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.

SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer, and inactivation of the VHL tumor suppressor gene is found in almost all cases of hereditary and sporadic ccRCCs. CcRCC is associated with the reprogramming of fatty acid metabolism, and stearoyl-CoA desaturases (SCDs) are the main enzymes controlling fatty acid composition in cells. In this study, we report that mRNA and protein expression of the stearoyl-CoA desaturase SCD5 is downregulated in VHL-deficient cell lines. Similarly, in C. elegans vhl-1 mutants, FAT-7/SCD5 activity is repressed, supporting an evolutionary conservation. SCD5 regulation by VHL depends on HIF, and loss of SCD5 promotes cell proliferation and a metabolic shift towards ceramide production. In summary, we identify a novel regulatory function of VHL in relation to SCD5 and fatty acid metabolism, and propose a new mechanism of how loss of VHL may contribute to ccRCC tumor formation and progression.

ATR represents a therapeutic vulnerability in clear cell renal cell carcinoma.

Metastatic clear cell renal cell carcinomas (ccRCCs) are resistant to DNA-damaging chemotherapies, limiting therapeutic options for patients whose tumors are resistant to tyrosine kinase inhibitors and/or immune checkpoint therapies. Here we show that mouse and human ccRCCs were frequently characterized by high levels of endogenous DNA damage and that cultured ccRCC cells exhibited intact cellular responses to chemotherapy-induced DNA damage. We identify that pharmacological inhibition of the DNA damage-sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) with the orally administered, potent, and selective drug M4344 (gartisertib) induced antiproliferative effects in ccRCC cells. This effect was due to replication stress and accumulation of DNA damage in S phase. In some cells, DNA damage persisted into subsequent G2/M and G1 phases, leading to the frequent accumulation of micronuclei. Daily single-agent treatment with M4344 inhibited the growth of ccRCC xenograft tumors. M4344 synergized with chemotherapeutic drugs including cisplatin and carboplatin and the poly(ADP-ribose) polymerase inhibitor olaparib in mouse and human ccRCC cells. Weekly M4344 plus cisplatin treatment showed therapeutic synergy in ccRCC xenografts and was efficacious in an autochthonous mouse ccRCC model. These studies identify ATR inhibition as a potential novel therapeutic option for ccRCC.

MARVEL domain containing CMTM4 affects CXCR4 trafficking.

The MARVEL proteins CMTM4 and CMTM6 control PD-L1, thereby influencing tumor immunity. We found that defective zebrafish cmtm4 slowed the development of the posterior lateral line (pLL) by altering the Cxcr4b gradient across the pLL primordium (pLLP). Analysis in mammalian cells uncovered that CMTM4 interacted with CXCR4, altering its glycosylation pattern, but did not affect internalization or degradation of CXCR4 in the absence of its ligand CXCL12. Synchronized release of CXCR4 from the endoplasmic reticulum revealed that CMTM4 slowed CXCR4 trafficking from the endoplasmic reticulum to the plasma membrane without affecting overall cell surface expression. Altered CXCR4 trafficking reduced ligand-induced CXCR4 degradation and affected AKT but not ERK1/2 activation. CMTM4 expression, in contrast to that of CXCR4, correlated with the survival of patients with renal cell cancer in the TCGA cohort. Furthermore, we observed that cmtm4 depletion promotes the separation of cells from the pLLP cell cluster in zebrafish embryos. Collectively, our findings indicate that CMTM4 exerts general roles in the biosynthetic pathway of cell surface molecules and seems to affect CXCR4-dependent cell migration.

Therapeutic significance of ARID1A mutation in bladder cancer.

Bladder cancer (BC) develops from the tissues of the urinary bladder and is responsible for nearly 200,000 deaths annually. This review aims to integrate knowledge of recently discovered functions of the chromatin remodelling tumour suppressor protein ARID1A in bladder urothelial carcinoma with a focus on highlighting potential new avenues for the development of personalised therapies for ARID1A mutant bladder tumours. ARID1A is a component of the SWI/SNF chromatin remodelling complex and functions to control many important biological processes such as transcriptional regulation, DNA damage repair (DDR), cell cycle control, regulation of the tumour microenvironment and anti-cancer immunity. ARID1A mutation is emerging as a truncal driver mutation that underlies the development of a sub-set of urothelial carcinomas, in cooperation with other driver mutations, to cause dysregulation of a number of key cellular processes. These processes represent tumour drivers but also represent potentially attractive therapeutic targets.

Therapeutic Effects of Inhibition of Sphingosine-1-Phosphate Signaling in HIF-2α Inhibitor-Resistant Clear Cell Renal Cell Carcinoma.

Specific inhibitors of HIF-2α have recently been approved for the treatment of ccRCC in VHL disease patients and have shown encouraging results in clinical trials for metastatic sporadic ccRCC. However, not all patients respond to therapy and pre-clinical and clinical studies indicate that intrinsic as well as acquired resistance mechanisms to HIF-2α inhibitors are likely to represent upcoming clinical challenges. It would be desirable to have additional therapeutic options for the treatment of HIF-2α inhibitor resistant ccRCCs. Here we investigated the effects on tumor growth and on the tumor microenvironment of three different direct and indirect HIF-α inhibitors, namely the HIF-2α-specific inhibitor PT2399, the dual HIF-1α/HIF-2α inhibitor Acriflavine, and the S1P signaling pathway inhibitor FTY720, in the autochthonous Vhl/Trp53/Rb1 mutant ccRCC mouse model and validated these findings in human ccRCC cell culture models. We show that FTY720 and Acriflavine exhibit therapeutic activity in several different settings of HIF-2α inhibitor resistance. We also identify that HIF-2α inhibition strongly suppresses T cell activation in ccRCC. These findings suggest prioritization of sphingosine pathway inhibitors for clinical testing in ccRCC patients and also suggest that HIF-2α inhibitors may inhibit anti-tumor immunity and might therefore be contraindicated for combination therapies with immune checkpoint inhibitors.

Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells.

BACKGROUND: Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. METHODS: We investigated HIF's effect on ENaC expression in mpkCCD cl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. RESULTS: In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of ß ENaC and γ ENaC, as well as of Na,K-ATPase. HIF1 α silencing increased ß ENaC and γ ENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1 α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γ ENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γ ENaC abundance under high sodium intake. CONCLUSIONS: This study reveals that γ ENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

VHL suppresses RAPTOR and inhibits mTORC1 signaling in clear cell renal cell carcinoma.

Inactivation of the tumor suppressor von Hippel-Lindau (VHL) gene is a key event in hereditary and sporadic clear cell renal cell carcinomas (ccRCC). The mechanistic target of rapamycin (mTOR) signaling pathway is a fundamental regulator of cell growth and proliferation, and hyperactivation of mTOR signaling is a common finding in VHL-dependent ccRCC. Deregulation of mTOR signaling correlates with tumor progression and poor outcome in patients with ccRCC. Here, we report that the regulatory-associated protein of mTOR (RAPTOR) is strikingly repressed by VHL. VHL interacts with RAPTOR and increases RAPTOR degradation by ubiquitination, thereby inhibiting mTORC1 signaling. Consistent with hyperactivation of mTORC1 signaling in VHL-deficient ccRCC, we observed that loss of vhl-1 function in C. elegans increased mTORC1 activity, supporting an evolutionary conserved mechanism. Our work reveals important new mechanistic insight into deregulation of mTORC1 signaling in ccRCC and links VHL directly to the control of RAPTOR/mTORC1. This may represent a novel mechanism whereby loss of VHL affects organ integrity and tumor behavior.

Sensitivity and Resistance of Oncogenic RAS-Driven Tumors to Dual MEK and ERK Inhibition.

Oncogenic mutations in RAS family genes arise frequently in metastatic human cancers. Here we developed new mouse and cellular models of oncogenic HrasG12V-driven undifferentiated pleomorphic sarcoma metastasis and of KrasG12D-driven pancreatic ductal adenocarcinoma metastasis. Through analyses of these cells and of human oncogenic KRAS-, NRAS- and BRAF-driven cancer cell lines we identified that resistance to single MEK inhibitor and ERK inhibitor treatments arise rapidly but combination therapy completely blocks the emergence of resistance. The prior evolution of resistance to either single agent frequently leads to resistance to dual treatment. Dual MEK inhibitor plus ERK inhibitor therapy shows anti-tumor efficacy in an HrasG12V-driven autochthonous sarcoma model but features of drug resistance in vivo were also evident. Array-based kinome activity profiling revealed an absence of common patterns of signaling rewiring in single or double MEK and ERK inhibitor resistant cells, showing that the development of resistance to downstream signaling inhibition in oncogenic RAS-driven tumors represents a heterogeneous process. Nonetheless, in some single and double MEK and ERK inhibitor resistant cell lines we identified newly acquired drug sensitivities. These may represent additional therapeutic targets in oncogenic RAS-driven tumors and provide general proof-of-principle that therapeutic vulnerabilities of drug resistant cells can be identified.

HIF-1α and HIF-2α differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice.

Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8+ T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness.

Glucagon-like peptide 2 for intestinal stem cell and Paneth cell repair during graft-versus-host disease in mice and humans.

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2-based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

Tumor necrosis factor stimulates fibroblast growth factor 23 levels in chronic kidney disease and non-renal inflammation.

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis, and its early rise in patients with chronic kidney disease is independently associated with all-cause mortality. Since inflammation is characteristic of chronic kidney disease and associates with increased plasma FGF23 we examined whether inflammation directly stimulates FGF23. In a population-based cohort, plasma tumor necrosis factor (TNF) was the only inflammatory cytokine that independently and positively correlated with plasma FGF23. Mouse models of chronic kidney disease showed signs of renal inflammation, renal FGF23 expression and elevated systemic FGF23 levels. Renal FGF23 expression coincided with expression of the orphan nuclear receptor Nurr1 regulating FGF23 in other organs. Antibody-mediated neutralization of TNF normalized plasma FGF23 and suppressed ectopic renal Fgf23 expression. Conversely, TNF administration to control mice increased plasma FGF23 without altering plasma phosphate. Moreover, in Il10-deficient mice with inflammatory bowel disease and normal kidney function, plasma FGF23 was elevated and normalized upon TNF neutralization. Thus, the inflammatory cytokine TNF contributes to elevated systemic FGF23 levels and also triggers ectopic renal Fgf23 expression in animal models of chronic kidney disease.

IL-8 and CXCR1 expression is associated with cancer stem cell-like properties of clear cell renal cancer.

Recent studies suggest that clear cell renal cell carcinoma (ccRCC) possesses a rare population of cancer stem cells (CSCs) that might contribute to tumor heterogeneity, metastasis and therapeutic resistance. Nevertheless, their relevance for renal cancer is still unclear. In this study, we successfully isolated CSCs from established human ccRCC cell lines. CSCs displayed high expression of the chemokine IL-8 and its receptor CXCR1. While recombinant IL-8 significantly increased CSC number and properties in vitro, CXCR1 inhibition using an anti-CXCR1 antibody or repertaxin significantly reduced these features. After injection into immune-deficient mice, CSCs formed primary tumors that metastasized to the lung and liver. All xenografted tumors in mice expressed high levels of IL-8 and CXCR1. Furthermore, IL-8/CXCR1 expression significantly correlated with decreased overall survival in ccRCC patients. These results suggest that the IL-8/CXCR1 phenotype is associated with CSC-like properties in renal cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mouse genetic background influences whether HrasG12V expression plus Cdkn2a knockdown causes angiosarcoma or undifferentiated pleomorphic sarcoma.

Soft tissue sarcomas are rare mesenchymal tumours accounting for 1% of adult malignancies and are fatal in approximately one third of patients. Two of the most aggressive and lethal forms of soft tissue sarcomas are angiosarcomas and undifferentiated pleomorphic sarcomas (UPS). To examine sarcoma-relevant molecular pathways, we employed a lentiviral gene regulatory system to attempt to generate in vivo models that reflect common molecular alterations of human angiosarcoma and UPS. Mice were intraveneously injected with MuLE lentiviruses expressing combinations of shRNA against Cdkn2a, Trp53, Tsc2 and Pten with or without expression of HrasG12V , PIK3CAH1047R or Myc. The systemic injection of an ecotropic lentivirus expressing oncogenic HrasG12V together with the knockdown of Cdkn2a or Trp53 was sufficient to initiate angiosarcoma and/or UPS development, providing a flexible system to generate autochthonous mouse models of these diseases. Unexpectedly, different mouse strains developed different types of sarcoma in response to identical genetic drivers, implicating genetic background as a contributor to the genesis and spectrum of sarcomas.

Evidence of renal angiomyolipoma neoplastic stem cells arising from renal epithelial cells.

Renal angiomyolipomas (AML) contain an admixture of clonal tumour cells with features of several different mesenchymal lineages, implying the existence of an unidentified AML neoplastic stem cell. Biallelic inactivation of TSC2 or TSC1 is believed to represent the driving event in these tumours. Here we show that TSC2 knockdown transforms senescence-resistant cultured mouse and human renal epithelial cells into neoplastic stem cells that serially propagate renal AML-like tumours in mice. mTOR inhibitory therapy of mouse AML allografts mimics the clinical responses of human renal AMLs. Deletion of Tsc1 in mouse renal epithelia causes differentiation in vivo into cells expressing characteristic AML markers. Human renal AML and a renal AML cell line express proximal tubule markers. We describe the first mouse models of renal AML and provide evidence that these mesenchymal tumours originate from renal proximal tubule epithelial cells, uncovering an unexpected pathological differentiation plasticity of the proximal tubule.

Oncogenic HrasG12V expression plus knockdown of Cdkn2a using ecotropic lentiviral vectors induces high-grade endometrial stromal sarcoma.

The uterine corpus represents the most common site for tumour development in the female genital system. Uterine neoplasms are categorised as epithelial, mesenchymal, mixed epithelial-mesenchymal or trophoblastic tumours. In this study we employed a mouse genetic approach using the MuLE lentiviral gene regulatory system to functionally test the ability of ecotropic lentiviruses to model epithelial and mesenchymal uterine malignancies ex vivo and in vivo. We discovered that MuLE lentiviruses efficiently infect uterine stromal cells but not endometrial epithelial cells when injected into the uterus of cycling, pseudopregnant or ovarectomized mice. Consistent with this cellular infection spectrum, we show that intra-uterine injection of ecotropic MuLE viruses expressing oncogenic HrasG12V together with knockdown of Cdkn2a induce high-grade endometrial stromal sarcomas. These findings establish this approach as an efficient method of generating autochthonous mouse models of uterine sarcomas and in general for performing genetic manipulations of uterine stromal cells in vivo.

Combined mutation in Vhl, Trp53 and Rb1 causes clear cell renal cell carcinoma in mice.

Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel-Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Detecting circulating tumor DNA in renal cancer: An open challenge.

BACKGROUND: Detection of circulating tumor DNA (ctDNA) in blood of cancer patients is regarded as an important step towards personalized medicine and treatment monitoring. In the present study, we investigated the clinical applicability of ctDNA as liquid biopsy in renal cancer. METHODS: ctDNA in serum and plasma samples derived from ccRCC and colon cancer patients as well as ctDNA isolated from RCC xenografts with known VHL mutation status was investigated using next generation sequencing (NGS). Additionally, a Taqman mutation specific assay was used for specific VHL mutation detection in blood. RESULTS: In our study, we successfully identified KRAS mutation in colon cancer patients. We also confirmed the presence of specific VHL mutations in ctDNA derived from RCC xenografts indicating the capability of renal tumors to release DNA into the blood circulation. However, we could not detect any VHL mutation in plasma or serum samples derived from nine ccRCC patients. To increase the sensitivity, a VHL mutation specific Taqman assay was tested. With this approach, the pVHL mutation p.Val130Leu in exon 2 in one patient was successfully detected. CONCLUSION: These data suggest a reduced tumor DNA shedding and an increased clearance of the tumor DNA from the circulation in renal cancer patients independently of tumor size, metastases, and necrosis. This implies that highly sensitive detection methods for mutation calling and prior knowledge of the mutation are required for liquid biopsies in ccRCC.

Vhl deletion in renal epithelia causes HIF-1α-dependent, HIF-2α-independent angiogenesis and constitutive diuresis.

One of the earliest requirements for the formation of a solid tumor is the establishment of an adequate blood supply. Clear cell renal cell carcinomas (ccRCC) are highly vascularized tumors in which the earliest genetic event is most commonly the biallelic inactivation of the VHL tumor suppressor gene, leading to constitutive activation of the HIF-1α and HIF-2α transcription factors, which are known angiogenic factors. However it remains unclear whether either or both HIF-1α or HIF-2α stabilization in normal renal epithelial cells are necessary or sufficient for alterations in blood vessel formation. We show that renal epithelium-specific deletion of Vhl in mice causes increased medullary vascularization and that this phenotype is completely rescued by Hif1a co-deletion, but not by co-deletion of Hif2a. A physiological consequence of changes in the blood vessels of the vasa recta in Vhl-deficient mice is a diabetes insipidus phenotype of excretion of large amounts of highly diluted urine. This constitutive diuresis is fully compensated by increased water consumption and mice do not show any signs of dehydration, renal failure or salt wasting and blood electrolyte levels remain unchanged. Co-deletion of Hif1a, but not Hif2a, with Vhl, fully restored kidney morphology and function, correlating with the rescue of the vasculature. We hypothesize that the increased medullary vasculature alters salt uptake from the renal interstitium, resulting in a disruption of the osmotic gradient and impaired urinary concentration. Taken together, our study characterizes a new mouse model for a form of diabetes insipidus and non-obstructive hydronephrosis and provides new insights into the physiological and pathophysiological effects of HIF-1α stabilization on the vasculature in the kidney.