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.

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.

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.

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.

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.

Combined deletion of Vhl, Trp53 and Kif3a causes cystic and neoplastic renal lesions.

The von Hippel-Lindau (VHL) tumour suppressor gene is bi-allelically inactivated in the majority of cases of clear cell renal cell carcinoma (ccRCC); however, Vhl knockout mouse models do not recapitulate human ccRCC, implying that additional mutations are required for tumour formation. Mutational inactivation of VHL sensitises renal epithelial cells to lose the primary cilium in response to other mutations or extracellular stimuli. Loss of cilia is believed to represent a second hit in VHL mutant cells that causes the development of cystic lesions that, in some cases, can progress to ccRCC. Supporting this idea, genetic ablation of the primary cilium by deletion of the kinesin family member 3A (Kif3a) gene cooperates with loss of Vhl to accelerate cyst formation in mouse kidneys. Additionally, aged Vhl/Trp53 double-mutant mice develop renal cysts and tumours at a relatively low incidence, suggesting that there is a genetic cooperation between VHL and TP53 mutation in the development of ccRCC. Here we generated renal epithelium-specific Kif3a/Trp53 and Vhl/Kif3a/Trp53 mutant mice to investigate whether primary cilium deletion would accelerate the development of cystic precursor lesions or cause their progression to ccRCC. Longitudinal microcomputed tomography (µCT) imaging and histopathological analyses revealed an increased rate of cyst formation, increased proportion of cysts with proliferating cells, higher frequency of atypical cysts as well as the development of neoplasms in Vhl/Kif3a/Trp53 mutant kidneys compared to Kif3a/Trp53 or Vhl/Kif3a mutant kidneys. These findings demonstrate that primary cilium loss, in addition to Vhl and Trp53 losses, promotes the transition towards malignancy and provide further evidence that the primary cilium functions as a tumour suppressor organelle in the kidney. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Combined Deletion of Vhl and Kif3a Accelerates Renal Cyst Formation.

A subset of familial and sporadic clear cell renal cell carcinomas (ccRCCs) is believed to develop from cystic precursor lesions. Loss of function of the von Hippel-Lindau tumor suppressor gene (VHL) predisposes renal epithelial cells to loss of the primary cilium in response to specific signals. Because the primary cilium suppresses renal cyst formation, loss of the cilium may be an initiating event in the formation of ccRCC. To test this hypothesis, we analyzed the consequences of inducible renal epithelium-specific deletion of Vhl together with ablation of the primary cilium via deletion of the kinesin family member 3A (Kif3a) gene. We developed a microcomputed tomography-based imaging approach to allow quantitative longitudinal monitoring of cystic burden, revealing that combined loss of Vhl and Kif3a shortened the latency of cyst initiation, increased the number of cysts per kidney, and increased the total cystic burden. In contrast with findings in other cystic models, cysts in Kif3a mutant mice did not display accumulation of hypoxia-inducible factor 1-α (HIF1α), and deletion of both Hif1a and Kif3a did not affect cyst development or progression. Vhl/Kif3a double mutation also increased the frequency of cysts that displayed multilayered epithelial growth, which correlated with an increased frequency of misoriented cystic epithelial cell divisions. These results argue against the involvement of HIF1α in promoting renal cyst growth and suggest that the formation of simple and atypical renal cysts that resemble ccRCC precursor lesions is greatly accelerated by the combined loss of Vhl and the primary cilium.

KPNA2 is overexpressed in human and mouse endometrial cancers and promotes cellular proliferation.

Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.

Multitasking by pVHL in tumour suppression.

Functional inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product, pVHL, leads to cancer in humans. It is widely accepted that pVHL functions to destabilise hypoxia inducible factor alpha (HIFalpha) subunits, key effectors of the hypoxia signalling pathway. However, growing evidence indicates that tumour suppression by pVHL also involves the control of a wide variety of HIFalpha-independent processes including microtubule dynamics, primary cilium maintenance, cell proliferation, neuronal apoptosis, extracellular matrix deposition and responses to DNA damage. Moreover, it is becoming apparent that tumour initiation requires not only VHL mutation but also the alteration of additional cooperating cancer pathways. These studies are beginning to provide insights into the signalling networks involving pVHL that normally control diverse cellular processes and how disruption of these networks leads to tumour formation.

pVHL and GSK3beta are components of a primary cilium-maintenance signalling network.

Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3beta are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3beta alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3beta is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3beta function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.

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.

pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation.

In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.

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.

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.

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.

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.

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.

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.