Wild-type VHL Research Articles

Abstract 3159: VHL1 activation with a CRISPR-based gene activation platform causes increased MLKL expression and triggers necroptosis in renal cell carcinoma

Abstract Background: Kidney cancer is a common and deadly disease. Over 90% of all kidney cancers are classified as renal cell carcinoma (RCC), of which clear-cell RCC (ccRCC) is the most predominant subtype. VHL is a tumor suppressor and plays a pivotal role in ccRCC development. While >50% of ccRCC patients present with pathogenic VHL mutations, deep deletions and low VHL expression are also common. Over the past two decades, therapeutic advances have been made for metastatic ccRCC; however, none directly target VHL, and the five-year overall survival for metastatic ccRCC remains ~12%. Here, we used a fusion protein, consisting of a deactivated Cas9 (dCas9) linked to the histone acetyltransferase protein p300, to determine if VHL activation initiates programmed cell death in a preclinical model of ccRCC. Methods: Caki-1 cells are a human ccRCC line, are wild-type VHL with low-moderate expression. Caki-1 cells were stably transduced with our dCas9-p300 system via lentivirus, and then transfected with either sgRNAs targeting VHL or a non-targeting (NT) sgRNA. Ten targeting sgRNAs were designed, with eight starting within -120 bp from the VHL1 transcriptional start site (chr3:10141778), and two starting within +50 bp after the TSS. VHL gene expression changes from baseline were evaluated by RT-qPCR at 8h, 24h and 48h after sgRNA transfection. At 24-72h, Caki-1 cells were harvested and stained with Annexin V and propidium iodide; apoptosis and necrosis were then assessed by flow cytometry. Last, to determine whether VHL reactivation triggered necroptosis, MLKL gene expression changes were evaluated by RT-qPCR at 8h, 24h and 48h. Results: Four targeting sgRNAs met internal thresholds for evaluation within our dCas9-300 system: sgRNA3 bp), sgRNA4, sgRNA9, and sgRNA10. Significant increases in VHL expression were observed at 8h for sgRNA3 and sgRNA10 (62.4% and 56.5%, respectively; both P<0.0001, n=3), at 24h for all four sgRNAs (range 91.1-236.1%; all P<0.0001, n=3), and again at 48h for all four sgRNAs (range 84.4-194.4%; P<0.0001, n=3). At 48h and 72h, sgRNA4 (6.78% and 7.35%) and sgRNA9 (6.71% and 6.76%) showed evidence of necroptosis activation. sgRNA4 demonstrated significantly increased MLKL expression at 24h and 48h (199.6% and 144.6%; both P<0.0001, n=3) as did sgRNA9 (192.7% and 235.6%; both P<0.0001, n=3) suggesting necrosome activation. Conclusions: To our knowledge, this is the first study to associate VHL reactivation with increased MLKL expression and necroptosis initiation. These data support continued development of an experimental system we have pioneered (PMID: 31712774), which will leverage chemical-induced proximity to reactivate VHL. Citation Format: Manfred S. Meng, Ryan M. Kemper, Travis J. Nelson, Nathaniel A. Hathaway, Daniel James Crona. VHL1 activation with a CRISPR-based gene activation platform causes increased MLKL expression and triggers necroptosis in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3159.

Abstract 3177: Angiopoietin-like 4 suppress clear cell renal cell carcinoma via inhibition lysosomal acid lipase

Abstract Renal cell carcinomas(RCC) account for about 76,000 new cancer cases annually in the U.S. and 403,000 cases world wide. Clear cell RCC (ccRCC) is both the most common and aggressive subtype, accounting for 75-85% of cases with a 5-year survival rate of 50-69% for all cases and only 10% for metastatic disease. In our study, we report thatANGPTL4 is highly upregulated in ccRCC compared to other types of cancers and normal kidney tissues. However, we identified a cohort of patients with significantly lower expression of ANGPTL4 which had a worse prognosis compared to rest of the patients with higher expression of ANGPTL4. This subset of patients had a lower frequency of mutated VHL and were enriched for genes related to lipid metabolism. In transplant tumor models using RCC cell lines with wild-type VHL, we found that ANGPTL4 suppresses tumor growth. Moreover, loss of ANGPTL4 in ccRCC cell lines reduces colony formation in a lysosomal acid lipase (LAL) dependent manner, a phenotype that is rescued by expression of nANGPTL4. This data indicates that ANGPTL4 suppresses ccRCC tumors in a cancer cell intrinsic manner via regulation of LAL activity. This cohort of patients with low ANGPTL4 may be more dependent on lipid metabolism and less likely to respond to anti-angiogenic TKI plus immune checkpoint inhibitor therapy. Citation Format: Zeng Jin, Umasankar De, Tanzia Tithi, Jeremy Kleberg, Akhila Nataraj, Elena Jolley, Brandon Davies, Weizhou Zhang, Ryan Kolb. Angiopoietin-like 4 suppress clear cell renal cell carcinoma via inhibition lysosomal acid lipase [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 3177.

Abstract B028: Tipifarnib synergizes with axitinib in renal cell carcinoma models

Abstract Clear cell renal cell carcinoma (ccRCC) is a highly vascularized tumor type, primarily due to loss of the Von Hippel-Lindau (VHL) gene that stabilizes hypoxia-inducible factor alpha proteins (HIF1α and HIF2a), primary drivers of the angiogenic response in this cell type. This includes production of angiogenic factors, such as VEGFA, which promotes the formation of blood vessels that provide tumors oxygen, nutrients, and growth factors. Anti-angiogenic tyrosine kinase inhibitors (TKIs) such as the VEGFR1/2/3 inhibitor axitinib have demonstrated therapeutic benefit in RCC patients by exploiting the critical dependency of RCC tumors on their vasculature. However, resistance to TKIs is commonly observed, leading to clinical relapse. Here, we report that the farnesyltransferase inhibitor (FTI), tipifarnib synergizes with axitinib, resulting in tumor regressions in the VHL-mutant RCC cell line-derived xenograft (CDX) models, 786-O and A498, and stasis in VHL-wild type Caki1 CDX and KI-9310 PDX models, despite the modest antitumor activity of either drug as monotherapy. To investigate the mechanism underlying this synergism, we performed immunohistochemical analysis of angiogenic markers in 786-O tumors treated with tipifarnib, axitinib, or the combination. Consistent with its mechanism of action, axitinib alone decreased angiogenesis compared to vehicle, which was evident as reduced expression of endothelial cell (EC) markers in the tumors. The combination of tipifarnib and axitinib, however, led to a further two-fold reduction in EC staining, suggesting that tipifarnib is enhancing the anti-angiogenic effect of axitinib. Further in vitro and in vivo studies are underway to establish the scope and mechanistic underpinnings of the axitinib-tipifarnib effects in RCC. Citation Format: Jovylyn Gatchalian, Linda Kessler, Hetika Vora Patel, Stacia Chan, Francis Burrows, Shivani Malik. Tipifarnib synergizes with axitinib in renal cell carcinoma models [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 B028.

Differential Activation of NRF2 Signaling Pathway in Renal-Cell Carcinoma Caki Cell Lines

Renal-cell carcinoma (RCC) is a heterogeneous disease consisting of several subtypes based on specific genomic profiles and histological and clinical characteristics. The subtype with the highest prevalence is clear-cell RCC (ccRCC), next is papillary RCC (pRCC), and then chromophobe RCC (chRCC). The ccRCC cell lines are further subdivided into prognostic expression-based subtypes ccA or ccB. This heterogeneity necessitates the development, availability, and utilization of cell line models with the correct disease phenotypic characteristics for RCC research. In this study, we focused on characterizing proteomic differences between the Caki-1 and Caki-2 cell lines that are commonly used in ccRCC research. Both cells are primarily defined as human ccRCC cell lines. Caki-1 cell lines are metastatic, harboring wild-type VHL, whereas Caki-2 are considered as the primary ccRCC cell lines expressing wild-type von Hippel-Lindau protein (pVHL). Here, we performed a comprehensive comparative proteomic analysis of Caki-1 and Caki-2 cells using tandem mass-tag reagents together with liquid chromatography mass spectrometry (LC/MS) for the identification and quantitation of proteins in the two cell lines. Differential regulation of a subset of the proteins identified was validated using orthogonal methods including western blot, q-PCR, and immunofluorescence assays. Integrative bioinformatic analysis identifies the activation/inhibition of specific molecular pathways, upstream regulators, and causal networks that are uniquely regulated and associated with the two cell lines and RCC subtypes, and potentially the disease stage. Altogether, we have identified multiple molecular pathways, including NRF2 signaling, which is the most significantly activated pathway in Caki-2 versus Caki-1 cells. Some of the differentially regulated molecules and signaling pathways could serve as potential diagnostic and prognostic biomarkers and therapeutic targets amongst ccRCC subtypes.

PIN1 and CDK1 cooperatively govern pVHL stability and suppressive functions

The VHL protein (pVHL) functions as a tumor suppressor by regulating the degradation or activation of protein substrates such as HIF1α and Akt. In human cancers harboring wild-type VHL, the aberrant downregulation of pVHL is frequently detected and critically contributes to tumor progression. However, the underlying mechanism by which the stability of pVHL is deregulated in these cancers remains elusive. Here, we identify cyclin-dependent kinase 1 (CDK1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) as two previously uncharacterized regulators of pVHL in multiple types of human cancers harboring wild-type VHL including triple-negative breast cancer (TNBC). PIN1 and CDK1 cooperatively modulate the protein turnover of pVHL, thereby conferring tumor growth, chemotherapeutic resistance and metastasis both in vitro and in vivo. Mechanistically, CDK1 directly phosphorylates pVHL at Ser80, which primes the recognition of pVHL by PIN1. PIN1 then binds to phosphorylated pVHL and facilitates the recruitment of the E3 ligase WSB1, therefore targeting pVHL for ubiquitination and degradation. Furthermore, the genetic ablation or pharmacological inhibition of CDK1 by RO-3306 and PIN1 by all-trans retinoic acid (ATRA), the standard care for Acute Promyelocytic Leukemia could markedly suppress tumor growth, metastasis and sensitize cancer cells to chemotherapeutic drugs in a pVHL dependent manner. The histological analyses show that PIN1 and CDK1 are highly expressed in TNBC samples, which negatively correlate with the expression of pVHL. Taken together, our findings reveal the previous unrecognized tumor-promoting function of CDK1/PIN1 axis through destabilizing pVHL and provide the preclinical evidence that targeting CDK1/PIN1 is an appealing strategy in the treatment of multiple cancers with wild-type VHL.

Genomic alterations in metastatic renal cell carcinoma (mRCC): Impact on survival and clinical outcomes.

700 Background: Genomic alterations in RCC can serve as biomarkers for response to therapy. Wild type VHL has shown inferior response to VEGF inhibitors, while patients (pts) with mutated VHL have improved outcomes with immunotherapy (IO)/tyrosine kinase inhibitor (TKI) therapy. We describe the impact of genomic alterations on outcomes and response to front-line therapy. Methods: Pts with mRCC who underwent genomic sequencing between 2015-2022 at Indiana University and were treated with front-line therapy were included. Kaplan-Meier method was used to analyze progression free survival (mPFS) and overall survival (OS) using the log rank test to compare groups. Results: 141 pts were included. Median age was 58yrs (range, 24-81). Tumor histology was 68% clear cell, 10% papillary, 9% unclassified, 1% chromophobe, 1% poorly differentiated, and 9% other. 18% of pts had sarcomatoid features; 13% had rhabdoid. 47% pts had metastasis at diagnosis and 53% at relapse. Metastasis sites were lungs 60%, regional lymph nodes (LNs) 41%, bone 36%, distant LNs 21%, liver 17%, and brain 8%. IMDC risk was 18% good, 48% intermediate, 17% poor, and 17% unknown. Front-line therapy was IO/IO in 21%, IO/TKI in 18%, single-agent TKI in 29%, single-agent IO in 6%, and other in 26%. Genetic alterations included VHL in 50%, PBRM1 in 29%, SETD2 in 23%, BAP1 in 16%, and TP53 in 15%. Median follow-up was 2.74yrs (range 0.1-18.6). Overall mPFS with first line therapy was 1.2yrs (95%CI 0.9-1.5). 2-yr OS was 80% (95%CI: 71-86). For pts with a VHL mutation, mPFS was 1.4yrs (95% CI; 0.9-1.8) compared to 0.9yrs (0.5-1.4) for pts without (p=0.38). 2-yr OS for pts with a VHL mutation was 93% (95% CI: 82-97) vs. 68% (95%CI: 54-78) for pts without (p=0.01). No other mutations impacted overall mPFS or 2-yr OS. mPFS for pts treated with IO/IO was 0.6yrs (95%CI 0.3-0.9) vs. 0.9yrs (95%CI 0.5-1.4) for IO/TKI (p=0.09). 2-yr OS for pts treated with IO/IO was 70% (95% CI 44-85) compared to 94% (95% CI 65-99) for IO/TKI (p=0.29). For pts with SETD2 mutations treated with IO/IO, mPFS was 0.7yrs (95%CI 0.3-1.2) vs. 1.4yrs (95%CI 0.8-1.9) for IO/TKI (p=0.01). For pts with BAP1 mutations treated with IO/IO, mPFS was 0.4yrs (95%CI 0.2-1.2) vs. NE (95%CI 0.2-NE) for IO/TKI (p=0.04). The table outlines outcomes by genomic mutation and therapy. Conclusions: VHL mutation was associated with improved 2-yr OS. Pts with SETD2 and BAP1 mutations treated with IO/TKI had improved mPFS, though no differences in OS. [Table: see text]

Abstract 3293: In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma

Abstract Oncolytic vaccinia virus has several advantages over other OV platforms, including an efficient life cycle, multiple mechanisms of viral spread, large genome, allowing acceptance of foreign therapeutic DNA inserts, and proven safety in humans, due to its use as smallpox vaccine. JX-594 (Pexavec, SillaJen) is an attenuated (TKdeactivated ) oncolytic vaccinia VV, engineered to express GM-CSF and to selectively target tumor over non-tumor tissues. It is currently being investigated as a novel therapeutic option in patients with advanced Renal Cell Carcinoma (RCC, NCT03294083). The present study was undertaken to investigate the effects of JX-954 in a panel of clear and non-clear cell RCC cell lines and characterize the potential mechanisms of JX-594 oncolysis in vitro. We evaluated the oncolytic potency of JX-594 in human clear cell RCCs (VHL mutant 786-0, A498, VHL wild type Caki-1), non-clear cell RCC (ACHN, papillary RCC, and UOK-262, an FH deficient papillary RCC cell) and murine renal cancer cells (RENCA). Potent and persistent cytotoxic effects were induced by JX-594 in all human RCC cells, regardless of the histological subtype, and by mJX-594 (a Western Reserve strain of vaccinia virus expressing murine GMCSF) in RENCA cells. Moreover, we observed efficient viral replication in human (786-0, ACHN) and murine (RENCA) cells, confirming RCC permissiveness to viral infection. Functional proteomics analysis of JX-594 treated 786-0 cells was performed by reverse phase protein array (RPPA) to gain knowledge on potential mechanisms of viral oncolysis. JX-594 treatment had significant effects on the expression levels of proteins related to inducing cell cycle, apoptosis, necrosis, Akt signaling, MAPK signaling, autophagy and stress induction. In particular, the proteins related to cell cycle (Wee1, Cdc25c, CHK1/2, and Cyclin B1) and Akt signaling (PI3K, mTOR, Rictor and FOXO), MAPK signaling (JAK2, PAK1, FAK and MEK1) were down regulated, while proteins involved in apoptosis and necrosis were upregulated. In summary, our studies show that human and murine renal cancer cells are permissive to infection and replication by JX-594 and mJX-594, which induce potent oncolytic effects in clear and non-clear cell RCC, effects associated with significant modulation of RCC pathways associated with cell cycle, proliferation, and survival and stress responses. Citation Format: Yuqi Jing, F Bustamante Guerrero, Jaime Merchan. In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma [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 3293.

Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel–Lindau syndrome

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel–Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel–Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

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.

The Most Common VHL Point Mutation R167Q in Hereditary VHL Disease Interferes with Cell Plasticity Regulation.

Simple SummaryVon Hippel–Lindau (VHL) disease is characterized by mutations in the VHL gene, which can induce numerous benign and malignant tumors in different organs, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). The aim of this study was to examine whether the VHL-R167Q mutation, which is associated with a high risk of developing ccRCC (type 2B VHL disease), could impact the plasticity of renal carcinoma cells. Our transcriptomic results show that VHL-R167Q regulates hypoxia-, plasticity-, and stemness-related genes. Moreover, analysis in a ccRCCs TCGA dataset highlighted that a number of genes regulated by hypoxia and related to stemness in VHL-R167Q-expressing tumors are associated with a poor survival in ccRCCs patients.Von Hippel–Lindau disease (VHL) is a rare hereditary syndrome due to mutations of the VHL tumor suppressor gene. Patients harboring the R167Q mutation of the VHL gene have a high risk of developing ccRCCs. We asked whether the R167Q mutation with critical aspects of pseudo-hypoxia interferes with tumor plasticity. For this purpose, we used wild-type VHL (WT-VHL) and VHL-R167Q reconstituted cells. We showed that WT-VHL and VHL-R167Q expression had a similar effect on cell morphology and colony formation. However, cells transfected with VHL-R167Q display an intermediate, HIF2-dependent, epithelial–mesenchymal phenotype. Using RNA sequencing, we showed that this mutation upregulates the expression of genes involved in the hypoxia pathway, indicating that such mutation is conferring an enhanced pseudo-hypoxic state. Importantly, this hypoxic state correlates with the induction of genes belonging to epithelial–mesenchymal transition (EMT) and stemness pathways, as revealed by GSEA TCGA analysis. Moreover, among these deregulated genes, we identified nine genes specifically associated with a poor patient survival in the TCGA KIRC dataset. Together, these observations support the hypothesis that a discrete VHL point mutation interferes with tumor plasticity and may impact cell behavior by exacerbating phenotypic switching. A better understanding of the role of this mutation might guide the search for more effective treatments to combat ccRCCs.

GIPC2 is an endocrine-specific tumor suppressor gene for both sporadic and hereditary tumors of RET- and SDHB-, but not VHL-associated clusters of pheochromocytoma/paraganglioma

Pheochromocytoma/paraganglioma (PPGL) is an endocrine tumor of the chromaffin cells in the adrenal medulla or the paraganglia. Currently, about 70% of PPGLs can be explained by germline or somatic mutations in several broadly expressed susceptibility genes including RET, VHL, and SDHB, while for the remaining, mainly sporadic cases, the pathogenesis is still unclear. Even for known susceptible genes, how mutations in these mostly ubiquitous genes result in tissue-specific pathogenesis remains unanswered, and why RET-mutated tumors almost always occur in the adrenal while SDHB-mutated tumors mostly occur extra-adrenal remains a mystery. By analyzing 22 sporadic PPGLs using SNP 6.0 genotyping arrays combined with expression profiling of 4 normal and 4 tumor tissues, we identified GIPC2, a gene located at 1p31.1 with preferential expression in adrenal and inducible by adrenal glucocorticoid, as a novel putative tumor suppressor gene for PPGLs. Copy number deletion and GIPC2 promoter hypermethylation but not GIPC2 mutation, accompanied with reduced GIPC2 expression, were observed in 39 of 55 PPGLs in our cohort. Examination of a published expression database consisting of 188 PPGLs found little GIPC2 expression in Cluster 1A (SDHx-associated) and Cluster 2A (NF1/RET-associated) tumors, but less pronounced reduction of GIPC2 expression in Cluster 1B (VHL-associated) and Cluster 2B/2C tumors. GIPC2 induced p27, suppressed MAPK/ERK and HIF-1ɑ pathways as well as cancer cell proliferation. Overexpressing GIPC2 in PC12 cells inhibited tumor growth in nude mice. We found GIPC2 interacted with the nucleoprotein NONO and both proteins regulated p27 transcription through the same GGCC box on p27 promoter. Significantly, low expression of both GIPC2 and p27 was associated with shorter disease-free survival time of PPGLs patients in the TCGA database. We found that PPGL-causing mutations in RET and in SDHB could lead to primary rat adrenal chromaffin cell proliferation, ERK activation, and p27 downregulation, all requiring downregulating GIPC2. Notably, the RET-mutant effect required the presence of dexamethasone while the SDHB-mutant effect required its absence, providing a plausible explanation for the tumor location preference. In contrast, the PPGL-predisposing VHL mutations had no effect on proliferation and GIPC2 expression but caused p53 downregulation and reduced apoptosis in chromaffin cells compared with wild-type VHL. Thus, our study raises the importance of cortical hormone in PPGL development, and GIPC2 as a novel tumor suppressor provides a unified molecular mechanism for the tumorigenesis of both sporadic and hereditary tumors of Clusters 1A and 2A concerning SDHB and RET, but not tumors of Cluster 1B concerning VHL and other clusters.

High VHL Expression Reverses Warburg Phenotype and Enhances Immunogenicity in Kidney Tumor Cells

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. The Von Hippel-Lindau disease tumor suppressor VHL, a known tumor suppressor gene, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1)-regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes, including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins, thus rendering ccRCC cells more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) composed of ten interferon-responsive proteins, whose mRNA expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

Differential sensitivity of renal carcinoma cells to doxorubicin and epigenetic therapeutics depends on the genetic background.

Differential sensitivity to chemotherapeutics is a limitation in chemotherapy of kidney cancer patients. Role of genetic background in chemotherapy is not fully understood. Therefore, this study evaluated the influence of genetic/epigenetic background of renal cancer cells on the sensitivity to chemotherapeutics. Two renal cell carcinoma (RCC) cell lines, Caki-1 and 786-0, with different genetic makeup of p53 and VHL were treated with doxorubicin either alone or in combination with epigenetic therapeutics 5-aza-2-dc and TSA. Sensitivity of RCC cells to these drugs was evaluated by cell viability and cell cycle analysis and was further confirmed by analysis of selected genes expression. Cell viability data revealed that 786-0 cells were more sensitive than Caki-1 to doxorubicin. Combination of doxorubicin with 5-aza-2-dc or TSA was more effective to inhibit growth of Caki-1 cells but not the 786-0. Data of cell cycle analysis and expression of representative genes for tumor suppressor, cell cycle and survival, drug transporter and DNA repair further provided the molecular basis for differential sensitivity of Caki-1 and 786-0 cell lines to doxorubicin. Important findings of this study suggest that doxorubicin is more cytotoxic to primary renal cancer 786-0 cells with mutant VHL and p53 than the metastatic Caki-1 cells with wild-type VHL and p53, and this differential response was independent of p53 expression level. This study suggests that combination of doxorubicin with epigenetic therapeutics could potentially be beneficial in clinical treatment of renal cancer patients with wild-type VHL and p53 but not in patients with mutant VHL and p53.

Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction.

Simple SummaryRenal cell carcinoma (RCC) is the eighth leading malignancy in the world, accounting for 4% of all cancers with poor outcome when metastatic. Protein kinases are highly druggable proteins, which are often aberrantly activated in cancers. The aim of our study was to identify candidate targets for metastatic clear cell renal cell carcinoma therapy, using chemo-genomic-based high-throughput screening. We found that the combined inhibition of the CK2 and ATM kinases in renal tumor cells and patient-derived tumor samples induces synthetic lethality. Mechanistic investigations unveil that this drug combination triggers apoptosis through HIF-2α-(Hypoxic inducible factor HIF-2α) dependent reactive oxygen species (ROS) overproduction, giving a new option for patient care in metastatic RCC.Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

Squalene deters drivers of RCC disease progression beyond VHL status

Identifying drug candidates to target cellular events/signaling that evades von Hippel-Lindau tumor suppressor (VHL) gene interaction is critical for the cure of renal cell carcinoma (RCC). Recently, we characterized a triterpene-squalene derived from marine brown alga. Herein, we investigated the potential of squalene in targeting HIF-signaling and other drivers of RCC progression. Squalene inhibited cell proliferation, induced cell dealth and reverted the cells' metastatic state (migration, clonal expansion) independent of their VHL status. Near-identical inhibition of HIF-1α and HIF-2α and the regulation of downstream targets in VHL wild type and mutant cell lines demonstrated squalene efficacy beyond VHL-HIF interaction. In a rat model of chemically induced RCC, squalene displayed chemopreventive capabilities by substantial reversal of lipid peroxidation, mitochondrial redox regulation, maintaining ∆ψm, inflammation [Akt, nuclear factor κB (NF-κB)], angiogenesis (VEGFα), metastasis [matrix metalloproteinase 2 (MMP-2)], and survival (Bax/Bcl2, cytochrome-c, Casp3). Squalene restored glutathione, glutathione reductase, glutathione-s-transferase, catalase, and superoxide dismutase and stabilized alkaline phosphatase, alkaline transaminase, and aspartate transaminase. The correlation of thiobarbituric acid reactive substance with VEGF/NF-κB and negative association of GSH with Casp3 show that squalene employs reduction in ROS regulation. Cytokinesis-block micronuclei (CBMN) assay in VHLwt/mut cells revealed both direct and bystander effects of squalene with increased micronucleus (MN) frequency. Clastogenicity analysis of rat bone marrow cells demonstrated an anti-clastogenic effect of squalene, with increased polychromatic erythrocytes (PCEs), decreased MNPCE,s and MN normochromatic erythrocytes. Squalene could effectively target HIF signaling that orchestrate RCC evolution. The efficacy of squalene is similar in VHLwt and VHLmut RCC cells, and hence, squalene could serve as a promising drug candidate for an RCC cure beyond VHL status and VHL-HIF interaction dependency. Summary: Squalene derived from marine brown algae displays strong anti-cancer (RCC) activity, functionally targeting HIF-signaling pathway, and affects various cellular process. The significance of squalene effect for RCC is highlighted by its efficiency beyond VHL status, designating itself a promising drug candidate. Graphical abstract.

203P Characterization of renal cell carcinoma (RCC) with VHL mutation

VHL is one of the most commonly mutated oncogene in RCC. However, different co-mutated genes and VHL mutation types are associated with different prognoses and treatments. A more comprehensive understanding of the genomic landscape relative to different VHL variant subsets will help guide therapeutic development. Molecular profiles of 322 RCC samples were obtained using next-generation sequencing of 808 genes (Acornmed Corporation) and classified based on the presence and types of VHL variant. Incidence of VHL mutations was noted across the cohort. Co-occurring genomic alterations and tumor mutational burden (TMB) were analyzed by VHL mutation type. Across the entire cohort, 163 VHL mutations were detected among 154 patients(47.8%). Frameshift mutations were the most prevalent mutation type, accounting for the 42.9%(70/163), followed by missense, nonsense, splicing mutation, copy number loss(42.4%(69/163), 9.2%(15/163), 4.3%(7/163) and 1.2%(2/163) respectively). These mutations were classified into structural variant(58.4%) and non-structural variant(41.6%) based on the effects of variants on protein structure. PBRM1, SETD2, BAP1, KDM5C were commonly co-occurring with VHL(all p<0.05). SETD2 and KDM5C mutations were more common in VHL-nosv RCC, followed by VHL-sv and the VHL-wt. BAP1 has a similar mutation frequency in cases of VHL-sv and VHL-nosv, while VHL-wt had a lower frequency. PBRM1 was mutated in 10.1% of VHL wild type RCC but more frequently noted in both VHL subtype, with the higher rate in VHL-sv (43.3%) and lower in VHL-nosv(29.7%). TMB-H was defined by >12.8 mutations/Mb(upper quartile of the cohort). TMB-H varied across the different VHL mutation subset, most common in VHL-sv (31.1%) and least common in VHL wild type(VHL-wt) (22.2%). VHL mutations are relatively common in Renal cell Carcinoma and VHL structural variant is the most common type. The different VHL mutation has different co-occurring mutations and a different genomic landscape. VHL-sv was associated with the highest rate of TMB and PBRM1 frequency. These different clinical correlates in terms of therapeutic interventions need to be investigated.

Androgen receptor modulates metastatic routes of VHL wild-type clear cell renal cell carcinoma in an oxygen-dependent manner.

Recent studies indicated that the androgen receptor (AR) plays important roles in modulating metastasis of VHL-mutant clear cell renal cell carcinoma (ccRCC). However, the precise mechanisms of AR roles in VHL wild-type (VHL-wt) ccRCC, remain unclear. Here we found that AR interacted with VHL to modulate the metastasis of VHL-wt ccRCC via an oxygen-dependent manner. Mechanism dissection revealed that AR could transcriptionally suppress the miR-185-5p expression in the presence of functional VHL-wt protein under a normoxic condition, which might then result in increasing the expression of VEGF-A and VEGF-C via targeting the 3'UTR of mRNAs at a post-transcriptional level. In contrast, under a hypoxic condition, AR could increase miR-185-5p expression to suppress VEGF-C expression, yet this miR-185-5p effect on VEGF-A was reversed via AR's positive regulation on the HIF2α-increased VEGF-A expression that resulted in increasing VEGF-A in the VHL-wt RCC cells. These distinct AR functions under different oxygen conditions may involve the VHL-impacted ubiquitination and nuclear localization of AR. The differential regulation of VEGF-A vs VEGF-C by AR may then result in differential impacts on the ccRCC metastatic destinations of VHL-wt ccRCC cells under different oxygen conditions. These finer mechanisms may help in the development of a novel therapy to better suppress the ccRCC progression under different oxygenization conditions.

Case report: a synonymous VHL mutation (c.414A\u2009>\u2009G, p.Pro138Pro) causes pathogenic familial hemangioblastoma through dysregulated splicing

Backgroundvon Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood.Case presentationWe report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband’s children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level.ConclusionsOur study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease.

Von Hippel-Lindau Protein Maintains Metabolic Balance to Regulate the Survival of Naive B Lymphocytes.

SummaryB lymphocytes undergo metabolic reprogramming upon activation to meet the bioenergetic demands for proliferation and differentiation. Yet, little is known if and how the fate of naive B cells is metabolically regulated. Here, we specifically delete von Hippel-Lindau protein (VHL) in B cells using CD19-Cre and demonstrate that metabolic balance is essential for naive B cell survival. Loss of VHL disturbs glycolytic and oxidative metabolic balance and causes severe reduction in mature B cells. Mechanistically, the metabolic imbalance in VHL-deficient B cells, arising from over-stabilization of hypoxia-inducible factor-1α (HIF-1α), triggers reductive glutamine metabolism leading to increased Fas palmitoylation and caspase-8-mediated apoptosis. Blockade of reductive glutamine metabolic flux by lactate supplementation and ATP citrate lyase inhibition restores the metabolic balance and rectifies the impaired survival of VHL-deficient B cells. Hence, we unravel that the VHL/HIF-1α pathway is required to maintain the metabolic balance of naive B cells and ensure their survival.

Predictive genomic markers of response to VEGF targeted therapy in metastatic renal cell carcinoma.

BackgroundFirst-line treatment for metastatic renal cell carcinoma (mRCC) is rapidly changing. It currently includes VEGF targeted therapies (TT), multi-target tyrosine kinase inhibitors (TKIs), mTOR inhibitors, and immunotherapy. To optimize outcomes for individual patients, genomic markers of response to therapy are needed. Here, we aim to identify tumor-based genomic markers of response to VEGF TT to optimize treatment selection.MethodsFrom an institutional database, primary tumor tissue was obtained from 79 patients with clear cell mRCC, and targeted sequencing was performed. Clinical outcomes were obtained retrospectively. Progression-free survival (PFS) on first-line VEGF TT was correlated to genomic alterations (GAs) using Kaplan-Meier methodology and Cox proportional hazard models. A composite model of significant GAs predicting PFS in the first-line setting was developed.ResultsAbsence of VHL mutation was associated with inferior PFS on first-line VEGF TT. A trend for inferior PFS was observed with GAs in TP53 and FLT1 C/C variant. A composite model of these 3 GAs was associated with inferior PFS in a dose-dependent manner.ConclusionIn mRCC, a composite model of TP53 mutation, wild type VHL, and FLT1 C/C variant strongly predicted PFS on first-line VEGF TT in a dose-dependent manner. These findings require external validation.