Genome-scale CRISPR screening in tRCC cell lines subsequently revealed tRCC-selective vulnerabilities in components of the OXPHOS machinery and in the NRF2 pathway, a distinction from most other kidney cancers in which aerobic glycolysis predominates. Overall, our genomic and functional studies implicate an essential transcriptional program driven by TFE3 fusions that converges on activation of the OXPHOS and NRF2 pathways. These molecular features provide mechanistic rationale for exploring the therapeutic targeting of OXPHOS and/or redox homeostasis pathways in tRCC. Citation Format: Jiao Li, Danny S. Gallant, Ananthan Sadagopan, Bingchen Li, Qingru Xu, Srinivas R. Viswanathan. An integrative genomics approach converges on a critical OXPHOS program driven by MiT/TFE fusions in translocation renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR013.

Abstract PR004: Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities

Abstract Introduction. Translocation Renal Cell Carcinoma (tRCC) is a distinct and aggressive molecular subtype of kidney cancer unusually prevalent in children and adolescents. tRCC has a poor prognosis with no specific therapies and is incurable in the metastatic setting. tRCC is characterized by chromosomal translocations involving three out of the four Microphthalmia (MiT) family transcription factors, TFE3, TFE3, and MITF. The most commonly involved gene is TFE3, which most often fuses with ASPSCR1. However, the molecular mechanisms by which ASPSCR1-TFE3 (and more broadly MiT/TFE fusion proteins) dictates tRCC tumorigenesis remain unclear and advances are hampered by the paucity of animal models. Herein, we sought to model tRCC and performed integrative genomic analyses to dissect molecular mechanisms underpinning tRCC tumorigenesis with the goal of translating this knowledge into therapeutic opportunities. Methods. We conditionally expressed a human ASPSCR1-TFE3 fusion in the Pax8 and Sglt2 lineages and performed phenotypic analyses as well as WES and RNAseq of emerging tRCC tumors. Similar genomic analyses of a human tRCC cohort were performed. The mutational landscape, cooperating pathways, and potential therapeutic targets were identified through comparative genomic and transcriptomic analyses. Emerging hypotheses were tested through in vitro and in vivo assays. Results. We show that human ASPSCR1-TFE3 driven by Pax8-Cre, a credentialled RCC driver, disrupted nephron development causing neonatal death, whilst Sglt2-Cre resulted in tRCC with complete penetrance and short latency. In both models, ASPSCR1-TFE3 induced characteristic morphological changes, loss of epithelial markers, and an EMT program, but only in the Sglt2-Cre model was proliferation observed. Given that Pax8-Cre, but not Sglt2-Cre¸ induce clear cell RCC (ccRCC), these data show that tRCC and ccRCC originate from different cell types. Comparative murine/human genomic analyses show that tumorigenesis by MiT/TFE fusion proteins is broadly cooperative (i.e. p53, ATM/ATR, Fanconi anemia, SWI/SNF, Hippo, and Wnt). Gene expression and functional studies implicate EPHA5 as well as Met/Ret in tRCC tumorigenesis. Antitumor/antiproliferative activity was observed with the therapeutic targeting of these pathways. Conclusion. This study provides insight into the tRCC mutational landscape, the cell of origin, and MiT/TFE-driven transcriptional programs offering several avenues for therapeutic development. Citation Format: Gopinath Prakasam, Akhilesh Mishra, Alana Christie, Jeffrey Miyata, Vanina T. Tcheuyap, Hui Ye, Quyen N. Do, Kevin B. Jones, Mai-Carmen Requena-Komuro, Ivan Pedrosa, Dinesh Rakheja, Payal Kapur, James Brugarolas. Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR004.

Abstract B020: A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma

Abstract TFE3 is a member of basic helix-loop-helix leucine zipper MiT transcription factor family and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of genes fusions, most of TFE3 fusions partner genes are linked to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, molecular cloning, next generation transcriptomics, FRET technology, mutagenesis, proteomics, and high-throughput high-content screening (HTHCS) we interrogated a series of oncogenic mechanisms of TFE3-SF-containing fusions, including spontaneous nuclear translocation, nuclear paraspeckle occupancy, enhanced in vitro proliferation and in vivo growth, transcriptome remodeling, alternative splicing reprogramming, and novel dimer partner recruitment. Molecular inhibition of TFE3-SF dimerization reverses its oncogenic activity and represents a potential new target for therapeutic intervention. Using HTHCS combined with FRET technology we screened FDA approved drugs library (LOPAC) and small molecule library (Microsource) and identified hits compounds which inhibit TFE3-SF dimerization. Hits compounds were validated in 2D and 3D models utilizing patient derived xenolines expressing TFE3-SF. Ouabain and terfenadine demonstrated decreased cell proliferation reduced spheroid growth and in vivo tumor growth. Overall, our results unmask synthetic vulnerabilities of TFE3-SF dimerization for novel therapeutic strategies in patients with this aggressive type of kidney cancer. Citation Format: Ilaria Delle Fontane, Nuri Damayanti, Christopher Rupert, David E. Heppner, Roberto Pili. A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B020.

Abstract IA027: New advances in the pathogenesis of renal cell carcinoma in tuberous sclerosis and Birt-Hogg-Dubé syndrome

Abstract Tuberous Sclerosis Complex (TSC) is caused by either TSC1 or TSC2 mutations, resulting in tumors of the brain, heart, skin, and kidney with hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). Renal cell carcinomas (RCC) in TSC have chromophobe-like histology features. In TSC2-deficient cells, mTORC1 activity toward its canonical (Raptor-dependent) substrates is increased, while the phosphorylation of non-canonical substrates such as TFEB is decreased, leading to nuclear localization and increased activity of TFEB. Remarkably, treatment with the mTORC1 inhibitor Rapamycin or downregulation of Rheb or Raptor shifts the balance of mTORC1 activity toward the non-canonical substrates in TSC2-deficient cells, leading to increased phosphorylation of TFEB and decreased TFEB activity. Knockout of Tfeb abolishes renal pathology in two mouse models of TSC, indicating that this substrate specificity is the critical mediator of renal disease in TSC. Together, our results indicate that the TSC protein complex controls a critical switch in mTORC1 substrate specificity upstream of Rheb and the Rags, and the effects of rapamycin in TSC are mediated by the paradoxical increased activity of mTORC1 towards the non-canonical target TFEB. These findings may shed light on the sporadic form of chromophobe RCC, which is derived from the renal intercalated cells and has several mysterious features including mitochondrial abundance, whole chromosome losses, and high levels of glutathione (50-100 times higher than matched normal kidney). We hypothesize that hyperactivation of TFEB and/or TFE3 may represent a “missing link” to chromophobe RCC, which highly express lysosomal genes and proteins, thereby connecting chromophobe RCC to TSC-associated and BHD-associated RCC and to translocation RCC. Citation Format: Elizabeth Henske. New advances in the pathogenesis of renal cell carcinoma in tuberous sclerosis and Birt-Hogg-Dubé syndrome [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA027.

AKR1B10 Is a New Sensitive and Specific Marker for Fumarate Hydratase-Deficient Renal Cell Carcinoma

Fumarate hydratase (FH)–deficient renal cell carcinoma (RCC) is a rare and distinct subtype of renal cancer caused by FH gene mutations. FH negativity and s-2-succinocysteine (2SC) positivity on immunohistochemistry can be used to screen for FH-deficient RCC, but their sensitivity and specificity are not perfect. The expression of AKR1B10, an aldo-keto reductase that catalyzes cofactor-dependent oxidation-reduction reactions, in RCC is unclear. We compared AKR1B10, 2SC, and FH as diagnostic biomarkers for FH-deficient RCC. We included genetically confirmed FH-deficient RCCs (n = 58), genetically confirmed TFE3 translocation RCCs (TFE3-tRCC) (n = 83), clear cell RCCs (n = 188), chromophobe RCCs (n = 128), and papillary RCCs (pRCC) (n = 97). AKR1B10, 2SC, and FH were informative diagnostic markers. AKR1B10 had 100% sensitivity and 91.4% specificity for FH-deficient RCC. The nonspecificity of AKR1B10 was shown in 26.5% of TFE3-tRCCs and 21.6% of pRCCs. 2SC showed 100% sensitivity and 88.9% specificity. However, nonspecificity for 2SC was evident in multiple RCCs, including pRCC, TFE3-tRCC, clear cell RCCs, and chromophobe RCCs. FH was 100% specific but 84.5% sensitive. AKR1B10 served as a highly sensitive and specific diagnostic biomarker. Our findings suggest the value of combining AKR1B10 and 2SC to screen for FH-deficient RCC. AKR1B10+/2SC+/FH− cases can be diagnosed as FH-deficient RCC. Patients with AKR1B10+/2SC+/FH+ are highly suspicious of FH-deficient RCC and should be referred for FH genetic tests.