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While the mutational and transcriptional landscapes of renal cell carcinoma (RCC) are well-known, the epigenome is poorly understood. We characterize the epigenome of clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC) by using ChIP-seq, ATAC-Seq, RNA-seq, and SNP arrays. We integrate 153 individual data sets from 42 patients and nominate 50 histology-specific master transcription factors (MTF) to define RCC histologic subtypes, including EPAS1 and ETS-1 in ccRCC, HNF1B in pRCC, and FOXI1 in chRCC. We confirm histology-specific MTFs via immunohistochemistry including a ccRCC-specific TF, BHLHE41. FOXI1 overexpression with knock-down of EPAS1 in the 786-O ccRCC cell line induces transcriptional upregulation of chRCC-specific genes, TFCP2L1 , ATP6V0D2 , KIT , and INSRR , implicating FOXI1 as a MTF for chRCC. Integrating RCC GWAS risk SNPs with H3K27ac ChIP-seq and ATAC-seq data reveals that risk-variants are significantly enriched in allelically-imbalanced peaks. This epigenomic atlas in primary human samples provides a resource for future investigation. The epigenomic landscape of renal cell carcinoma (RCC) remains to be explored. Here, integrative epigenomic analysis of primary human RCC samples and RCC GWAS risk SNPs identifies transcription-factor specific subtypes and enrichment of risk variants in allelically-imbalanced peaks.

Epigenetic dysregulation, including accumulation of Histone H3 lysine 27 acetylation (H3K27ac), is a hallmark of pVHL-deficient clear cell Renal Cell Carcinomas (ccRCCs). Using an in vivo positive selection ORF screen in poorly tumorigenic pVHL-proficient cells and mechanistic studies in pVHL-deficient cells, we discovered that the aspartate (Asp) and glutamate (Glu) transporter, SLC1A1/EAAT3, is a metabolic dependency in ccRCC. pVHL loss promotes Hypoxia Inducible Factor (HIF)-independent SLC1A1 expression via H3K27ac dysregulation. SLC1A1 inactivation, genetically or pharmacologically, depletes Asp/Glu-derived metabolites (e.g., Tricarboxylic acid cycle and nucleotide intermediates), impedes ccRCC growth, and sensitizes ccRCCs to anti-metabolite drugs (e.g., glutaminase blockers). In human tumors, higher SLC1A1 expression is associated with reduced immune infiltration, oncogenic metabolic programs, and advanced stage/metastatic disease. Finally, in ccRCC animal models, SLC1A1 inactivation diminishes lung metastasis and the outgrowth of established renal tumors. Altogether, our studies credential SLC1A1 as an actionable, HIF-independent, metabolic dependency in pVHL-deficient ccRCCs. Clear cell renal cell carcinoma (ccRCC) bears the hallmark loss of VHL but remains incurable. Here, the authors identify the SLC1A1 dicarboxylic amino acid transporter as an actionable, oncogenic, HIF-independent, metabolic dependency in VHL-deficient ccRCCs.

Key Clinical Messages Behçet's disease (BD) or syndrome is a chronic, recurrent, multisystem, inflammatory vasculitis disorder with findings of oral aphthous ulcers, genital ulcers, and uveitis. Gastrointestinal (GI) involvement can be the initial presentation as presented in this case. Behçet's disease (BD) or syndrome is a chronic, recurrent, multisystem, inflammatory vasculitis disorder of unknown etiology with classical findings of oral aphthous ulcers, genital ulcers, and ocular involvements including chronic anterior, intermediate, posterior, and even panuveitis. Gastrointestinal involvement in BD usually presents with chronic diarrhea, hematochezia as the disease affects ileocecal area which might be similar to presentation of inflammatory bowel diseases. Here, we report a case of undiagnosed BD who presented with chronic diarrhea for 4 months, leading to the diagnosis of BD and responded well to corticosteroid therapy. Pathology slides showing inflammatory changes.

  68Ga Prostate-specific membrane antigen (PSMA) is an increasingly popular radiopharmaceutical tracer in prostate cancer and is becoming increasingly researched in other cancers such as breast cancer, renal cell carcinoma, glioblastoma multiforme, among others. Cholangiocarcinoma is the second most common primary hepatic malignant tumor; it is an aggressive tumor with a 5-year survival rate of less than 5 %. We herein report a case of primary cholangiocarcinoma detected on 68Ga-PSMA PET-CT conducted as part of follow up for prostate cancer and confirmed by biopsy and immunohistochemistry.

Abstract Background Renal cell carcinoma (RCC) comprises a heterogeneous group of cancers with diverse molecular drivers and clinical behaviors. Despite this diversity, the majority of RCC cases are treated using therapeutic strategies developed for the most common subtype, clear cell RCC (ccRCC), due to limited understanding of subtype-specific biology. Translocation RCC (tRCC) is a rare and clinically aggressive RCC subtype defined by 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 other 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 The study employed an integrative multi-omics approach to dissect the cellular and molecular landscape of tRCC. The tRCC cohort consisted of 16 tumor samples from 15 patients, analyzed using single-nucleus RNA sequencing (snRNA-seq), single-nucleus ATAC sequencing (snATAC-seq), spatial transcriptomics, and T cell receptor (TCR) sequencing. Comparative analyses were performed against single-cell datasets from ccRCC samples, and bulk RNA-sequencing datasets were integrated for validation. Differential gene expression and chromatin accessibility analyses were conducted to identify tRCC-specific transcriptional programs and regulatory elements. The tumor cell-of-origin was inferred by comparing tumor transcriptomes to a single-cell atlas of normal human kidney tissue. Tumor-intrinsic transcriptional programs and intra-tumoral heterogeneity were delineated using non-negative matrix factorization methods. Finally, we characterized the immune landscape and cellular crosstalk within the TME by integrating snRNA-seq data, immune deconvolution of bulk RNA-seq data, TCR sequencing, and spatial transcriptomics. Results Our study revealed that tRCC and ccRCC likely share a common cell of origin in the VCAM1-positive proximal tubule cells but diverge significantly at the molecular level due to their distinct oncogenic drivers. Tumors from tRCC demonstrated profound differences in transcriptional pathways compared to ccRCC, including upregulation of oxidative phosphorylation, respiration, and lysosomal pathways driven by TFE3 fusions (Figure A). Notably, tRCC tumors showed higher activity of a pigmentation-related MITF-like transcriptional program (Figure B). Chromatin accessibility profiling revealed selective enrichment of regulatory elements associated with TFE3 and epithelial-mesenchymal transition (EMT), underscoring the unique epigenomic landscape of tRCC. Despite the genetically quiescent landscape, we identified six conserved oncogenic meta-programs shared across tRCC tumors, describing the intra-tumoral heterogeneity. Among them, EMT and proximal tubule programs were largely mutually exclusive and modulated by the level of TFE3 activity, with high TFE3 expression driving EMT and suppressing epithelial identity (Figure C). The tRCC tumor microenvironment was characterized by limited infiltration of cytotoxic CD8+ T cells compared to ccRCC, many of which displayed dysfunctional phenotypes. Additionally, the TME was enriched for immunosuppressive tumor-associated macrophages and matrix-associated fibroblasts. Spatial and ligand-receptor interaction analyses revealed several suppressive cellular interactions, including those involving COL1A1-ITGAV and SPP1-ITGAV axes, which may promote EMT and reinforce local immune suppression (Figure D). Conclusions This study defines the fusion-driven oncogenic programs, intratumoral heterogeneity, and a profoundly immunosuppressive TME of tRCC, which likely underlie its poor response to immunotherapy. Our findings provide a framework for rational therapeutic development, including strategies targeting fusion-mediated transcriptional programs and reprogramming the tumor microenvironment to overcome immune resistance.

Abstract Background Despite the success of immune checkpoint inhibitors (ICI) for the treatment of renal cell carcinoma (RCC), many patients do not receive durable clinical benefit. Therefore, an understanding of resistance to ICIs is critical for the treatment of this disease. Using scRNA-seq, we previously found increased tissue-resident ZNF683 + (Hobit) SLAMF7+ CD8+ exhausted T cells (T-exh-SLAMF7) in human RCC resistant to PD-1 blockade in the HCRN GU16-260 trial. A T-exh-SLAMF7 gene expression signature (GES) was associated with worse clinical outcomes with PD-1 blockade in multiple validation cohorts. Here, through bulk RNA-seq of RCC biospecimens from 90 patients enrolled in this trial, we identified higher tertiary lymphoid structures (TLS) in patients responsive to PD-1 blockade and investigated the interplay between TLS and T-exh-SLAMF7 in shaping therapeutic responses. Methods Bulk RNA sequencing was performed on tumor samples from 90 RCC patients enrolled in the HCRN GU16-260 clinical trial. GES scores were calculated for each sample by z-scoring all genes and then computing the average expression of the genes comprising each signature of interest. To validate TLS presence at the protein level, 19 FFPE RCC tumor samples were analyzed using a 7-plex multiplex immunofluorescence panel targeting DAPI, CD20, CD3, CD21, CD4, PD-1, and FOXP3. TLS were manually quantified based on the colocalization of B and T cell markers in organized structures. Patients were stratified by high versus low TLS and T-exh-SLAMF7 GES scores (≥ or < median) for downstream analyses of clinical response and progression-free survival. Results Patients with complete/partial response had a higher (≥ median) TLS GES score compared to patients with progressive disease (P = .0004). Similarly, high TLS signature scores were associated with improved progression-free survival (PFS; HR = 2.08, 95% CI: 1.28–3.4, P = .0032), indicating significantly higher risk of progression in patients with low TLS scores. We confirmed that tumors with a high TLS GES score had a higher number of TLS detected by multiplex immunofluorescence (P = .028). Finally, we analyzed the interplay between TLS and tissue-resident exhausted CD8+ T cells. We divided patients into four categories based on median split TLS and T-exh-SLAMF7 GES score values: TLS high SLAMF7 low (n = 28), TLS high SLAMF7 high (n = 15), TLS low SLAMF7 high (n = 28), and TLS low SLAMF7 low (n = 15). Patients with both high TLS and low T-exh-SLAMF7 GES scores had substantially improved PFS compared to all other patients (HR = 0.45, 95% CI: 0.26-0.79, P = .0052), with 60.7% PFS at 12 months compared to 25.9% in the remaining groups. Conclusions These findings support a paradigm where both high TLS and low T-exh-SLAMF7 cells are required for optimal response to PD-1 blockade in RCC. Ongoing studies will use spatial transcriptomics and functional assays to evaluate the interaction between TLS and T-exh-SLAMF7 cells and to define the roles of SLAMF7 and Hobit in regulating CD8+ T cell effector functions within the RCC tumor microenvironment. Note: Encore Presentation; recently published in Cancer Discovery (PMID: 39992403)

Abstract Background Epigenetic dysregulation, including accumulation of Histone H3 lysine 27 acetylation (H3K27ac), is a hallmark of pVHL-deficient clear cell Renal Cell Carcinomas (ccRCCs). H3K27ac is associated with transcriptional activation and its accumulation at cis-regulatory elements (eg, promoters and enhancers/super-enhancers) marks key oncogenes and regulators of cellular identity in many cancers. In ccRCC, specific alterations in H3K27ac have been linked to tumorigenesis and metastatic progression. Importantly, these earlier studies largely relied on the HIF2α-dependent 786-O cells (or their metastatic derivatives), perhaps, missing the importance of HIF-independent epigenetic programs. Altogether, we hypothesized that H3K27ac marks critical genes in pVHL-deficient ccRCCs that sustain tumorigenic and metastatic programs via both HIF-dependent and independent mechanisms. Methods Using an in vivo positive selection ORF screen in poorly tumorigenic pVHL-proficient cells and cell-based mechanistic studies in pVHL-deficient cells, we discovered that the aspartate (Asp) and glutamate (Glu) transporter, SLC1A1/EAAT3, is a metabolic oncogenic dependency in ccRCC. Results pVHL loss promotes HIF-independent SLC1A1 expression via H3K27ac dysregulation. SLC1A1 inactivation, using either genetic or pharmacological approaches, depletes Asp/Glu-derived metabolites [eg, Tricarboxylic acid (TCA) cycle and nucleotide intermediates], impedes ccRCC growth, and sensitizes ccRCCs to anti-metabolite drugs (eg, glutaminase blockers). In human tumors, higher SLC1A1 expression is associated with reduced immune infiltration, oncogenic metabolic programs, and advanced stage/metastatic disease. Finally, in ccRCC animal models, SLC1A1 inactivation diminishes lung metastasis and the outgrowth of established renal tumors. Conclusions Altogether, our studies credential SLC1A1 as a novel, actionable, HIF-independent, metabolic dependency in pVHL-deficient ccRCCs.

Abstract Background Tumor-associated macrophage (TAM) infiltration has been shown to modulate response to immune checkpoint inhibitors in various cancers, but its role in metastatic clear cell renal cell carcinoma (mccRCC) remains unclear. Here, we investigated the role of CD163+ TAMs as a potential determinant of clinical outcomes to first-line anti-PD-1 therapy (nivolumab) in patients with mccRCC enrolled in the HCRN GU16-260 trial. Moreover, as recent data suggest that the interaction between TAMs and tumor infiltrating lymphocytes (TILs) promotes T cell exhaustion, we explored the spatial relationship between CD163+ TAMs and CD8+ TILs in different states of exhaustion (ie, terminally exhausted (TE) and non-terminally exhausted (NTE) CD8+ TILs). Methods Pre-treatment tumor samples from 67 patients were analyzed by multiplex immunofluorescence to identify CD163+ TAMs, CD8+PD-1+TIM-3+ and/or LAG-3 + (TE CD8+), and CD8+PD-1+TIM-3−LAG-3− (NTE CD8+) TILs. Associations between the natural log of density of CD163+ TAMs with progression-free survival (PFS) and objective response rate (ORR) were assessed using univariable Cox and logistic regression models, respectively. An optimized cutoff was determined using minimum p value for ORR. For each tumor, the density of TE CD8+ TILs and the density of NTE CD8+ TILs were calculated within a 30 µm radius area centered on CD163+ TAMs (proximal area) and outside of this area (non-proximal area), using the ‘sf’ package within R software. The densities of TE and NTE CD8+ TILs were compared in proximal versus non-proximal areas across all tumor samples using the Wilcoxon signed-rank test. For each CD8+ TIL population (TE and NTE), the enrichment in proximity of CD163+ TAMs was assessed by calculating the difference in densities in proximal and non-proximal areas normalized by the density in the overall tumor area. The level of enrichment in TE CD8+ TILs versus NTE CD8+ TILs in proximity of CD163+ TAMs was compared using the Wilcoxon signed-rank test. Results The density of CD163+ TAMs, analyzed as a continuous variable was positively associated with ORR (OR: 2.21, 95% CI: 1.33 to 3.69, P = .002) and PFS (HR: 0.77, 95% CI: 0.61 to 0.97, P = .028). At an optimized cutoff, patients with high density of CD163+ TAMs (n = 34, 50.7%) had higher ORR (65% vs. 15%, P < .001) and longer median PFS (16.6 months, 95% CI: 5.5-32.9 vs. 5.5 months, 95% CI: 4.1-10.6, P = .009) compared to patients with low density of CD163+ TAMs (n = 33, 49.3%). The density of CD163+ TAMs was moderately correlated with the density of TE CD8+ TILs (Spearman correlation, r = 0.55) and weakly correlated with the density of NTE CD8+ TILs (r = 0.32). Proximity analysis showed that the density of TE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 123.3/mm2 vs. 37.2/mm2; P < .001). Similarly, the density of NTE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 127.2/mm2 vs. 66.8/mm2; P < .001). The level of enrichment in proximity of CD163+ TAMs was higher for TE CD8+ TILs compared to NTE CD8+ TILs (0.77 vs. 0.58; p = 0.0011). Conclusions High levels of CD163+ TAMs are associated with improved outcomes to anti-PD-1 therapy in mccRCC. In addition, exhausted CD8+ TILs preferentially localize in proximity of CD163+ TAMs in ccRCC tissues, supporting that TAM-T cell interactions are critical for driving T cell dysfunction. Taken together, our data are consistent with the hypothesis that the efficacy of PD-1 blockade may be in part mediated by reprogramming TAMs from a pro-tumorigenic to an anti-tumorigenic state.

Abstract Background Sarcomatoid differentiation of RCC (sRCC) is associated with poor survival. Recent studies showed marked response of sRCC to immune checkpoint blockade (ICB). While distinctive patterns of gene expression in sRCC have been identified, the gene regulatory programs and TFs that drive SD remain unknown. The aim of this study is to nominate TFs responsible for SD and to investigate their association with the clinical outcomes of patients with RCC. Methods Chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac – a histone modification associated with active regulatory elements – was performed on pathologically reviewed sRCC and non-sRCC samples collected at the Dana-Farber Cancer Institute. Regulatory elements that were differentially active between the two groups were identified based on levels of H3K27ac (Benjamini-Hochberg q<0.01, log-fold change (LFC) threshold=1). Enrichment of specific TF binding motifs at activated regulatory elements in sRCC was assessed using HOMER. Differential gene expression analysis of TFs was performed using DESeq2 on RNA-seq data from TCGA. A Mann-Whitney U test was performed on RNA-seq data from the IMmotion151 and Javelin Renal 101 clinical trials to compare mean expression level of TFs in transcript per million (TPM) in these trials. Patients with non-sRCC enrolled in the IMmotion151 trials were divided into quartiles based on gene expression levels of candidate TFs. Progression-free survival (PFS) was compared between non-sRCC patients stratified by expression quartiles as well as patients with sRCC using a multivariable Cox proportional-hazards model accounting for age and IMDC risk score. To validate these findings, a similar analysis was performed in the Javelin Renal 101 trial. Results We obtained high-quality H3K27ac ChIP-seq profiles for 9 sRCC and 17 non-sRCC samples. We identified 278 candidate regulatory elements with increased H3K27ac levels in sRCC vs. non-sRCC. These regulatory elements were enriched for nucleotide motifs bound by the TFs FOSL1 and E2F7. Differential expression analysis between 48 sRCC vs. 493 non-sRCC samples showed that FOSL1 and E2F7 were significantly upregulated in sRCC vs. non-sRCC (LFC=1.7, q=5e-11; LFC=1.8, q=1.3e-20; resp.). Mean TPMs of FOSL1 and E2F7 were significantly increased in sRCC vs. non-sRCC in IMmotion151 cohort and Javelin Renal 101 (all p<0.001). Among patients who received sunitinib, those with the highest quartile of FOSL1 and E2F7 expression showed significantly shorter PFS in IMmotion151 patients compared to patients with the lowest quartile of expression (HR=1.6, 95%CI=1.3-2.2, p=0.008 & HR=2.6, 95%CI=1.8-3.7, p<0.001; resp.). Furthermore, patients with highest quartile of expression showed similar PFS compared to patients with sRCC (p=0.56 and p=0.64; resp.). These results were validated in the sunitinib arm of the Javelin Renal 101 cohort (Figure). Figure: Kaplan-Meier curves of progression-free survival (PFS) in patients with in the sunitinib arm of Javelin Renal 101 by sarcomatoid differentiation and FOSL1 expression levels. Conclusions This is the first study to characterize the epigenomic landscape of sRCC by integrating ChIP-seq and RNA-seq data. Our findings implicated FOSL1 and E2F7 as transcriptional regulators of SD with prognostic relevance. These TFs seems to be associated with aggressive behavior in non-sRCC as well. Further studies are underway to functionally validate these results.

Translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer driven by TFE3 gene fusions, which act via poorly characterized downstream mechanisms. Here we report that TFE3 fusions transcriptionally rewire tRCCs toward oxidative phosphorylation (OXPHOS), contrasting with the highly glycolytic nature of most other renal cancers. Reliance on this TFE3 fusion-driven OXPHOS programme renders tRCCs vulnerable to NADH reductive stress, a metabolic stress induced by an imbalance of reducing equivalents. Genome-scale CRISPR screening identifies tRCC-selective vulnerabilities linked to this metabolic state, including EGLN1 , which hydroxylates HIF-1α and targets it for proteolysis. Inhibition of EGLN1 compromises tRCC cell growth by stabilizing HIF-1α and promoting metabolic reprogramming away from OXPHOS, thus representing a vulnerability for OXPHOS-dependent tRCC cells. Our study defines tRCC as being dependent on a mitochondria-centred metabolic programme driven by TFE3 fusions and nominates EGLN1 inhibition as a therapeutic strategy in this cancer. Li et al. show that the TFE3 driver fusion transcriptionally upregulates an OXPHOS programme in translocation renal cell carcinoma that renders this cancer vulnerable to reductive stress, induced by imbalance of reducing equivalents.