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Circadian disruption enhances cancer risk, and many tumors exhibit disordered circadian gene expression. We show rhythmic gene expression is unexpectedly robust in clear cell renal cell carcinoma (ccRCC). Furthermore, the clock gene BMAL1 is higher in ccRCC than in healthy kidneys, unlike in other tumor types. BMAL1 is closely related to ARNT, and we show that BMAL1-HIF2α regulates a subset of HIF2α target genes in ccRCC cells. Depletion of BMAL1 reprograms HIF2α chromatin association and target gene expression and reduces ccRCC growth in culture and in xenografts. Analysis of pre-existing data reveals higher BMAL1 in patient-derived xenografts that are sensitive to growth suppression by a HIF2α antagonist (PT2399). We show that BMAL1-HIF2α is more sensitive than ARNT-HIF2α to suppression by PT2399, and increasing BMAL1 sensitizes 786O cells to growth inhibition by PT2399. Together, these findings indicate that an alternate HIF2α heterodimer containing the circadian partner BMAL1 contributes to HIF2α activity, growth, and sensitivity to HIF2α antagonist drugs in ccRCC cells.

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Circadian disruption enhances cancer risk, and many tumors exhibit disordered circadian gene expression. We show rhythmic gene expression is unexpectedly robust in clear cell renal cell carcinoma (ccRCC). Furthermore, the clock gene BMAL1 is higher in ccRCC than in healthy kidneys, unlike in other tumor types. BMAL1 is closely related to ARNT, and we show that BMAL1-HIF2α regulates a subset of HIF2α target genes in ccRCC cells. Depletion of BMAL1 reprograms HIF2α chromatin association and target gene expression and reduces ccRCC growth in culture and in xenografts. Analysis of pre-existing data reveals higher BMAL1 in patient-derived xenografts that are sensitive to growth suppression by a HIF2α antagonist (PT2399). We show that BMAL1-HIF2α is more sensitive than ARNT-HIF2α to suppression by PT2399, and increasing BMAL1 sensitizes 786O cells to growth inhibition by PT2399. Together, these findings indicate that an alternate HIF2α heterodimer containing the circadian partner BMAL1 contributes to HIF2α activity, growth, and sensitivity to HIF2α antagonist drugs in ccRCC cells.

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Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows great but incomplete benefits, underscoring the need to better understand the roles of glutamine and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria across diverse cell types, excluding ccRCC. GLS1 clustering is rapid (1-3 hours) and reversible, is specifically driven by the level of intracellular glutamate, and is mediated by mitochondrial fission. Clustered GLS1 has markedly enhanced glutaminase activity and promotes cell death under glutamine-deprived conditions. We further show that HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby protecting ccRCC cells from cell death induced by glutamine deprivation. Reversing this protection, by genetic expression of GLS1 mutants that constitutively cluster, enhances ccRCC cell death in culture and suppresses ccRCC growth in vivo . These finding provide multiple insights into cellular glutamine handling, including a novel metabolic pathway by which HIF2α promotes ccRCC, and reveals a potential therapeutic avenue to synergize with HIF2α inhibition in the treatment of ccRCC.

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The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell autonomous EMT in PDAC cells, which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest that hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multidrug therapies, providing a potential strategy for overcoming chemoresistance. SIGNIFICANCE: Integrated regulation of histone methylation and MAPK signaling by the low-oxygen environment of pancreatic cancer drives long-lasting EMT that promotes chemoresistance and shortens patient survival and that can be pharmacologically inhibited. See related commentary by Wirth and Schneider, p. 1739.

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Intratumoral hypoxia correlates with metastasis and poor survival in patients with sarcoma. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated here that the hypoxia-inducible collagen-modifying enzyme lysyl hydroxylase PLOD2 and its substrate collagen type VI (COLVI) weaken the lung endothelial barrier and promote transendothelial migration. Mechanistically, hypoxia-induced PLOD2 in sarcoma cells modified COLVI, which was then secreted into the vasculature. Upon reaching the apical surface of lung endothelial cells, modified COLVI from tumor cells activated integrin ß1 (ITGß1). Furthermore, activated ITGß1 colocalized with Kindlin2, initiating their interaction with F-actin and prompting its polymerization. Polymerized F-actin disrupted endothelial adherens junctions and induced barrier dysfunction. Consistently, modified and secreted COLVI was required for the late stages of lung metastasis in vivo. Analysis of patient gene expression and survival data from The Cancer Genome Atlas (TCGA) revealed an association between the expression of both PLOD2 and COLVI and patient survival. Furthermore, high levels of COLVI were detected in surgically resected sarcoma metastases from patient lungs and in the blood of tumor-bearing mice. Together, these data identify a mechanism of sarcoma lung metastasis, revealing opportunities for therapeutic intervention. SIGNIFICANCE: Collagen type VI modified by hypoxia-induced PLOD2 is secreted by sarcoma cells and binds to integrin ß1 on endothelial cells to induce barrier dysfunction, which promotes sarcoma vascular dissemination and metastasis.

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Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target. SIGNIFICANCE: The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.

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Availability of the essential amino acid methionine affects cellular metabolism and growth, and dietary methionine restriction has been implicated as a cancer therapeutic strategy. Nevertheless, how liver cancer cells respond to methionine deprivation and underlying mechanisms remain unclear. Here we find that human liver cancer cells undergo irreversible cell cycle arrest upon methionine deprivation in vitro. Blocking methionine adenosyl transferase 2A (MAT2A)-dependent methionine catabolism induces cell cycle arrest and DNA damage in liver cancer cells, resulting in cellular senescence. A pharmacological screen further identified GSK3 inhibitors as senolytics that selectively kill MAT2A-inhibited senescent liver cancer cells. Importantly, combined treatment with MAT2A and GSK3 inhibitors therapeutically blunts liver tumor growth in vitro and in vivo across multiple models. Together, methionine catabolism is essential for liver tumor growth, and its inhibition can be exploited as an improved pro-senescence strategy for combination with senolytic agents to treat liver cancer.

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Kidney cancer is the seventh leading cause of cancer in the world, and its incidence is on the rise. Renal cell carcinoma (RCC) is the most common form and is a heterogeneous disease comprising three major subtypes that vary in their histology, clinical course and driver mutations. These subtypes include clear cell RCC, papillary RCC and chromophobe RCC. Molecular analyses of hereditary and sporadic forms of RCC have revealed that this complex and deadly disease is characterized by metabolic pathway alterations in cancer cells that lead to deregulated oxygen and nutrient sensing, as well as impaired tricarboxylic acid cycle activity. These metabolic changes facilitate tumour growth and survival. Specifically, studies of the metabolic features of RCC have led to the discovery of oncometabolites - fumarate and succinate - that can promote tumorigenesis, moonlighting functions of enzymes, and substrate auxotrophy owing to the disruption of pathways that enable the production of arginine and cholesterol. These metabolic alterations within RCC can be exploited to identify new therapeutic targets and interventions, in combination with novel approaches that minimize the systemic toxicity of metabolic inhibitors and reduce the risk of drug resistance owing to metabolic plasticity.

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Molecular oxygen (O2) is essential for cellular bioenergetics and numerous biochemical reactions necessary for life. Solid tumors outgrow the native blood supply and diffusion limits of O2, and therefore must engage hypoxia response pathways that evolved to withstand acute periods of low O2 Hypoxia activates coordinated gene expression programs, primarily through hypoxia inducible factors (HIFs), to support survival. Many of these changes involve metabolic rewiring such as increasing glycolysis to support ATP generation while suppressing mitochondrial metabolism. Since low O2 is often coupled with nutrient stress in the tumor microenvironment, other responses to hypoxia include activation of nutrient uptake pathways, metabolite scavenging, and regulation of stress and growth signaling cascades. Continued development of models that better recapitulate tumors and their microenvironments will lead to greater understanding of oxygen-dependent metabolic reprogramming and lead to more effective cancer therapies.

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Globally, hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related death. We previously identified an immune evasion pathway whereby tumor cells produce retinoic acid (RA) to promote differentiation of intratumoral monocytes into protumor macrophages. Retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, and RALDH3 are the three isozymes that catalyze RA biosynthesis. In this study, we have identified RALDH1 as the key driver of RA production in HCC and demonstrated the efficacy of RALDH1-selective inhibitors (Raldh1-INH) in suppressing RA production by HCC cells. Raldh1-INH restrained tumor growth in multiple mouse models of HCC by reducing the number and tumor-supporting functions of intratumoral macrophages as well as increasing T-cell infiltration and activation within tumors. Raldh1-INH also displayed favorable pharmacokinetic, pharmacodynamic, and toxicity profiles in mice thereby establishing them as promising new drug candidates for HCC immunotherapy.

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Gene expression states persist for varying lengths of time at the single-cell level, a phenomenon known as gene expression memory. When cells switch states, losing memory of their prior state, this transition can occur in the absence of genetic changes. However, we lack robust methods to find regulators of memory or track state switching. Here, we develop a lineage tracing-based technique to quantify memory and identify cells that switch states. Applied to melanoma cells without therapy, we quantify long-lived fluctuations in gene expression that are predictive of later resistance to targeted therapy. We also identify the PI3K and TGF-ß pathways as state switching modulators. We propose a pretreatment model, first applying a PI3K inhibitor to modulate gene expression states, then applying targeted therapy, which leads to less resistance than targeted therapy alone. Together, we present a method for finding modulators of gene expression memory and their associated cell fates.

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Over the last two decades, the rapidly expanding field of tumor metabolism has enhanced our knowledge of the impact of nutrient availability on metabolic reprogramming in cancer. Apart from established roles in cancer cells themselves, various nutrients, metabolic enzymes, and stress responses are key to the activities of tumor microenvironmental immune, fibroblastic, endothelial, and other cell types that support malignant transformation. In this article, we review our current understanding of how nutrient availability affects metabolic pathways and responses in both cancer and "stromal" cells, by dissecting major examples and their regulation of cellular activity. Understanding the relationship of nutrient availability to cellular behaviors in the tumor ecosystem will broaden the horizon of exploiting novel therapeutic vulnerabilities in cancer.

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Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2α transcription factor, and we demonstrate here that HIF-2α drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2α-driven speckle association via deletion of its speckle targeting motifs (STMs)-defined in this study-led to defective induction of speckle-associating HIF-2α target genes without impacting non-speckle-associating HIF-2α target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2α gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2α. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2α-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation.