VHL Inactivation Research Articles

Abstract 3024: HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney

Abstract The von Hippel Lindau (VHL) tumor suppressor gene is a classical tumor suppressor whose biallelic inactivation is a truncal event in clear cell renal cell carcinoma (ccRCC) evolution. VHL is best characterized as the recognition component of an E3 ubiquitin ligase complex that targets hypoxia inducible transcription factors (HIF1A and HIF2A) for conditional oxygen-dependent degradation. HIF activation following VHL inactivation is an early event in ccRCC progression. While the functions of HIF1A and HIF2A in ccRCC biology and progression have been studied, those in VHL-null non-neoplastic cells of the renal tubular epithelium (RTE) and their contribution to tumorigenesis have not been fully explored. Here, using a combination of lineage marking and single-cell RNA sequencing (scRNA-seq), we provide the first description of HIF1A- and/or HIF2A-dependent and -independent consequences of in vivo Vhl inactivation in the RTE with single-cell resolution. We have previously reported on the development of a novel mouse model designed to mark and track cells that have undergone Vhl inactivation in vivo. Using this model, we have shown that Vhl inactivation has extensive, cell-type specific effects on gene expression in cells of the RTE within 3 weeks of recombination. Furthermore, we have shown that over a course of 4-8 months after Vhl inactivation, Vhl-null cells undergo specific adaptive changes in gene expression that encompass genes not regulated early after Vhl inactivation. In the work reported here, we have coupled this mouse model to a tamoxifen-inducible RTE-specific Pax8-CreERT2 and conditional alleles for Hif1a and/or Epas1 (codes for HIF2A). We have analyzed ‘marked’ recombined cells at early (1-3 weeks) or late (4-8 months) timepoints after recombination and compared these cells to time-matched Vhl-null cells using immunohistochemistry, in situ RNA hybridization and scRNA-seq. We found that though both HIF isoforms were present in Vhl-null cells at the early timepoint, expression of genes regulated early after Vhl inactivation was affected more by Hif1a inactivation than Epas1 inactivation. In contrast, expression of genes that are regulated in Vhl-null cells over time was drastically impacted by Epas1 deletion but was unaffected by Hif1a deletion. Interestingly, most of these Epas1-dependent, time-dependent changes in Vhl-null cells involved genes that have not been identified as direct transcriptional targets of HIF. Collectively, our work suggests that HIF1A and HIF2A have temporally distinct contributions to gene expression in Vhl-null cells in the RTE. We are currently analyzing the cell-type specificity of these contributions and ascertaining their relevance to the adoption of ccRCC-associated gene expression profiles. Citation Format: Samvid Kurlekar, Joanna Lima, Ayslan B. Barros, Norma Masson, Sophia Zhai, Christopher W. Pugh, Julie Adam, Peter J. Ratcliffe. HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney [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 3024.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [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 6944.

Abstract 2096: A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy

Abstract Background: Ferroptosis is a novel form of cell death driven by iron-dependent lipid peroxidation, which disrupts cellular membranes resulting in immune-activating death. A variety of tumor types, particularly those associated with enhanced lipid and/or iron content, or which experience increased oxidative stress (including drug tolerant persister cells), are sensitive to ferroptosis, rendering ferroptosis a promising therapeutic strategy for cancer. However, currently available ferroptosis inducers show limited activity in vivo, suggesting a need for novel molecular targets to validate the clinical utility of ferroptosis induction. Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of VHL resulting in constitutive activation of HIF-1α and HIF-2α, which contribute to disease progression, including by promoting lipid accumulation that sensitizes ccRCC cells to ferroptosis. Methods: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α followed by structure-activity-relationship studies for validation. The molecular target of these inhibitors was identified using the Drug Affinity Responsive Target Stability assay, and target validation was confirmed using siRNA and NMR. In vitro efficacy was determined in a panel of cancer cell lines and in vivo anti-tumor efficacy was verified using mouse models of cancer. Results: We identified a novel molecule, KD061, which decreases HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation. These studies reveal a novel function of ISCA2 in extra-mitochondrial Fe-S assembly that may be limiting in cancer due to the increased need for these proteins in DNA replication and maintenance. Outside of ccRCC, a 92-cell line screen reveals a variety of tumor types sensitive to ISCA2 inhibition including melanoma and clear cell ovarian cancer. Treatment of tumor-bearing mice with KD061 resulted in > 65% tumor growth inhibition, associated with significantly increased lipid peroxidation, thus confirming ferroptosis in vivo, and with decreased HIF-1/2α. The anti-tumor efficacy of KD061 exceeded that of α-PD1 and was well tolerated at the therapeutic dose. Conclusions: We describe the first orally available inducer of ferroptosis with significant anti-tumor efficacy in ccRCC and other tumor types. Citation Format: Yangsook Song Green, Maria Ferreira dos Santos, Simone Ciofi-Baffoni, Xiaohui Liu, Mei Yee Koh. A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy [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 2096.

Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney.

Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.

Activation of HIF-1α/ACLY signaling axis promotes progression of clear cell renal cell carcinoma with VHL inactivation mutation

Objective: To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1α/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. Methods: Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1α in ccRCC cell lines. HeLa cells were treated with CoCl2 and hypoxia (1%O2) to activate HIF-1α and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. Results: Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1α expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1α in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1α in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1α in Caki-2 cells inhibited the mRNA and protein levels of ACLY (P<0.001 for all). CoCl2 and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α (P<0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1α could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (P<0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1α and ACLY. The depletion of HIF-1α could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1α knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (P<0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (P<0.001). Conclusions: VHL mutation-mediated HIF-1α overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.

Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [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 A011.

Abstract PR002: Injury-associated transcriptional state transitions in early renal carcinogenesis

Abstract Most epithelial cells with cancer driver mutations fail to form tumors. This has been particularly well established by genetic analysis of the renal epithelium, where cells with tumor-initiating mutations in the von Hippel-Lindau tumor suppressor (VHL) only rarely develop into clear cell renal carcinomas (ccRCC). The progression of VHLmutant cells to malignancy can take decades, offering a wide window of opportunity for preventative intervention. However, the cellular programs that facilitate the malignant transformation of VHL null ccRCC precursor clones remain elusive. Using a genetically engineered mouse model that allows conditional inactivation of Vhl and Polybromo 1 (Pbrm1), the second most commonly mutated ccRCC gene after VHL, in the renal epithelium of adult mice, we have analyzed the phenotypic evolution of the earliest stages of renal carcinoma formation over months at single cell resolution. Transcriptomic analysis by single cell RNA-sequencing (scRNA-seq) revealed that the acute activation of the hypoxia signaling response, a predicted consequence of genetic Vhl inactivation, is followed by distinct transcriptomic state transitions that precede the emergence of early carcinoma cells. These transitions are associated with constitutive activation of transcriptional programs that normally mediate acute and transient responses to renal injury, and are progressively activated at a low level in aging normal renal epithelium. Analysis of DNA accessibility by the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) identified widespread opening of chromatin regions in association with progression towards malignancy, revealing a chromatin footprint of tissue repair pathway activation. Analysis of human ccRCC samples, including early-stage cases from VHL mutation carriers, validated our observations at the transcriptomic and protein level. Furthermore, genetic perturbation experiments in transplantable ccRCC models demonstrated the functional role of the renal injury pathway in tumor re-initiation. These results are compatible with a model whereby activation of the renal epithelial injury pathway facilitates the early progression of mutant ccRCC precursor clones towards malignancy. That the same repair pathway is increasingly activated over time in normal renal epithelium points at a link between ageing and ccRCC development. An understanding of the causes of ageing-associated renal damage could provide opportunities for early intervention in ccRCC, especially in high-risk individuals. Citation Format: Shoko Hirosue, Jianfeng Ge, Leticia Castillon, Paulo Rodrigues, Dóra Bihary, Alyson Speed, Ludovic Wesolowski, Veronica Caraffini, Anne Y. Warren, Grant D. Stewart, Sarah J. Welsh, Thomas J. Mitchell, Athena Matakidou, Fatemeh S. Seyednasrollah, Shamith A. Samarajiwa, Sakari Vanharanta. Injury-associated transcriptional state transitions in early renal carcinogenesis [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 PR002.

Abstract IA001: Evidence for a ‘pathway tuning’ model of VHL-associated renal carcinoma

Abstract In clear cell renal carcinoma (RCC), the hypoxia inducible factor (HIF) transcriptional response is unphysiologically activated by mutations that impair or inactivate the von Hippel-Lindau tumour suppressor, which normally acts as a ubiquitin E3 ligase promoting the oxygen dependent proteolysis of hypoxia inducible factor (HIF). The lecture will review evidence for pro- and anti-tumourigenic actions arising from different components of HIF pathway activation in the setting of VHL-associated RCC. This includes evidence from genome-wide association studies (GWAS) that demonstrate a striking association of GWAS signals with cis-acting sequences in the HIF transcriptional system. This supports a ‘pathway tuning’ model in which multiple small selective effects are important in enabling cancer progression in the face of unphysiological activation of very extensive biological pathways. To assess this hypothesis a new experimental model has been developed, which enables the precise tracking of Vhl-inactivated cells. Results on the temporal and spatial effects of Vhl inactivation in kidneys will be presented. Citation Format: Peter J. Ratcliffe. Evidence for a ‘pathway tuning’ model of VHL-associated renal 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 IA001.

Abstract PR005: An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas

Abstract Renal cell carcinoma (RCC) tumors harbor various layers of intra-tumor heterogeneity at the genetic, transcriptomic, and cellular levels. A series of recent studies have underlined the importance of charting this cellular variability to understand the cell of origin of various RCCs and the mechanisms that underlie their response to therapy. However, the regulatory factors driving intra-tumor heterogeneity in RCCs, the role of this heterogeneity in driving metastasis, and its association with clinical outcomes are mostly unknown. To address these questions, we have generated a cellular map of transcriptional heterogeneity in RCCs, which consists of &amp;gt;100,000 single-cell expression profiles from six patient-derived xenografts (PDX) models, including models derived from primary and brain metastatic tumors, as well as ten primary and six metastatic patient tumor tissues. We also developed a computational method for Gene Expression Decomposition and Integration (GEDI) that enables seamless integration of single-cell transcriptomic data across heterogeneous cancer samples while providing interpretable axes of variation. Using GEDI, we performed a fine-grained analysis of the heterogeneity of cancer cells and found that, despite patient-specific differences, common sources of intra-tumor heterogeneity exist across samples, driven by variable activity of pathways such as TNF-α/NF-κB signaling and oxidative phosphorylation (OxPhos). Analysis of data from our PDX models confirmed that this heterogeneity was stable and reproducible. A major source of intra-tumor heterogeneity was hypoxia signaling, even in VHL-deficient clear cell RCC samples (ccRCC), challenging the traditional view that ccRCC cells have a uniform pseudo-hypoxic status due to VHL inactivation. Regulatory network activity projection allowed us to disentangle the contributions of Hypoxia Inducible Factors HIF1A and HIF2A, revealing their divergent regulatory programs: while HIF1A is associated with cell-cycle and proliferation signatures, the activity of HIF2A correlates with epithelial-mesenchymal transition (EMT) within tumors, which we validated by RNA sequencing of HIF1A and EPAS1 knockdown in VHL-negative cells. Next, we used GEDI to study the cell state changes that occur during metastasis in both neoplastic and non-neoplastic tumor-infiltrating cells. By examining the differences in gene expression patterns between primary and metastatic samples, we identified various pathways that are transcriptionally activated during this transition, including EMT and Oxphos. We also uncovered distinct subpopulations of cancer cells with high gene expression changes associated with a metastatic profile. Analysis of the ligand-receptor interactions in the tumor microenvironment revealed that interactions from malignant cells to immune cells were over-represented in metastatic tumors. Overall, our cellular atlas has uncovered a complex continuum of cell states in RCCs, highlighting various drivers of the intra-tumor heterogeneity and establishing various cell states associated with metastasis. Citation Format: Ariel Madrigal, Minjun Kim, Adrien Osakwe, Tianyuan Lu, Zohreh Mehrjoo, Elham Moslemi, Rick Farouni, Larisa Morales-Soto, Yu Chang Wang, Matthew Dankner, Haig Djambazian, Kevin Petrecca, Jonathan Spicer, Fadi Brimo, Peter Siegel, Morag Park, Jiannis Ragoussis, Simon Tanguay, Yasser Riazalhosseini, Hamed S. Najafabadi. An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas [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 PR005.

VON HIPPEL-LINDAU DISEASE, DESCRIPTION, GENETICS, MOLECULAR BASIS, CLASSIFICATION AND MANIFESTATIONS OF THE DISEASE

Introduction: Von Hippel-Lindau disease (VHL) is autosomal dominant tumor syndrome that debuts mostly in young adults , patients with this disease are linked to the triggering of various types of benign and malignant neoplasms in multiple locations, systems and organs, in particular affecting more the nervous system and other internal organs. Approximately this tumor syndrome shows an incidence rate of 1 in 36,000 live births with a penetrance greater than 90%. The molecular basis of VHL disease is the impairment of VHL protein function and the consequent clustering of hypoxia-inducible factors with subsequent consequences on cell differentiation and metabolism. Objective: to present current information related to Von Hippel-Lindau disease, description, genetics, molecular basis, classification and manifestations of the disease. Methodology: a total of 33 articles were analyzed in this review, including review and original articles, as well as clinical cases, of which 26 bibliographies were used because the other articles were not relevant to this study. The sources of information were PubMed, Google Scholar and Cochrane; the terms used to search for information in Spanish, Portuguese and English were: Von Hippel-Lindau, VHL, tumor suppressor gene, pheochromocytoma, hemangioblastomas. Results: VHL has an incidence of approximately 1 in 36,000 live births, with a penetrance of over 90%. These present tumors are initiated by inactivation of biallelic VHL and are related to pathologic activation of hypoxic gene response pathways. Conclusions: Von Hippel-Lindau disease is an autosomal dominant disorder that is generated by mutations in the VHL tumor suppressor gene. Within the field intrafamilial variation may evidence correctly shaped genotype-phenotype connections for renal cancer and pheochromocytoma risks. Visceral cysts (renal, pancreatic and epididymal) are frequent, however organ function involvement is rare. They usually occur with hemangioblastomas of the central nervous system and retina, as well as renal cancers. Unusually it includes non-functioning pancreatic endocrine cancers, adrenal and extra-adrenal pheochromocytomas, endolymphatic sac tumors, as well as head and neck paragangliomas. KEYWORDS: Von Hippel-Lindau, VHL, gene, suppressor, tumor, pheochromocytoma, hemangioblastomas

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Diagnostic and Prognostic Biomarkers in Renal Clear Cell Carcinoma.

Renal clear cell carcinoma (ccRCC) comprises over 75% of all renal tumors and arises in the epithelial cells of the proximal convoluted tubule. Molecularly ccRCC is characterized by copy number alterations (CNAs) such as the loss of chromosome 3p and VHL inactivation. Additional driver mutations (SETD2, PBRM1, BAP1, and others) promote genomic instability and tumor cell metastasis through the dysregulation of various metabolic and immune-response pathways. Many researchers identified mutation, gene expression, and proteomic signatures for early diagnosis and prognostics for ccRCC. Despite a tremendous influx of data regarding DNA alterations, gene expression, and protein expression, the incorporation of these analyses for diagnosis and prognosis of RCC into the clinical application has not been implemented yet. In this review, we focused on the molecular changes associated with ccRCC development, along with gene expression and protein signatures, to emphasize the utilization of these molecular profiles in clinical practice. These findings, in the context of machine learning and precision medicine, may help to overcome some of the barriers encountered for implementing molecular profiles of tumors into the diagnosis and treatment of ccRCC.

Abstract 939: A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney

Abstract Clear cell renal cell carcinoma (ccRCC) is driven by biallelic inactivation of von Hippel Lindau (VHL) tumour suppressor gene. While ccRCC genesis has been associated with several signaling and metabolic pathways, the exact process by which VHL inactivation becomes tumorigenic remains unknown. We surmised that observing the behaviour and gene expression profiles of Vhl-inactivated cells in vivo with spatiotemporal resolution would help define cell types and conditions in which this genetic lesion is on balance tumorigenic. Here we report a novel lineage marking model of in vivo Vhl inactivation and describe the immediate consequences of Vhl loss in different nephron segments at cellular resolution. Studying Vhl inactivation with single-cell resolution requires a system in which Vhl-inactivated cells can be accurately identified. We developed a mouse model in which Vhl recombination is structurally linked to the expression of a tdTomato reporter cassette. This allows recombined cells to be visualised and isolated based on tdTomato expression. We coupled this model with the renal tubular epithelium (RTE) restricted Pax8-CreERT2 to induce biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl and observed the immediate fates of affected cells. Vhl is thought to be haplosufficient; thus, the wt/KO mice act as lineage-marked Vhl-competent controls to lineage-marked Vhl-null cells. We first observed the behavior of Vhl KO cells in the RTE over time. Vhl was inactivated by a controlled dose of tamoxifen that induced recombination in a subset of cells across the nephron. Three weeks after recombination, Vhl KO cells were morphologically normal but had stabilized HIF-2, confirming a reported ‘HIF-isoform’ switch. There were equal numbers of tdTomato-positive cells in the KO/KO and wt/KO kidneys, suggesting that initial recombination is equal between the genotypes and that there is no immediate distortion in cell fates following Vhl KO. However, four months after recombination, there were fewer tdTomato-positive cells in the KO/KO kidneys, suggesting a gradual removal or loss of Vhl KO from tissue. This was most prominent in the renal papilla and cortex. Additionally, some Vhl KO cells presented with morphological signs of cellular stress and altered differentiation. Taken together, we believe Vhl KO cells are subjected to profound selection in the nephron and that cell-type-specific, negative, and heterogenous consequences of Vhl KO impact on cell survival. One corollary is that many cells which ultimately survive in Vhl KO organs are in fact Vhl-competent. By intrinsically coupling Vhl-inactivation and lineage marking, our model allows these populations to be distinguished and followed over time. We are now studying the differential gene expression patterns of Vhl KO cells in cells of the RTE and aim to define genetic signatures for contexts in which Vhl KO cells survive and contribute to ccRCC genesis. Citation Format: Joanna D C Lima, Samvid Kurlekar, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 939.

Abstract 862: Negative cellular outcomes following acute in vivo Vhl inactivation in mice

Abstract Clear cell renal cell carcinoma (ccRCC) is driven by the biallelic inactivation of the von Hippel Lindau tumor suppressor (VHL) gene, which results directly in the normoxic stabilization of hypoxia inducible factors (HIF-1 and -2). While VHL is expressed ubiquitously, its inactivation is tumorigenic only in specific tissues. We hypothesize that this tissue-specificity arises from a negative selective pressure on VHL KO cells that is tolerated in certain contexts. To assess this phenomenon, we induced Vhl deletion in vivo simultaneously across the mouse body and observed cell fate with single-cell resolution. Here we show that inducing widespread inter-organ Vhl deletion results in a massive removal of affected cells from tissue. Our study required technology that allowed us to visualize and isolate Vhl-recombined cells from tissue. For this, we developed a novel lineage marking model in which Cre-mediated excision of Vhl is coupled to knock-in of a tdTomato reporter cassette under the endogenous Vhl promoter. We used this model in conjunction with the ubiquitously-expressed UBC-CreERT2 to induce marked biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl. One week after inactivation was induced by administration of tamoxifen, there were equal numbers of tdTomato-positive cells in both genotypes, implying that initial recombination was similar and that Vhl KO did not affect immediate cell survival. Surprisingly, there was a massive reduction in the number of tdTomato-positive cells in multiple KO/KO organs after four weeks. This loss was accompanied by proliferation of tdTomato-negative cells, as assayed by BrdU incorporation, suggesting that tissues can at least partially compensate for loss of Vhl-inactivated cells. Finally, we found that the removal of cells was preceded by signs of cellular damage and stress, such as accumulation of p27, cytoplasmic translocation of HMGB1, and TUNEL-positivity in the kidney and liver. Taken together, we conclude that Vhl inactivation is inherently deleterious to cells and entrains a negative cell selection process in multiple organs. One corollary is that there must exist some conditions under which such selection might not occur; secondary mutations found in ccRCC, such as in Pbrm1, or loss of individual HIF isoforms, might provide such conditions to Vhl KO cells. Currently we are investigating the role of these additional perturbations in modifying the cellular response to Vhl loss. Our study will provide new insights into the genesis of ccRCC by redefining the mechanisms underlying the creation of pro-tumorigenic niches following Vhl inactivation. Citation Format: Samvid Kurlekar, Joanna D C Lima, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. Negative cellular outcomes following acute in vivo Vhl inactivation in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 862.

Abstract 1587: Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors

Abstract Kidney cancer is the seventh most common malignancy in the Western world and 80% of these are clear cell renal cell cancers (ccRCC). ccRCC arises following inactivation of the VHL gene, which negatively regulates HIF (hypoxia-inducible factor) transcription factors. Therefore, in ccRCC, HIF is constitutively active and drives a gene expression program that is central to tumorigenesis. Whilst this is necessary for ccRCC formation, it is not sufficient, and other mutations must also occur in combination with VHL inactivation to drive ccRCC tumorigenesis. The mechanisms underlying their synergy with VHL inactivation remain poorly understood. These secondary driver mutations are often subclonal in nature, suggesting the presence of phenotypically distinct malignant cells within an individual tumor. This intratumor heterogeneity renders it difficult to study the functional impact of driver mutations in ccRCC by conventional ‘bulk’ analyses such as RNA-seq. Consequently, the molecular mechanisms underlying ccRCC initiation are not known. Furthermore, non-malignant stromal cells (including the vasculature and immune response) can support tumor growth, and little is known as to how they interact with malignant cells in ccRCC. This is particularly important since current therapeutic strategies in kidney cancer target the stromal compartment. Therefore, there is an unmet need to better-understand transcriptional heterogeneity and cellular diversity in ccRCC. To that end, we have sampled multiple regions from ccRCC tumors (and tumor-adjacent kidney tissue) and performed single cell RNA-seq (10x Genomics) on approximately 150,000 individual cells. These encompass malignant cells as well as non-malignant cells (including fibroblasts, endothelial cells, pericytes and immune cells), both of which exhibit pronounced transcriptional heterogeneity within and between tumors. Analysis of these transcriptomes has identified evolutionary gene expression programs underlying cancer cell dedifferentiation. In addition, we observe concurrent heterogeneity in angiogenic signaling between malignant cells and non-malignant blood vessel cells within these tumors. Lastly, we have explored the effect of microenvironmental hypoxia across different ccRCC tumor cell types. In summary, we present a single cell atlas derived from multi-region heterogenous ccRCC samples. This sheds light on the mechanisms underlying tumor evolution and progression, as well as angiogenic heterogeneity (which may contribute to the variable response to receptor tyrosine kinase inhibition) and the effect of hypoxia on ccRCC biology. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Peter J. Ratcliffe, David R. Mole. Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1587.

HLA-I-restricted CD8+ T cell immunity may accelerate tumorigenesis in conjunction with VHL inactivation

SummaryCD8+ T cells recognize and kill tumor cells with HLA-I tumor antigens in early tumorigenesis, the efficiency of which differs according to antigen-recognition coverage, as shown in earlier tumor onset in HLA-I homozygosity. However, the universality of these associations remains unknown. Here, we assessed the tumor type and driver mutation specificity in the association between tumor onset age and HLA-I zygosity. Statistical analyses identified an unexpected negative relationship in tumors with VHL biallelic loss, wherein HLA-I heterozygosity was associated with earlier tumor onset, while all others showed either no or a positive association. Testing on an independent dataset reproduced the VHL-dependent acceleration of tumor onset in the HLA-I heterozygous group, confirming the association. Further speculation proposed VEGF-A-mediated T cell exhaustion under VHL inactivation as a potential mechanism. Our findings suggest that CD8+ T cell immunity in early tumor suppression can be conditional to the genetic status of tumors and may even lead to adverse consequences.

A phase II study of talazoparib and avelumab in VHL deficient clear cell renal cell carcinoma.

347 Background: VHL inactivation may lead to impairments in the DNA damage response pathway and increased replication stress, which consequently can increase the genomic instability of clear cell renal cell carcinoma (RCC). Other molecularly driven RCC subtypes including FH and SDH deficient RCC show impaired DNA repair through oncometabolite accumulation. Synthetic lethality with PARP inhibitors (PARPi) has been suggested in preclinical models, and PARPi may potentiate the effects of immune checkpoint blockade (ICB) therapy. Methods: We conducted a single center, investigator-initiated phase II trial to evaluate combined PARPi + PD-L1 inhibition in two genomically selected advanced RCC cohorts: 1) VHL altered RCC; 2) FH, SDH-RCC and renal medullary carcinoma. For Cohort 1, patients had to have previously been treated with VEGFR TKI and ICB therapy with maximum 3 prior lines of therapy. Patients received talazoparib 1mg daily plus avelumab 800mg IV every 14 days. The primary endpoint was objective response rate (ORR) by iRECIST, and secondary endpoints included progression-free survival (PFS), safety and tolerability. Results from Cohort 1 are reported here. Results: From 2019-2021, 10 advanced RCC patients were enrolled per the first stage of a Simon 2-stage design for cohort 1. The median age was 63 years (R: 42-71), and median number of prior therapies was 2 (R: 1-3). All patients had VHL loss detected by tissue NGS sequencing via MSK-IMPACT, and 3/10 (30%) of patients had co-occurring somatic or germline alterations in DDR specific genes. No objective tumor responses were seen, and the disease control rate was 60% (6/10) with those patients achieving stable disease as best response. All patients experienced disease progression and the median PFS was 3.6 months (95% CI 1.4-4.9). Adverse events were in keeping with reported toxicities for individual agents without emerging safety signals. Conclusions: This is the first clinical study of combination PARPi and ICB therapy in advanced clear cell RCC. In our cohort of VHL deficient TKI/ICB refractory RCC, the study did not meet its pre-defined efficacy threshold to continue enrollment, and our results do not support further study of this regimen in this population. Exploratory efforts to evaluate this treatment approach in other biomarker selected populations of RCC, including cohort 2 of this study, are ongoing. Clinical trial information: NCT04068831.

An IDH-independent mechanism of DNA hypermethylation upon VHL inactivation in cancer

ABSTRACT Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate – a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.

Abstract 62: Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC)

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. Treatment of RCC has improved dramatically over the last decade, however, a curative treatment for advanced RCC remains rare. Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug”. The FDA approval of CAR-T cell therapies have ushered this new type of cellular immunotherapy into mainstream cancer therapy for hematologic malignancies. To date, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the immunosuppressive tumor microenvironment (TME), and on-target off-tumor toxicities due to shared antigens on normal tissues. Carbonic anhydrase IX (CAIX) is a therapeutic target against ccRCC that is under the control of HIF1α and becomes overexpressed on the surface of ccRCC cells because of VHL inactivation. The first anti-CAIX CAR-T study using the 1st generation G250-CD3 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC was terminated due to hepatitis that developed after CAR-T cell infusions. The on-target off-tumor side effects were attributed to low expression of CAIX on healthy bile duct cells. To translate CAR-T cell therapy to ccRCC, we designed dual-targeted fine-tuned immune restoring (DFIR) CAR-T cells. The DFIR-CAR T cells secreted immune checkpoint inhibitor (ICI) monoclonal antibodies at the tumor site and exhibited superior efficacy and safety profiling. Increased efficacy is achieved through anti-CD70/CAIX dual-targeting CAR which allows the CAR-T cell activation in response to either antigen to mitigate solid tumor heterogeneity. Elevated safety is addressed through fine-tuned CARs which have the affinities of the scFv targeting moieties tailored so that they are activated only by high density tumor associated antigens (TAAs) but not the same antigens expressed at physiologic levels on normal tissues. The ICI payloads act globally on the TME, not only to prevent CAR-T cell exhaustion but also to restore anti-tumor activity of the educated tumor infiltrated lymphocytes (TILs) that have accumulated in the TME. In summary, our DFIR CAR-T cell therapy holds the promise to achieve cures of ccRCC by killing heterogenous ccRCC cells, mitigating against on-target off-tumor toxicity, reversing TME immunosuppression and restoring host anti-tumor immunity. We believe that DFIR-CAR T cells are ready to be translated to the clinic upon completion of pre-IND studies. Citation Format: Yufei Wang, Alicia Buck, Marion Grimaud, Sreekumar Kodangattil, Cecile Razimbaud, Atef Fayed, Matthew Chang, Aedin Culhane, David A. Braun, Toni K. Choueiri, Catherine J. Wu, Kevin S. Wei, Leo L. Chan, Brandon P. Piel, Elena V. Ivanova, Cloud P. Paweletz, David A. Barbie, Rebecca Jennings, Miriam Ficial, Maura Aliezah Sticco-Ivins, Sabina Signoretti, Gordon J. Freeman, Quan K. Zhu, Wayne A. Marasco. Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 62.

High nuclear expression of HIF1α, synergizing with inactivation of LIMD1 and VHL, portray worst prognosis among the bladder cancer patients: association with arsenic prevalence.

Our study was aimed to understand the importance of LIMD1-VHL-HIF1α pathway in development of bladder carcinoma (BlCa) in association with arsenic prevalence. At first, the mRNA expression pattern of the genes of this pathway (LIMD1, VHL and HIF1α) was checked in GEO datasets and in our samples. Next, genetic and epigenetic profiling of LIMD1 and VHL was done in our sample pool, validated in T24 BlCa cell line. The results were next correlated with various clinico-pathological parameters. Differential under-expression of LIMD1 and VHL genes was found in muscle-invasive BlCa (MIBC) in comparison to non-muscle-invasive BlCa (NMIBC). However, HIF1α protein, but mRNA, was found to be overexpressed among the MIBC samples; depicting the probability of HIF1α protein stabilization. Analysis of genetic and epigenetic profiles of LIMD1 and VHL exposed a frequent promoter methylation of LIMD1 gene in MIBC samples. Further, in-depth look into the results unveiled that the high nuclear expression of HIF1α was significantly correlated with genetic alterations of LIMD1, alone or in combination with VHL. Moreover, treating the T24 cells with a de-methylating agent (5-aza-2'-deoxycytidine) re-expressed the methylated LIMD1 and VHL genes, which in turn, reduced the HIF1α protein level significantly. Additionally, patients with high arsenic content (> 112ng/g, AsH) seemed to have recurrent promoter methylation in LIMD1, as well as co-methylation/alteration of LIMD1 and VHL gene. Lastly, high nuclear expression of HIF1α in association with co-alteration of VHL and LIMD1 showed the worst overall survival (OS) among the patients. To conclude, MIBC samples portrayed higher alterations in VHL and LIMD1, thereby, stabilizing HIF1α protein and lowering the OS of patients.