Lipid dysregulation in renal cancer: Drivers of tumor growth and determinants of treatment-induced toxicity.

Proteostasis Modulators Prolong Missense VHL Protein Activity and Halt Tumor Progression

The Tumor Suppressor von Hippel-Lindau Gene Product and Metastasis: New Thoughts on an Old Molecule

PD64-01 THE STATUS OF THE TUMOR SUPPRESSORS VHL AND CDKN2A IMPACTS CLEAR CELL RENAL CELL CARCINOMA SENSITIVITY TO CDK 4/6 INHIBITORS

Patients with von Hippel-Lindau (VHL) disease often develop VHL-/- kidney cysts, which possibly progress into clear-cell renal carcinomas (ccRCCs). Recent data link the VHL gene product to formation of the primary cilium, an organelle that extends apically into the renal lumen. Exactly how VHL induces ciliogenesis or function is unknown. Here, we demonstrate that ciliary assembly and mechanotransduction is rapidly restored in VHL-/- ccRCC cells upon ectopic reconstitution of wild-type - but not variant alleles of - VHL. These data support and expand recent studies implicating a role for VHL in the initiation of ciliogenesis. Furthermore, reduction of cellular levels of VHL in this cell system was associated with fewer ciliated cells, suggesting a role for VHL in ciliary maintenance.

Focus on kidney cancer

Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models. Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis. 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment. Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.

In clear-cell renal cell carcinoma (ccRCC), inactivation of the tumor suppressor von Hippel-Lindau (VHL) occurs in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently, a large-scale genomic sequencing study of ccRCC tumors revealed that enzymes that regulate histone H3 lysine 4 trimethylation (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might have an important role in regulating gene expression and tumorigenesis. In this study we report that in VHL-deficient ccRCC cells, the overall H3K4Me3 levels were significantly lower than that of VHL+/+ counterparts. Furthermore, this was hypoxia-inducible factor (HIF) dependent, as depletion of HIF subunits by small hairpin RNA in VHL-deficient ccRCC cells restored H3K4Me3 levels. In addition, we demonstrated that only loss of JARID1C, not JARID1A or JARID1B, abolished the difference of H3K4Me3 levels between VHL-/- and VHL+/+ cells, and JARID1C displayed HIF-dependent expression pattern. JARID1C in VHL-/- cells was responsible for the suppression of HIF-responsive genes insulin-like growth factor-binding protein 3 (IGFBP3), DNAJC12, COL6A1, growth and differentiation factor 15 (GDF15) and density-enhanced phosphatase 1. Consistent with these findings, the H3K4Me3 levels at the promoters of IGFBP3, DNAJC12, COL6A1 and GDF15 were lower in VHL-/- cells than in VHL+/+ cells, and the differences disappeared after JARID1C depletion. Although HIF2α is an oncogene in ccRCC, some of its targets might have tumor suppressive activity. Consistent with this, knockdown of JARID1C in 786-O VHL-/- ccRCC cells significantly enhanced tumor growth in a xenograft model, suggesting that JARID1C is tumor suppressive and its mutations are tumor promoting in ccRCC. Thus, VHL inactivation decreases H3K4Me3 levels through JARID1C, which alters gene expression and suppresses tumor growth.

Background: Von Hippel-Lindau (VHL) disease is an autosomal-dominant hereditary cancer syndrome. Currently, studies on tyrosine kinase inhibitor (TKI) therapy for VHL disease are scarce. In this study, we retrospectively evaluated the efficacy and safety of four TKIs in patients with VHL disease.Methods: Patients diagnosed with VHL disease who were receiving TKIs were recruited. Patients were treated with sunitinib (n = 12), sorafenib (n = 11), axitinib (n = 6), or pazopanib (n = 3). The therapeutic response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.Results: From July 2009 to September 2018, 32 patients with VHL disease were eligible and included in this study. The median duration of TKI therapy was 22 months (IQR 8.5–44.75), and the median follow-up period was 31.5 months (IQR 13.5–63.5). According to the RECIST, 9 (28%) of 32 patients showed a partial response, 15 (47%) achieved stable disease, and eight exhibited continued disease progression. A partial response was observed in 11 (31%) of 36 renal cell carcinomas, 4 (27%) of 15 pancreatic lesions, and 1 (20%) of five central nervous system (CNS) hemangioblastomas. The average tumor size decreased significantly for renal cell carcinomas (P = 0.0001), renal cysts (P = 0.027), and pancreatic lesions (P = 0.003) after TKI therapy. Common side effects included hand–foot skin reactions, diarrhea, alopecia, thrombocytopenia, and fatigue.Conclusions: Partial alleviation of VHL disease-related tumors can be achieved by TKI therapies in some patients, providing an alternative treatment strategy, and the side effects of TKIs are acceptable. Larger prospective studies are warranted to further evaluate the efficacy and safety of TKIs in patients with VHL disease.

von Hippel Lindau (VHL) disease is a hereditary cancer syndrome caused by biallelic inactivation of the VHL tumor suppressor gene. The most widely known function of VHL is to limit normoxic protein expression of hypoxia-inducible factor-alpha (HIF-alpha). Loss of the functional VHL gene causes constitutive stabilization of HIF-alpha that primarily up-regulates hypoxia-inducible genes even at normal oxygen concentration, which in turn contribute to VHL tumor progression. We report on the novel function of VHL in hepatic glucose storage and disposal. VHL deletion in adult mouse liver quickly leads to increased accumulation of glycogen granules as well as lipid droplets. This abnormal glycogen storage in VHL-inactivated liver arises at least in part from significantly reduced expression of two key liver-specific glucose metabolism genes, glucose transporter-2 (GLUT2) and glucose-6-phosphatase (G-6-Pase). The expression pattern of these genes in VHL knock-out liver was in contrast to that of well-known HIF target genes, such as PGK, Glut-1, VEGF, and EPO, all of which are highly elevated upon VHL inactivation. Our findings suggest that two distinct signaling pathways exist at the downstream of VHL controlling different sets of gene expression. Following VHL inactivation, one pathway causes oxygen-independent overexpression of classic hypoxia-inducible genes and the other one described here suppresses expression of the genes important for liver glucose metabolism.

INTRODUCTION: Clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, carries a poor prognosis, with an estimated median overall survival time of only two years. This is often a clinically silent disease and about one third of patients present with metastases. Although inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) is a well-characterized driver event in ccRCC, the exact molecular underpinnings of this disease remain unclear. To address this gap, we performed a transcriptomic analysis, focusing on VHL-dependent long noncoding RNAs (lncRNAs). LncRNAs are known to be involved in cancer progression, but their role in ccRCC oncogenesis has not been extensively studied. Accordingly, the aim of the present study is to characterize lncRNA transcripts that are differentially associated with VHL inactivation status. We anticipate that characterization of the lncRNA expression landscape in ccRCC will enable the identification of biomarkers and novel therapeutic targets for this disease. METHODS: Transcriptome-wide array-based analyses were performed on total RNA derived from the 786-O (VHL-/-) ccRCC cell line, stably reconstituted with either wild-type VHL (786-O-VHL) or mutant VHL (786-O-C162F). Differential lncRNA analysis was conducted using the Arraystar Human LncRNA V4.0 array. Statistical analyses were performed using Agilent GeneSpring GX v12.1 software, with a false discovery rate (FDR) adjusted p-value < 0.05 used as a threshold for significance of differential lncRNA expression. LncRNAs were cross-referenced to data generated from The Cancer Genome Atlas (TCGA), a publicly available pan-cancer database, to identify those predictive of overall survival. Finally, qRT-PCR was used to validate the most highly differentially dysregulated lncRNAs in another primary ccRCC cell type (RCC4) and in paired kidney tissue. RESULTS: A total of 360 lncRNA transcripts were differentially expressed four-fold or greater in 786-O-C162F cells relative to 786-O-VHL cells. Of these lncRNAs, 269 were upregulated and 91 were downregulated. Cross-referencing to TCGA, 52 of the upregulated lncRNAs and 23 of the downregulated lncRNAs were predictive of overall survival in ccRCC patients, as evidenced by statistically significant Cox regression and Log-rank p-values (p < 0.05). The top five up- and downregulated lncRNAs were validated with qRT-PCR. CONCLUSION: To our knowledge, this is one of the only analyses to systematically assess lncRNA expression in ccRCC, and the first to assess lncRNA expression in a VHL-dependent manner. We anticipate that these molecular and clinical findings will provide a framework for utilizing VHL inactivation status as a biomarker to stratify patients with ccRCC and associated molecular targets for further study. Future inter-institutional collaborations are needed to validate these findings in a large cohort of primary ccRCC samples. Citation Format: Joseph N. Samuel, Philip A. Marsden. Transcriptomic profiling of VHL-dependent long noncoding RNAs in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1815.

ImportanceRenal cell carcinoma (RCC) is a common malignancy, with an estimated 434 840 incident cases worldwide in 2022. In the US, it is the sixth most common cancer among males and ninth among females.ObservationsClear cell RCC is the most common histologic subtype (75%-80% of cases) and is characterized by inactivation of the von Hippel Lindau (VHL) tumor suppressor gene. Many patients (37%-61%) are diagnosed with RCC incidentally on an abdominal imaging study such as ultrasound or computed tomographic scan, and 70% of patients have stage I RCC at diagnosis. Although its incidence has increased approximately 1% per year from 2015 through 2019, the mortality rate of RCC has declined about 2% per year in the US from 2016 through 2020. Patients with a solid renal mass or complex cystic renal mass should be referred to urology. Treatment options for RCC confined to the kidney include surgical resection with partial or radical nephrectomy, ablative techniques (eg, cryoablation, radiofrequency ablation, radiation), or active surveillance for some patients (especially those with renal masses <2 cm). For patients with renal masses less than 4 cm in size (48% of patients), partial nephrectomy can result in a 5-year cancer-specific survival of more than 94%. For advanced or metastatic RCC, combinations of immune checkpoint inhibitors or the combination of immune checkpoint inhibitors with tyrosine kinase inhibitors are associated with tumor response of 42% to 71%, with a median overall survival of 46 to 56 months.Conclusions and RelevanceRCC is a common malignancy that is often diagnosed incidentally on an abdominal imaging study. Seventy percent of patients are diagnosed with stage I RCC and 11% of patients with stage IV. First-line treatments for early-stage RCC are partial or radical nephrectomy, which can result in 5-year cancer-specific survival of more than 94%, ablative techniques, or active surveillance. New treatment options for patients with metastatic RCC include immune checkpoint inhibitors and tyrosine kinase inhibitors.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.

The von Hippel-Lindau (VHL) tumor suppressor gene has been shown to be mutated frequently not only in neoplasms from von Hippel-Lindau disease, but also in sporadic clear cell renal carcinoma. In order to reveal the possible role of the VHL tumor suppressor gene in the development of ovarian carcinoma, a total of 71 primary sporadic ovarian carcinomas were analyzed for the presence of mutations in the exon 2 and 3 of the VHL tumor suppressor gene, using the polymerase chain reaction with single strand conformation polymorphism analysis. No mutations in the VHL gene were found in any of the tumors analyzed. This result shows that the VHL tumor suppressor gene does not play a major role in the tumorigenesis of sporadic ovarian carcinoma.

Various conversion therapy options have become available to patients with unresectable HCC, but which conversion therapy is optimal for which type of patient is controversial. This study compared the efficacy and safety of TACE alone or combined with immune checkpoint and tyrosine kinase inhibitors. Data were retrospectively compared for patients with initially unresectable HCC who underwent conversion therapy consisting of TACE alone (n=459) or combined with immune checkpoint and tyrosine kinase inhibitors (n=343). Compared to the group that received TACE alone, the group that received triple conversion therapy showed significantly higher rates of overall survival (HR 0.43, 95%CI 0.35-0.53). In addition, triple therapy was associated with significantly longer median progression-free survival (15.9 vs. 8.0mo, p <0.001). These results were confirmed in matched subsets of patients from each group. However, subgroup analysis confirmed the results only for patients with HCC in intermediate or advanced stages, not in an early stage. Those who received triple conversion therapy had a significantly higher rate of hepatectomy after conversion therapy (36.4 vs. 23.5%, p <0.001). Among those who underwent hepatectomy after conversion therapy, triple therapy was associated with a significantly higher rate of complete tumor response (32.1 vs. 11.1%, p <0.001). However, it was also associated with a significantly higher frequency of serious adverse events (35.6 vs. 27.0%, p =0.009). Combining TACE with immune checkpoint and tyrosine kinase inhibitors was associated with significantly better survival and conversion efficacy than TACE alone among patients with intermediate or advanced unresectable HCC.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene and its protein product, pVHL, occurs in ~90% of clear cell renal cell carcinoma (ccRCC) cases. pVHL is part of the ubiquitin ligase complex that targets the HIF2α transcription factor, an oncoprotein in ccRCC, for proteasomal degradation. Without functional pVHL, HIF2α accumulates and induces inappropriate transcription of angiogenic, invasive, and growth-promoting genes. Drugs inhibiting HIF2α or its downstream target VEGF are active against ccRCC, but both de novo and acquired resistance are common and no current therapies are curative. We hypothesized that loss of pVHL might create context-specific dependencies that could then be targeted therapeutically. To look for such synthetic lethal targets, we generated human and Drosophila cell pairs that were isogenic for pVHL (or its Drosophila ortholog) and screened using RNAi and chemical compound libraries. The overlap of the hits from these screens identified a hyperdependence on CDK4/6 activity in pVHL-defective cells compared to their pVHL-proficient counterparts. In secondary assays we confirmed that pharmacologic inhibition of CDK4/6 by either Abemaciclib or Palbociclib preferentially reduced viability of VHL-/-cells as compared to VHL+/+cells across a variety of human ccRCC cell lines. Importantly, inhibition of VHL-/-ccRCC cells by Palbociclib was abrogated by expressing a Palbociclib-resistant CDK6 cDNA or by knockout of the canonical CDK4/6 target pRB. Sensitivity to Palbociclib could be reversed by expressing exogenous pVHL, but not a pVHL mutant lacking its known substrate docking site. HIF2aknockout in VHL-/-cells did not eliminate the effect of CDK4/6 inhibition, thus HIF2ais not necessary for the synthetic lethal relationship between VHLand CDK4/6. Moreover, the combination of the HIF2ainhibitor PT2399 and the CDK4/6 inhibitor Palbociclib synergistically suppressed proliferation of VHL-/-ccRCC in vitro in HIF2a-sensitive cell lines. Both Palbociclib and Abemaciclib suppress VHL-/-ccRCC tumor growth in nude mice, including tumors that are PT2399-resistant. My future work will aim to identify the mechanism that underlies the synthetic lethality between VHLand CDK4/6 activity. Citation Format: Hilary Nicholson, Benjamin Housden, Norbert Perrimon, William G Kaelin. HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C124. doi:10.1158/1535-7163.TARG-19-C124