Von Hippel-Lindau Tumor Suppressor Research Articles

VHL L169P Variant Does Not Alter Cellular Hypoxia Tension in Clear Cell Renal Cell Carcinoma.

In the current era of tumor genome sequencing, single amino acid missense variants in the von Hippel-Lindau (VHL) tumor suppressor gene are frequently identified in clear cell renal carcinoma (ccRCC). Due to the incomplete knowledge of the structural architecture of VHL protein, the functional significance of many missense mutations cannot be assigned. L169P is one such missense mutation identified in the case of aggressive, metastatic ccRCC. Here, we characterized the biochemical activity, transcriptomic hypoxia signature and biological functions of the L169P variant. Lentiviral vector expressing either wildtype (WT) or L169P VHL were used to transduce two VHL-deficient human ccRCC cell lines, 786-O and RCC4. The stability of the VHL protein and the expression level of VHL, HIF1α and HIF2α were analyzed. The impact of restoring L169P or WT VHL on the hypoxia gene expression program in 786-O cells was assessed by mRNA sequencing (RNAseq) and computed hypoxic scores. The impact of restoring VHL expression on the growth of ccRCC models was assessed in cell cultures and in chorioallantoic membrane (CAM) xenografts. In the 786-O cells, the protein stability of L169P VHL was comparable to WT VHL. No obvious difference in the capability of degrading HIF1α and HIF2α was observed between WT and L169P VHL in the 786-O or RCC4 cells. The hypoxic scores were not significantly different in the 786-O cells expressing either wildtype or L169P VHL. From the cellular function perspective, both WT and L169P VHL slowed cell proliferation in vitro and in vivo. The L169P VHL variant is comparable to WT VHL in terms of protein stability, ability to degrade HIF1α factors and ability to regulate hypoxia gene expression, as well as in the suppression of ccRCC tumor cell growth. Taken together, our data indicate that the L169P VHL variant alone is unlikely to drive the oncogenesis of sporadic ccRCC.

CT-based radiomics model for the prediction of genomic alterations in renal cell carcinoma (RCC)

Abstract Background Radiogenomics is an emerging tool with applications in screening for molecular biomarkers in diagnostic and prognostic assessment through the extraction of quantitative data from medical images (Shui et al., Front Oncol 2021). In this study, we aim to develop machine learning (ML) models to predict the most common genomic alterations present in our subset of renal cell carcinoma patients (pts). Methods Retrospectively, pts with tissue genomic testing from CT-guided biopsy samples were identified. Genomic testing was done via GEM ExTra assay, a CAP-accredited, CLIA-certified test encompassing tumor whole exome sequencing and whole transcriptome sequencing (TGen; Phoenix, AZ). Biopsy sample collection sites were identified from pre-biopsy contrast CT images, and the lesions were segmented with ITK-SNAP software, from which 510 radiomic features were extracted with Pyradiomics. The Least Absolute Shrinkage and Selection Operator (LASSO) regression was used to select the most relevant features. Logistic regression (LR) and support vector machine (SVM) classifiers were built for the prediction of PBRM1, VHL, and SETD2 gene alterations. Multiple metrics were used to evaluate the predictive performance via leave-one-out cross-validation, including the area under the receiver operating characteristic (AUROC) and the area under the precision-recall curve (AUPRC). Feature importance was evaluated with Shapley additive explanations (SHAP) method. Results A total of 14 RCC pts (10:4 M:F) with genomic testing from CT-guided biopsies were identified. The majority of pts were White (85.7%) and had clear cell histology (71.4%). The most common locations for the CT-guided biopsy were lung (18.2%), soft tissue (13.6%), kidney (9.1%), and bone (9.1%). The most common alterations were seen in PBRM1 (50%), VHL (43.9%), and SETD2 (35.7%) genes. The PBRM1 gene was predicted with the highest AUROC (0.84) and AUPRC (0.88) with the SVM classifier, followed by the SETD2 gene (AUROC=0.78 and AUPRC=0.66) with LR classifier and VHL gene (AUROC=0.56 and AUPRC=0.65) with SVM classifier. Notably, all three models showed good sensitivity in classifying gene mutation status (PBRM1: 0.86; VHL: 0.88; SETD2: 0.89). Among all radiomic features, first-order features, Gray Level Size Zone Matrix features, and Gray Level Dependence Matrix features were found to be the most important features for predictions. Conclusions Using a CT-based radiomics analysis of the biopsy area, we showed that SVM and LR prediction models could predict PBRM1, VHL, and SETD2 mutations with high accuracy. These models may assist in identifying potentially actionable alterations and yield ease in treatment selection for RCC. Further extensive studies are warranted to validate our findings and improve our model. CDMRP DOD Funding: no

A Phase 1/2, open label dose-escalation and expansion trial of NKT2152, an orally administered HIF2α inhibitor, to investigate safety, PK, PD and clinical activity in patients with advanced ccRCC

Abstract Background: Inactivation of the VHL gene leading to aberrant HIF2α activity is nearly universal in clear cell renal cell carcinoma (ccRCC). NKT2152 is a novel, potent, selective orally available HIF2α inhibitor optimized for enhanced PK exposure and sustained target inhibition which has demonstrated robust activity in both ccRCC cell line-derived and patient-derived xenograft RCC and other solid tumor models. This is a Phase 1/2 open label, multicenter, first in human study of NKT2152 in adults with advanced clear cell renal cell carcinoma (ccRCC) (NCT05119335). In Phase 1, up to ~60 patients will be enrolled according to a 3 + 3 design with backfill as permitted by the Safety Review Committee. The primary objective of phase 1 is to determine the recommended dose for expansion (RDE) based on the totality of clinical data. Phase 2 will enroll ~50 additional patients with the primary objective of determining investigator-assessed by RECIST v1.1. Key secondary objectives include safety, tolerability, PD effects, progression free survival, duration of response, and disease control rate. Exploratory objectives include evaluation of biomarkers predictive of tumor response. Adults who have advanced ccRCC without available standard therapy), ECOG PS 0-2, with measurable disease per RECIST 1.1 are eligible. Patients who have had prior HIF2a inhibitors, require supplemental oxygen, and with significant cardiac disease are excluded. Tumor assessments by CT or MRI are conducted every 8 weeks until 48 weeks, then every 12 weeks thereafter. Adverse events will be monitored and graded in severity using CTCAE v5.0. The Phase 1 study is currently actively accruing in the United States with Phase 2 dose expansion anticipated to start in Q3, 2023.

Abstract PR008: The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer

Abstract Significance: The clear cell renal cell carcinomas (ccRCCs), which harbor the hallmark inactivation of the von Hippel Lindau tumor suppressor protein (pVHL), represent the majority of adult kidney cancers. Although, early stage/localized ccRCC is effectively managed by surgery and surveillance, therapeutic options for advanced stage disease are limited, highlighting the unmet need for novel therapies. Chromatin dysregulation is routinely observed in ccRCC and our previous work identified a distinct histone modification signature associated with pVHL-deficient ccRCCs, versus the pVHL-proficient chromophobe and papillary sub-types, which included elevated levels of bulk Histone H3 K27 acetylation (H3K27ac). H3K27ac marks transcriptionally activated regions in the genome. We hypothesized that, as in other biological contexts, H3K27ac accumulation in pVHL-deficient ccRCCs drives the expression of key oncogenes. Here, we functionally probed this dysregulated epigenetic program to identify actionable targets in kidney cancer. Experimental Approach and Results: We identified genes marked by elevated H3K27ac (ChIP-Seq) and higher expression (RNA-Seq), specifically in pVHL-deficient cells and prioritized the top 100 genes that encoded druggable products. Using a library representing these ORFs, we did an in vivo ‘sufficiency’ screen, to identify candidates whose expression promoted tumor growth in otherwise poorly tumorigenic pVHL-proficient cells. Our screen (and downstream validation studies) identified SLC1A1, which encodes an Asp/Glu transporter named EAAT3, as an oncogenic driver in ccRCC. Results: SLC1A1 expression was elevated in pVHL-deficient cells – but in a HIF-independent manner. Inactivation of SLC1A1 (using CRISPR/Cas9) led to fitness defects in a panel of ccRCC cell lines, along with a notable depletion of nucleotide biosynthesis precursors, likely because of the importance of the Asp carbon skeleton in this pathway. Consistent with this, esterified-Asp rescued the fitness defects associated with SLC1A1 loss. Moreover, SLC1A1 upregulation (high Asp/Glu) diminished dependency on Glutaminase; whereas, SLC1A1 loss (low Asp/Glu) increased Glutaminase dependency and promoted sensitivity to Glutaminase blockade. Finally, using clinical cohorts and cell-based studies, we found that SLC1A1 can impact its own transcriptional expression, but also the expression of certain other metabolic transporters involved in Asp/Glu metabolism (e.g. SLC22A11) and nucleotide metabolism (e.g SLC28A1). We speculate, these transcriptional alterations are driven by the conversion of Glu into a-ketoglutarate, a key cofactor for KDMs. Our ongoing studies are addressing the mechanistic basis of this transcriptional regulation and exploring the feasibility of pharmacologically targeting SLC1A1 in ccRCC. Altogether, we show that ccRCCs hijack nutrient reabsorption mechanisms (e.g. SLC1A1) to drive oncogenic programs. SLC1A1 thus links epigenetic dysregulation to a HIF-independent metabolic program regulating nucleotide biosynthesis/uptake. Citation Format: Abhishek A. Chakraborty. The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer [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 PR008.

Abstract B031: Effect of macrophage inflammatory response and VHL mutation status on the development of clear cell renal carcinoma

Abstract Kidney[A1] cancer is a serious health care issue problem around the worldworldwide. It is symptomless asymptomatic in the early stage and is refractory to most [A2] of the cancer treatment modalities. The most prevalent form of the kidney cancer, renal cell carcinoma of the clear-cell type (ccRCC), is mainly caused by loss-of-function of the von Hippel-Lindau tumor suppressor gene (VHL). VHL protein is an E3 ubiquitin ligase and. iIts most prominentmain degradation target is the alpha subunit of the hypoxia-inducible factor (HIF). However, the etiology of ccRCC formation development remains unclear. Recent animal model studies in our laboratory have indicatedWe recently observed that tissue inflammation and fibrosis are the early events in ccRCC development is tissue inflammation and fibrosis. This interesting result provides important supports for the concept notion that chronic inflammation is can induce tumorigenesisic and. It is also consistent with the epidemiological evidence linking inflammatory kidney disease and kidney cancer. In the current present study, we explored the mechanistic detailsm by which changes within the VHL mutant cells orchestrateeffect[A3] a systemic inflammatory response[A4], and found that the metabolic abnormalities caused by VHL gene mutation lead to unresolved chronic endoplasmic reticular stress (ER stress), which in turn induces activation of JNK and NFκB, two major components of pro-inflammatory signaling pathways components[A5]. On the other hand, our previous study also demonstrated that LCN2[A6] sensitizes the inflammatory response of VHL mutant HK-2 cells in vhl mutant condition and the chemotactic abilityies of macrophage RAW 264.7 cells. [A7] However, the wider implication of this tumorigenic process [A8] that of RAW 264.7 cells is stillremains unclear and then warrants further investigationswill be discussed. Accumulating evidence indicated that Lon protease (Lon) is a multifunctional protein that mediatesinvolved in protein quality control and oxidative stress responses in mitochondria. However, the molecular mechanisms underlying the effects of Lon on the progression of ccRCC progression and inflammatory response inof the vhl VHL mutant condition cells remain largely unknown. Therefore, in this study, we aimed to elucidate the inflammatory effect role of inflammatory RAW 264.7 macrophages on the development of ccRCC development inusing cultured vhl VHL mutant renal tubular cells and RAW264.7 cells. Moreover, d Decoding how the action involved signaling pathways drive or suppress signals cellular and inflammatory responses or preventing it is a major research challenge. Once elucidated, it will for future research and will require the definition ofprovide the rationale for drug design and application on anti-ccRCC formation therapeutic strategies based onvia targeting macrophages infiltration. Citation Format: Chan-Yen Kuo. Effect of macrophage inflammatory response and VHL mutation status on the development of clear cell 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 B031.

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 PR007: Von Hippel Lindau (VHL) tumor suppressor regulates m6A-dependent gene expression involved in renal tumorigenesis

Abstract Background: Renal cancer is the most common adult genitourinary cancer, of which ~85% is accounted for by the clear cell subtype (ccRCC). In the majority of ccRCC, as a result of VHL loss, HIF2a stabilization is sufficient and necessary for promoting ccRCC tumor growth. However, HIF-2 inhibitors have limited efficacy in cancer therapy, highlighting the importance of identifying additional therapeutic vulnerabilities. N6-Methyladenosine (m6A) is the most abundant internal post-translation modification, and its contribution to cancer evolution has just been appreciated. In renal cancer, previous research showed that m6A may play an important role but the molecular mechanism underlying m6A-regulated ccRCC tumorigenesis and aggression is still obscure. Approach and results: We demonstrate that VHL binds with m6A enzymatic complex proteins and regulates METTL3-METTL14 complex formation. VHL depletion suppresses mRNA m6A modification. By performing m6A RIP-Seq for renal cells with or without VHL coupled with RNA-Seq, we identified a selection of genes that their expression may be regulated by m6A. Specifically, PIK3R3 was identified to be one critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promoted renal cell growth and tumor growth while vice versa. Mechanistically, the VHL-m6A regulated PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Conclusion: Taken together, we propose a new mechanism by which VHL regulates m6A through modulating METTL3-METTL14 protein complex formation, therefore promoting PIK3R3 mRNA stability and protein level that is critical for regulating ccRCC tumorigenesis. Citation Format: Cheng Zhang, Zhao Zhang, Austin Hepperla, Miaomiao Yu, Jin Zhou, Lianxin Hu, Jun Fang, Qian Liang, Kexin Xu, Liwei Jia, Qing Zhang. Von Hippel Lindau (VHL) tumor suppressor regulates m6A-dependent gene expression involved in renal tumorigenesis [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 PR007.

Abstract IA013: A zebrafish model for VHL and hypoxia signaling

Abstract he von Hippel-Lindau (VHL) tumor suppressor gene encodes an adaptor protein that regulates an array of transcription-dependent and -independent cellular and physiological processes. Mutations in this gene cause VHL disease, congenital polycythemia, renal cell carcinoma (RCC), and several other sporadic tumor types. Although different animal models for VHL disease have been adapted, the zebrafish VHL model offers distinct opportunities. Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities resembling early stages of RCC. The zebrafish vhl model offers a platform for the identification of genetic pathways, modifiers, and interactors involved in the development of VHL-associated neoplasms. Vhl mutants represent a unique and clinically relevant in vivo model for studying genotype-phenotype correlations and the identification of prognostic biomarkers. The amenability of zebrafish for chemical genetic screens will not only be helpful to identify novel therapeutic agents but may also reveal novel processes that require regulation by VHL. Citation Format: Ellen van Rooijien, Stefan Schulte-Merker, Ive Logister, Emile Voest, Rachel Giles. A zebrafish model for VHL and hypoxia signaling [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 IA013.

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 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.

Cleavage fragments of the C-terminal tail of polycystin-1 are regulated by oxidative stress and induce mitochondrial dysfunction

Mutations in the gene encoding polycystin-1 (PC1) are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). Cysts in ADPKD exhibit a Warburg-like metabolism characterized by dysfunctional mitochondria and aerobic glycolysis. PC1 is an integral membrane protein with a large extracellular domain, a short C-terminal cytoplasmic tail and shares structural and functional similarities with G protein-coupled receptors. Its exact function remains unclear. The C-terminal cytoplasmic tail of PC1 undergoes proteolytic cleavage, generating soluble fragments that are overexpressed in ADPKD kidneys. The regulation, localization, and function of these fragments is poorly understood. Here, we show that a ∼30kDa cleavage fragment (PC1-p30), comprising the entire C-terminal tail, undergoes rapid proteasomal degradation by a mechanism involving the von Hippel-Lindau tumor suppressor protein. PC1-p30 is stabilized by reactive oxygen species, and the subcellular localization is regulated by reactive oxygen species in a dose-dependent manner. We found that a second, ∼15kDa fragment (PC1-p15), is generated by caspase cleavage at a conserved site (Asp-4195) on the PC1 C-terminal tail. PC1-p15 is not subject to degradation and constitutively localizes to the mitochondrial matrix. Both cleavage fragments induce mitochondrial fragmentation, and PC1-p15 expression causes impaired fatty acid oxidation and increased lactate production, indicative of a Warburg-like phenotype. Endogenous PC1 tail fragments accumulate in renal cyst-lining cells in a mouse model of PKD. Collectively, these results identify novel mechanisms regarding the regulation and function of PC1 and suggest that C-terminal PC1 fragments may be involved in the mitochondrial and metabolic abnormalities observed in ADPKD.

Tumor Suppressor Gene VHL, Mechanism and Related Therapy

Von Hippel-Lindau (VHL) syndrome is a rare hereditary disorder characterized by the development of multiple tumorsin various organs. This paper provides a comprehensive overview of the VHL gene, its mechanism of action, and itsimplications in VHL-related tumorigenesis. The first section explores the role of the VHL tumor suppressor gene inregulating angiogenesis and oxygen sensing, highlighting its significance in tumor suppression. The second sectiondiscusses the association between VHL gene mutations and the development of various tumor types, emphasizing thediverse clinical manifestations of VHL syndrome. The third section examines the diagnostic methods for identifyingVHL gene mutations, including genetic testing and clinical evaluations. The fourth section delves into therapeuticapproaches for managing VHL-related tumors, encompassing surgical interventions, targeted therapies, radiotherapy,and emerging treatment strategies. This paper aims to enhance understanding of VHL syndrome, facilitate earlydiagnosis, and guide treatment decisions for individuals affected by this complex disorder.

Potential Role of VHL, PTEN, and BAP1 Mutations in Renal Tumors.

The genetic profiling of renal tumors has revealed genomic regions commonly affected by structural changes and a general genetic heterogeneity. The VHL, PTEN, and BAP1 genes are often mutated in renal tumors. The frequency and clinical relevance of these mutations in renal tumors are still being researched. In our study, we investigated VHL, PTEN, and BAP1 genes and the sequencing of 24 samples of patients with renal tumors, revealing that VHL was mutated at a noticeable frequency (25%). Six of the investigated samples showed mutations, and one genetic polymorphism (rs779805) was detected in both heterozygote and homozygote forms. PTEN gene mutation was observed in only one sample, and one specimen showed genetic polymorphism. In the case of the BAP1 gene, all of the samples were wild types. Interestingly, VHL mutation was detected in two female patients diagnosed with AML and in one with oncocytoma. We assume that VHL or PTEN mutations may contribute to the development of human renal cancer. However, the overall mutation rate was low in all specimens investigated, and the development and prognosis of the disease were not exclusively associated with these types of genetic alterations.

NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.

ARC-20: A phase 1 dose-escalation and dose-expansion study to investigate the safety, tolerability, and pharmacology of HIF-2α inhibitor AB521 monotherapy in patients with clear cell renal cell carcinoma and other solid tumors.

TPS4602 Background: Renal cell carcinoma (RCC) is one of the 10 most common neoplasms in the United States. Patients with clear cell RCC (ccRCC) commonly have inactivation of the von Hippel-Lindau tumor suppressor protein, resulting in accumulation of hypoxia-inducible factor alpha (HIFα) subunits (especially HIF-2α) and leading to tumor growth. AB521, an orally bioavailable small molecule inhibitor of HIF-2α, potently inhibits transcription of HIF-2α-dependent genes in cell lines and preclinical species. Single doses of AB521 (3 mg, 10 mg, 30 mg, and 100 mg) and multiple ascending doses of AB521 (15 mg, 30 mg, and 50 mg) have been previously studied in healthy volunteers. ARC-20 is a first-in-patient study to investigate the safety, tolerability, and pharmacology of AB521 monotherapy in patients with solid tumors and evaluate the clinical activity of AB521 in previously treated patients with ccRCC. Methods: ARC-20 (NCT05536141) is a phase 1, multicenter, open-label study. Eligible patients are adults with solid tumors with ≥1 measurable lesion(s) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 and an Eastern Cooperative Oncology Group performance status of 0 or 1. ARC-20 has 2 stages, dose escalation and dose expansion. The dose escalation stage uses a standard 3+3 design and includes patients with pathologically confirmed solid tumors and no other available treatment options. Patients will be sequentially assigned to 1 of 4 cohorts evaluating doses of AB521 ranging from 20 to 200 mg/day. Intermediate dose cohorts may be evaluated based on emergent safety or pharmacologic data. The dose expansion stage will evaluate approximately 30 patients with histologically confirmed ccRCC who have received previous treatment for metastasis with an anti-programmed cell death protein 1 (anti-PD-1) therapy and a vascular endothelial growth factor (VEGF)-targeting tyrosine kinase inhibitor (TKI). Patients previously treated with a HIF-2α-targeting therapy are not eligible for dose expansion. Results from the dose-escalation stage will inform the dose and schedule used during dose expansion. Primary endpoints are the incidence of dose-limiting toxicity and adverse events. Secondary endpoints include objective response rate, AB521 plasma concentration, and AB521 pharmacokinetics. Exploratory endpoints include changes in expression of erythropoietin and other HIF-2α driven biomarkers in blood and tumor tissues, along with progression-free survival for ccRCC patients during dose expansion. Clinical trial information: NCT05536141 .

First-in-human clinical trial design of a first-in-class theranostic approach with a peptide-based radioligand targeting CA IX-expressing tumors.

TPS3160 Background: Expression of carbonic anhydrase IX (CA IX), a cell surface glycoprotein, can be induced by a state of hypoxia or by mutation of the Von Hippel-Lindau tumor suppressor gene. CAIX expression in tumors is often associated with progressive disease and overall poor outcomes in various solid cancers. Notably, high CA IX expression has been observed in 83%, 40%, and 29% of clear cell renal cell carcinoma (ccRCC), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) specimens, respectively1. CA IX may therefore represent a valuable therapeutic target. DPI-4452 is a first-in-class cyclic peptide that binds with high affinity to CA IX. Radiolabeling DPI-4452 with gallium-68 ([68Ga]Ga-DPI-4452) or lutetium-177 ([177Lu]Lu-DPI-4452) is an innovative theranostic approach for identifying and treating patients (pts) with CA IX-expressing tumors. Pre-clinical data has shown that [177Lu]Lu-DPI-4452 is taken up by tumors and induces dose-dependent growth inhibition in ccRCC and CRC xenograft mouse models1,2. Methods: We present the design of a Phase I/II study (NCT05706129) of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 in pts with unresectable locally advanced or metastatic ccRCC, CRC or PDAC. The study is comprised of three phases: imaging (Part A), dose-escalation (Part B), and expansion (Part C). In Part A, pts will receive a single intravenous (IV) 185 MBq dose of [68Ga]Ga-DPI-4452. Safety, tolerability, pharmacokinetics (PK), dosimetry and imaging characteristics of [68Ga]Ga-DPI-4452 will be evaluated using positron emission tomography/computed tomography (CT) at four timepoints on Day 1. All pts will be followed for seven days post-injection for safety observations. In Part B, based on [68Ga]Ga-DPI-4452 uptake, pts will receive sequential, escalating doses of IV [177Lu]Lu-DPI-4452 on Day 1 of each 28-day cycle for up to eight cycles. During Cycle 1, each pt will undergo up to four whole-body planar imaging and single photon emission CT/CT analyses for dosimetry over seven days. In Part B, the recommended Phase 2 dose of [177Lu]Lu-DPI-4452 will be determined, guided by a Bayesian Logistic Regression model. Safety, PK, and dosimetry will be assessed to minimize any radiation-induced toxicity and to optimize absorbed radiation dose in the tumor. Upon completion of Part B, Part C will enroll up to three cohorts of pts with CA IX-positive ccRCC, CRC or PDAC to assess preliminary efficacy of [177Lu]Lu-DPI-4452, with a primary endpoint of objective response rate, as measured by RECIST v1.1. References Wiedemann N et al. J Nucl Med 2022;63(Suppl 2):4048. Attinger A et al. Eur J Nucl Med Mol Imaging 2022;49(Suppl 1):S1–S751. Clinical trial information: NCT05706129 .

The impact of surgery on patients with VHL-associated tumors: An international patient survey.

4517 Background: Von Hippel-Lindau syndrome (VHL) is a rare hereditary neoplastic disorder caused by mutations in the VHL gene. Treatment (tx) options for patients are limited to multiple surgeries dispersed between regular scans, watchful waiting and txs that preserve organ function. Methods: An international, cross-sectional survey comprising patients in the United States (US), Canada (CA), the United Kingdom (UK), France (FR), and Germany (DE) was conducted. Patients were recruited via the VHL Alliance; data were collected between Dec 2021 and May 2022. For inclusion, patients must have renal cell carcinoma (RCC), pancreatic neuroendocrine tumors (pNET), and/or central nervous system hemangioblastoma (CNS-Hb). Data collected included demographics, impact of most recent surgery on aspects of condition (1-7 scale 1 being greatly worsened and 7 being greatly improved), tx goals (top 3 goals from a list of 11), and patient preference in treatment. Patients were presented two scenarios “watch and wait” and potentially need surgery, or “take a once-daily pill” to delay a potential surgery. Results: 220 patients (68.2% female, median age 40.0, median disease duration 15.8 years) in the US (n=108), CA (n=37), the UK (n=21), FR (n=3), and DE (n=51) completed the study. 146 patients reported RCC (66.4%), n=122 reported pNET (55.5%), and n=190 reported CNS-Hb (86.4%). Among the top 4 symptoms experienced by patients were kidney cysts (n=82, 37.3%), numbness (n=59, 26.8%), dizziness (n=56, 25.5%), and fatigue/low energy (n=54, 24.5%). 205 (93.2%) patients had experienced surgery; n=171 (77.7%) had experienced multiple surgeries (median number of surgeries, 4.0). 166 patients recorded data on their most recent surgery. Of these, patients reported that their most recent surgery worsened (scored 1-3) their fatigue (51.8%), mental health (51.2%), and ability to go about daily life (45.2%). 47.3% of patients selected reduce the number of surgeries as a top tx goal, 46.4% selected being able to go about life as usual, 36.8% delay the need for surgery, and 33.2% improved quality of life. 73% of patients indicated they would prefer to take a pill which would possibly delay the time until surgery, rather than watch and wait to see if the tumor would grow. Conclusions: Surgery negatively impacts the lives of patients, leading to a worsening in their fatigue, mental health, and ability to go about daily life. Almost half of patients would like a treatment that reduces the number of surgeries, and over a third would like a treatment that delays the need for surgery. Nearly three quarters of patients would prefer to take a pill that might delay the need for surgery. Tx options that reduce the need for surgery would benefit this patient group.

Von Hippel-Lindau is Associated to Pancreatic Neuroendocrine Tumors: A Comprehensive Review.

Multiorgan tumors are a hallmark of the autosomal dominant genetic disorder known as Von Hippel-Lindau syndrome (VHL), which is typically the result of inherited aberrations of the VHL tumor suppressor gene. The most frequent cancer is retinoblastoma, which can also occur in the brain and spinal cord, renal clear cell carcinoma (RCCC), paraganglioma, and neuroendocrine tumors. There may also be lymphangiomas, epididymal cysts, and pancreatic cysts or pancreatic neuroendocrine tumors (pNETs). The most frequent causes of death are metastasis from RCCC and neurological complications from retinoblastoma or central nervous system (CNS). Pancreatic cysts are present in 35-70% of VHL patients. Simple cysts, serous cysts, or pNETs are possible presentations, and the likelihood of malignant degeneration or metastasis is no greater than 8%. Although VHL has been associated with pNETs, their pathological characteristics are unknown. Furthermore, it is unknown whether variations in the VHL gene cause the development of pNETs. Hence, this retrospective study was undertaken with the main aim to examine whether pNETs are connected to VHL from a surgical perspective.

Clear cell renal cell carcinoma molecular variations in non-Hispanic White and Hispanic patients.

The United States is becoming increasingly diverse, but few molecular studies have assessed the progression of clear cell renal cell carcinoma (ccRCC) in diverse patient populations. This study examined ccRCC molecular variations in non-Hispanic White (NHW) and Hispanic patients and their effect on the association of gene expression with high-grade (Grade 3 or 4) ccRCC and overall mortality. A total of 156 patients were included in VHL sequencing and/or TempO-Seq analysis. DESeq2 was used to identify the genes associated with high-grade ccRCC. Logistic regression analysis was performed to assess whether race and ethnicity was associated with high/moderate impact VHL somatic mutations and the ccA/ccB subtype. Cox regression analysis was performed to assess association of molecular subtype and gene expression with overall mortality. NHWs had moderate or high impact mutations in the VHL gene at a higher frequency than Hispanics (40.2% vs. 27.4%), while Hispanics had a higher frequency of the ccA subtype than NHWs (61.9% vs. 45.8%). ccA was more common in patients with BMI≥35 (65.2%) than in those with BMI < 25 (45.0%). There were 11 differentially expressed genes between high- and low-grade tumors. The Haptoglobin (HP) gene was most significantly overexpressed in high- compared to low-grade ccRCC in all samples (p-adj = 1.7 × 10-12 ). When stratified by subtype, the 11 genes were significantly differentially expressed in the ccB subtype, but none of them were significant after adjusting for multiple testing in ccA. Finally, patients with the ccB subtype had a significantly increased risk of overall mortality (HR 4.87; p = 0.01) compared to patients with ccA, and patients with high HP expression and ccB, had a significantly increased risk of mortality compared to those with low HP expression and ccA (HR 6.45, p = 0.04). This study reports ccRCC molecular variations in Hispanic patients who were previously underrepresented.

Tumor heterogeneity in VHL drives metastasis in clear cell renal cell carcinoma

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL+) cells. Renal tumors with either VHL+ or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL+ cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL−) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.