Von Hippel-Lindau Tumor Suppressor Research Articles

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.

Integrated Metabolomic and Transcriptomic Analysis of Modified Nucleosides for Biomarker Discovery in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2'-O-methylcytidine. The method's reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials.

Abstract 3956: Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene

Abstract Background. Clear cell renal cell carcinoma (ccRCC) is the most frequent type of cancer in the kidney. These tumors are highly vascular and correlate with poor prognosis. Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is a truncal event of ccRCC carcinogenesis, which yields new insights for targeted therapy. Our studies identified a small molecule, STF-62247, that is toxic to cells lacking VHL compared to RCC with a functional gene. We reported that STF-62247 blocks late stages of autophagy by targeting lysosome dynamics. Moreover, we identified lysosomal vulnerability in VHL-mutated ccRCCs, which could lead cells to death. Goal. This study aims to recognize and characterize STF-62247 potential targets. Results. Using biochemical approaches, our results indicated that STF-62247 cytotoxicity is driven by PIKfyve inhibition. Binding affinity between PIKfyve and STF-62247 is around 5 nM. Intracytoplasmic vacuoles and enlargement of endolysosomes were observed, which was prevented by adding exogenous PI(3,5)P2. Moreover, PIKfyve inhibitors such as apilimod, YM201636, Vacuolin-1, APY0201 sensitize ccRCC with a loss of VHL. Finally, genetic knockdown of PIKfyve was achieved by CRISPR/Cas9 leading VHL-inactivated cells to death. Conclusion. Altogether, our studies identified, for the first time, PIKfyve as potential target for kidney cancer cells with a loss of VHL. Citation Format: Nadia Bouhamdani, Sandra Turcotte. Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3956.

Abstract 2599: Targeting SETD2-inactivated cells with STF-62247 leads to cell cycle arrest

Abstract The SETD2 gene encodes a histone methyltransferase responsible for the trimethylation of histone 3 on lysine 36 (H3K36me3). While this canonic function plays a role in the regulation of cellular processes such as transcription, alternative splicing, and DNA damage reparation, SETD2 is also important for the methylation of other proteins like EZH2, STAT1, and the cytoskeletal proteins actin and α-tubulin. Mutations in SETD2 are found in a broad range of cancers, including Clear Cell Renal Cell Carcinoma (ccRCC). Loss of chromosome 3p followed by the inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) is a focal characteristic of ccRCC, however, additional mutations are common on other genes also found on chromosome 3p, including SETD2. Our previous studies demonstrated the possibility of targeting VHL-deficient cells by blocking the late stages of autophagy with the small molecule STF-62247. We recently identified PIKfyve, a phosphoinositide kinase important for the biosynthesis of phosphatidylinositol 3,5-biphosphate (PI(3,5)P2), as STF-62247’s target. In the mindset of personalized medicine, we hypothesize that other mutations on genes like SETD2 could influence cell response to STF-62247 and other PIKfyve inhibitors such as apilimod. The objectives of this project are to 1) evaluate whether SETD2 expression influences the response to STF-62247 and PIKfyve inhibitors in ccRCC and 2) investigate the mechanism by which STF-62247 is selectively targeting SETD2-inactivated cells. Molecular biology approaches such as CRISPR/Cas9 and the Gateway cloning system were used to repress or reintroduce the gene coding for SETD2 in ccRCC cells like 786-0 and RCC-ER. Western blots were used for the validation of our models. Cell response to STF-62247 and other agents was measured by clonogenic assays, cell counts, and XTT viability assay. Flow cytometry, in addition to western blots, was used to study the cell cycle. Our results indicate that repression of SETD2 in 786-0, especially in 786-0 where VHL was reintroduced (786-0/VHL), sensitized cells to STF-62247 and apilimod. When treated with STF-62247, sensitive cells stopped proliferating and the reintroduction of a truncated version of SETD2 partially rescued them. In RCC-ER, a SETD2-deficient cell line, the reintroduction of SETD2 or VHL increased the viability and proliferation of the cells when treated with STF-62447. That resistance was increased when both genes were reintroduced together. Flow cytometry results showed an increase in S-phase in SETD2-inactivated 786-0/VHL cells treated with STF-62247. Likewise, decreased expression of some proteins related to DNA replication and mitosis, such as CDT1 and Cyclin B1, also indicate alterations in the cell cycle. Altogether, our results put forward the potential of using PIKfyve inhibitors as a new therapeutic approach to treat cancer patients with inactivated SETD2. Citation Format: Mathieu Johnson, Sandra Turcotte. Targeting SETD2-inactivated cells with STF-62247 leads to cell cycle arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2599.

Abstract 494: BPI-452080: A potent and selective HIF-2α inhibitor for the treatment of clear cell renal cell carcinoma, von Hippel-Lindau disease, and other solid tumors

Abstract The most observed alteration in clear cell renal cell carcinoma (ccRCC) is the inactivation of the tumor suppressor von Hippel-Lindau (VHL) E3 ligase due to genetic predisposition, somatic mutations, or methylation. Loss of VHL can lead to hypoxia-inducible factor 2α (HIF-2α) accumulation and elevated expression of HIF target genes, such as VEGFA, many of which facilitate angiogenesis, proliferation, and metastasis of ccRCC. Therefore, inhibiting HIF-2α has become a new strategy for the treatment of ccRCC. Here, we present the discovery of BPI-452080, a selective small molecule HIF-2α inhibitor. In VHL defective ccRCC cell lines, BPI-452080 potently and selectively blocked the heterodimerization of HIF-2α with HIF-1β, but not that of HIF-1α with HIF-1β. It inhibited the downstream hypoxia-responsive gene transcription and VEGFA protein secretion. In multiple xenograft mouse models, oral administration of BPI-452080 significantly inhibited tumor growth in a dose-dependent manner. BPI-452080 demonstrated good PK/PD correlation in the 786-O xenograft model after a single dosing, as shown by a concentration-dependent inhibition of VEGFA secretion in plasma and a reduction of the mRNA levels of VEGFA, CXCR4, CCND1, and GLUT1 genes in tumor tissue. Moreover, BPI-452080 exhibited good bioavailability in multiple pre-clinical species, favorable ADME properties, and good tolerance in toxicology studies. In conclusion, BPI-452080 is a potent, selective, and orally bioavailable HIF-2α inhibitor which presents a novel therapeutic option for ccRCC, von Hippel-Lindau disease, and other solid tumors. BPI-452080 is planned to enter Phase I clinical trial in China in early 2023. Citation Format: Pingping Wang, Rongwen Yang, Yun Sun, Xuepeng Ju, Jiayu Zhao, Yanju Liu, Xiaoyun Liu, Zhengyao Zou, Jialin Ren, Min Wang, Haibo Chen, Xuegang Yi, Jian Zhang, Teng Ma, Gaoliang Cheng, Lijia Liu, Jing Guo, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-452080: A potent and selective HIF-2α inhibitor for the treatment of clear cell renal cell carcinoma, von Hippel-Lindau disease, and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 494.

LncRNA MALAT1 promotes growth and metastasis of head and neck squamous cell carcinoma by repressing VHL through a non-canonical function of EZH2

Long non-coding RNAs (LncRNAs) are implicated in malignant progression of human cancers. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-known lncRNA, has been reported to play crucial roles in multiple malignancies including head and neck squamous cell carcinoma (HNSCC). However, the underlying mechanisms of MALAT1 in HNSCC progression remain to be further investigated. Here, we elucidated that compared with normal squamous epithelium, MALAT1 was notably upregulated in HNSCC tissues, especially in which was poorly differentiated or with lymph nodes metastasis. Moreover, elevated MALAT1 predicted unfavorable prognosis of HNSCC patients. The results of in vitro and in vivo assays showed that targeting MALAT1 could significantly weaken the capacities of proliferation and metastasis in HNSCC. Mechanistically, MALAT1 inhibited von Hippel–Lindau tumor suppressor (VHL) by activating EZH2/STAT3/Akt axis, then promoted the stabilization and activation of β-catenin and NF-κB which could play crucial roles in HNSCC growth and metastasis. In conclusion, our findings reveal a novel mechanism for malignant progression of HNSCC and suggest that MALAT1 might be a promising therapeutic target for HNSCC treatment.

Prognostic Role of Long-Chain Acyl-Coenzyme A Synthetase Family Genes in Patients with Clear Cell Renal Cell Carcinoma: A Comprehensive Bioinformatics Analysis Confirmed with External Validation Cohorts

We aimed to determine the prognostic role of long-chain acyl-CoA synthetases (ACSLs) as a disease marker for kidney clear cell carcinoma (KIRC). The Cancer Genome Atlas (TCGA) data were accessed via open access LinkedOmics database for KIRC. Provisional datasets were used for analysis as previously described and gene expression quantification data were downloaded. The corresponding clinical information of patients also were obtained from the database. Five ACSL family members, ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, were investigated in the TCGA-KIRC cohort. Xena browser, cBioPortal and UALCAN, and Cancer Cell Line Encyclopedia (CCLE) databases were also used to confirm the results. External validation was performed using patient cohorts from the Gene Expression Omnibus (GEO-NCBI) database. Finally, the protein-protein interaction (PPI) was constructed based on the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized using Cytoscape software. Pathological T3-T4 stage tumors had significantly lower ACSL1 mRNA expression (P=.009). Patients with pathologically confirmed metastasis exhibited significantly lower expression, as well (P=.02). ACSL1 mRNA expression was associated with overall survival (OS) and negatively correlated with OS time. Univariate and multivariate analyses showed that lower ACSL1 mRNA expression level was associated with mortality. Moreover, ACSL1 mRNA expression was exhibited significant difference in some VHL gene region mutations and PBRM1_p.R1010 mutation, and negatively correlated with HIF1-alpha mRNA expression (P < .001). Confirmatory analyses and external validation also revealed similar findings. Lowered ACSL1 mRNA expression is associated with worse tumor histopathology and poor overall survival in KIRC. It may be used for prognostic marker for KIRC.