Targeting HIF-activated collagen prolyl 4-hydroxylase expression disrupts collagen deposition and blocks primary and metastatic uveal melanoma growth.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.

EZH2 targeting reduces medulloblastoma growth through epigenetic reactivation of the BAI1/p53 tumor suppressor pathway.

Medulloblastoma (MB) is a malignant pediatric brain tumor for which new therapies are urgently needed. We demonstrate that treatment with EPZ-6438 (Tazemetostat), an enhancer of zeste homolog 2 (EZH2) inhibitor approved for clinical trials, blocks MB cell growth in vitro and in vivo, and prolongs survival in orthotopic xenograft models. We show that the therapeutic effect is dependent on epigenetic reactivation of adhesion G-protein-coupled receptor B1 (BAI1/ADGRB1), a tumor suppressor that controls p53 stability by blocking Mdm2. Histone 3 trimethylated on lysine 27 (H3K27me3), a marker of silent chromatin conformation is present at the ADGRB1 promoter, and inhibition of EZH2, the catalytic component of the Polycomb Repressive complex 2 (PRC2) that methylates H3K27, switches the gene into an active chromatin status and reactivates BAI1 expression. Mechanistically, targeting EZH2 promotes transition from H3K27me3 to H3K27ac at the promoter, recruits the C/EBPß (CREB-binding protein) and CBP transcription factors and activates ADGRB1 gene transcription. Taken together, our results identify key molecular players that regulate ADGRB1 gene expression in MB, demonstrate that reactivation of BAI1 expression underlies EPZ-6438 antitumorigenic action, and provide preclinical proof-of-principle evidence for targeting EZH2 in patients with MB.

Correction: EZH2 targeting reduces medulloblastoma growth through epigenetic reactivation of the BAI1/p53 tumor suppressor pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Arylsulfonamide 64B Inhibits Hypoxia/HIF-Induced Expression of c-Met and CXCR4 and Reduces Primary Tumor Growth and Metastasis of Uveal Melanoma.

PURPOSE: Uveal melanoma (UM) is the most prevalent and lethal intraocular malignancy in adults. Here, we examined the importance of hypoxia in UM growth and tested the antitumor effects of arylsulfonamide 64B, an inhibitor of the hypoxia-induced factor (HIF) pathway in animal models of UM and investigated the related mechanisms. EXPERIMENTAL DESIGN: UM cells were implanted in the uvea of mice eyes and mice systemically treated with 64B. Drug effect on primary eye tumor growth, circulating tumor cells, metastasis formation in liver, and survival were examined. 64B effects on UM cell growth, invasion and hypoxia-induced expression of C-X-C chemokine receptor type 4 (CXCR4) and mesenchymal-epithelial transition factor (c-Met) were measured. Luciferase reporter assays, chromatin immunoprecipitation, co-immunoprecipitation, and cellular thermal shift assays were used to determine how 64B interferes with the HIF transcriptional complex. RESULTS: Systemic administration of 64B had potent antitumor effects against UM in several orthotopic mouse models, suppressing UM growth in the eye (∼70% reduction) and spontaneous liver metastasis (∼50% reduction), and extending mice survival (P < 0.001) while being well tolerated. 64B inhibited hypoxia-induced expression of CXCR4 and c-Met, 2 key drivers of tumor invasion and metastasis. 64B disrupted the HIF-1 complex by interfering with HIF-1α binding to p300/CBP co-factors, thus reducing p300 recruitment to the MET and CXCR4 gene promoters. 64B could thermostabilize p300, supporting direct 64B binding to p300. CONCLUSIONS: Our preclinical efficacy studies support the further optimization of the 64B chemical scaffold toward a clinical candidate for the treatment of UM.

A novel small-molecule arylsulfonamide causes energetic stress and suppresses breast and lung tumor growth and metastasis.

Neoplastic cells display reprogrammed metabolism due to the heightened energetic demands and the need for biomass synthesis of a growing tumor. Targeting metabolic vulnerabilities is thus an important goal for cancer therapy. Here, we describe a novel small-molecule arylsulfonamide (N-cyclobutyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-3,4-dimethoxybenzenesulfonamide) that exerts potent cytotoxicity and energetic stress on tumor cells while largely sparing non-cancerous human cells. In tumor cells, it stimulates glycolysis and accelerates glucose consumption. Consequently, intracellular ATP levels plummet, triggering activation of AMP-activated protein kinase (AMPK), and diminishing the mammalian target of rapamycin complex 1 (mTORC1) and hypoxia-inducible factor 1 (HIF-1) signaling. In orthotopic triple-negative breast cancer and subcutaneous lung cancer mouse models, this arylsulfonamide robustly suppresses primary tumor growth, inhibits the formation of distant metastases to the lung, and extends mouse survival while being very well tolerated. These therapeutic effects are further potentiated by co-administration of 2-deoxy-D-glucose (2-DG), a glucose analog and glycolysis inhibitor. Collectively, our findings provide preclinical proof of concept for the further development of this arylsulfonamide in combination with 2-DG towards cancer treatment.

Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example.

The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating zinc during expression and streamlining the purification time, resulting in a high yield of optimally folded protein (120 mg per 4 L expression media) that is suitable for structural NMR studies. The structural integrity of the final recombinant p300-CH1 has been verified to be optimal using onedimensional 1H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins.

Design and synthesis of benzopyran-based inhibitors of the hypoxia-inducible factor-1 pathway with improved water solubility.

While progress has been made in treating cancer, cytotoxic chemotherapeutic agents are still the most widely used drugs and are associated with severe side-effects. Drugs that target unique molecular signalling pathways are needed for treating cancer with low or no intrinsic toxicity to normal cells. Our goal is to target hypoxic tumours and specifically the hypoxia inducible factor (HIF) pathway for the development of new cancer therapies. To this end, we have previously developed benzopyran-based HIF-1 inhibitors such as arylsulfonamide KCN1. However, KCN1 and its earlier analogs have poor water solubility, which hamper their applications. Herein, we describe a series of KCN1 analogs that incorporate a morpholine moiety at various positions. We found that replacing the benzopyran group of KCN1 with a phenyl group with a morpholinomethyl moiety at the para positions had minimal effect on potency and improved the water solubility of two new compounds by more than 10-fold compared to KCN1, the lead compound.

A role for activated Cdc42 in glioblastoma multiforme invasion.

Cdc42 is a Rho-GTPase which plays a major role in regulating cell polarity and migration by specifying the localization of filopodia. However, the role of Cdc42 in GBM invasion has not been thoroughly investigated. We generated stable doxycycline-inducible clones expressing wild type (WT)-, constitutively active (CA)-, and dominant negative (DN)-Cdc42 in three different human glioma cell lines. Expression of CA-Cdc42 significantly increased the migration and invasive properties of malignant glioma cells compared to WT and DN-Cdc42 cell clones, and this was accompanied by a greater number of filopodia and focal adhesion structures which co-localize with phosphorylated focal adhesion kinase (FAK). By mass spectrometry and immunoprecipitation studies, we demonstrated that activated Cdc42 binds to IQGAP1. When implanted orthotopically in mice, the CA-Cdc42 expressing glioma cells exhibited enhanced local migration and invasion, and led to larger tumors, which significantly reduced survival. Using the Cancer Genome Atlas dataset, we determined that high Cdc42 expression is associated with poorer progression free survival, and that Cdc42 expression is highest in the proneural and neural subgroups of GBM. In summary, our studies demonstrate that activated Cdc42 is a critical determinant of the migratory and invasive phenotype of malignant gliomas, and that its effect may be mediated, at least in part, through its interaction with IQGAP1 and phosphorylated FAK.

Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex.

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.

Hypoxia inducible factor pathway inhibitors as anticancer therapeutics.

Hypoxia is a significant feature of solid tumor cancers. Hypoxia leads to a more malignant phenotype that is resistant to chemotherapy and radiation, is more invasive and has greater metastatic potential. Hypoxia activates the hypoxia inducible factor (HIF) pathway, which mediates the biological effects of hypoxia in tissues. The HIF complex acts as a transcription factor for many genes that increase tumor survival and proliferation. To date, many HIF pathway inhibitors indirectly affect HIF but there have been no clinically approved direct HIF inhibitors. This can be attributed to the complexity of the HIF pathway, as well as to the challenges of inhibiting protein-protein interactions.

Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1α interaction with cofactors p300/CBP.

PURPOSE: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action. EXPERIMENTAL DESIGN: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1ß/p300 transcription complex. RESULTS: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP. CONCLUSIONS: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression.

Binding Model for the Interaction of Anticancer Arylsulfonamides with the p300 Transcription Cofactor.

Hypoxia inducible factors (HIFs) are transcription factors that activate expression of multiple gene products and promote tumor adaptation to a hypoxic environment. To become transcriptionally active, HIFs associate with cofactors p300 or CBP. Previously, we found that arylsulfonamides can antagonize HIF transcription in a bioassay, block the p300/HIF-1α interaction, and exert potent anticancer activity in several animal models. In the present work, KCN1-bead affinity pull down, (14)C-labeled KCN1 binding, and KCN1-surface plasmon resonance measurements provide initial support for a mechanism in which KCN1 can bind to the CH1 domain of p300 and likely prevent the p300/HIF-1α assembly. Using a previously reported NMR structure of the p300/HIF-1α complex, we have identified potential binding sites in the p300-CH1 domain. A two-site binding model coupled with IC50 values has allowed establishment of a modest ROC-based enrichment and creation of a guide for future analogue synthesis.

Design and synthesis of novel small-molecule inhibitors of the hypoxia inducible factor pathway.

Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility, and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels but interfere with the ability of HIF-1α/HIF-1ß to interact with cofactors p300/CBP to form an active transcriptional complex.

Sulfonamides as a new scaffold for hypoxia inducible factor pathway inhibitors.

Solid tumors generally grow under hypoxic conditions, a pathophysiological change, which activates the expression of genes responsible for malignant, aggressive, and treatment-refractory properties. Hypoxia inducible factor (HIF) is the chief transcription factor regulating hypoxia-driven gene expression. Therefore, the HIF pathway has become a critical target for cancer therapeutics development. We screened a privileged library of about 10,000 natural-product-like compounds using a cell-based assay for HIF-dependent transcriptional activity and identified several arylsulfonamide HIF pathway inhibitors. Among these compounds, the most potent ones showed an IC(50) of ∼0.5 µM in the hypoxia-responsive element (HRE)-luciferase reporter system. Further studies are needed to fully elucidate the mechanism of action of this class of compounds and their structure-activity relationship.

Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.

Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: A one transcription factor (HIF-1) show?

Transcriptional activation by hypoxia is mediated by the hypoxia-inducible factor (HIF) via binding to the hypoxia-responsive element (HRE). Hypoxia in solid tumors associates with poorer outcome of the disease and reliable cellular markers of tumor hypoxia would represent a valuable diagnostic marker and a potential therapeutic target. In this category, carbonic anhydrase IX (CAIX) is one of the most promising candidates. Here, we summarize the knowledge about transcriptional regulation of CA9. The HRE is the central regulatory element in the CA9 promoter, whereas other elements are limited to lesser roles of amplification of signals received at the HRE. The analysis of known mechanisms of activation of CA9 reveals the prominent role of the HIF-1 pathway. Experimental paradigms with uncoupled HIF-1alpha stability and transcriptional activity (pericellular hypoxia, proteasomal inhibitor) provide evidence that CA9 expression monitors transcriptional activity of HIF-1, rather than the abundance of HIF-1alpha. Furthermore, these paradigms could provide a corollary to some of the apparently discordant cases (CAIX+, HIF-1alpha-) or (CAIX-, HIF-1alpha+) observed in vivo. In conclusion, the existing data support the notion that CA9, due to the unique structure of its promoter, is one of the most sensitive endogenous sensors of HIF-1 activity.

PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.

The phosphatidylinositol 3-kinase/Akt (PI3K) pathway regulates hypoxia-inducible factor (HIF) activity. Higher expression of HIF-1alpha and carbonic anhydrase IX (CAIX), a hypoxia-inducible gene, in HT10806TG fibrosarcoma cells (mutant N-ras allele), compared to derivative MCH603 cells (deleted mutant N-ras allele), correlated with increased PI3K activity. Constitutive activation of the PI3K pathway in MCH603/PI3K(act) cells increased HIF-1alpha but, surprisingly, decreased CAIX levels. The cell-type specific inhibitory effect on CAIX was confirmed at the transcriptional level whereas epigenetic modifications of CA9 were ruled out. In summary, our data do not substantiate the generalization that PI3K upregulation leads to increased HIF activity.

Regulation of gene expression by hypoxia: integration of the HIF-transduced hypoxic signal at the hypoxia-responsive element.

Cells experiencing lowered O(2) levels (hypoxia) undergo a variety of biological responses in order to adapt to these unfavorable conditions. The master switch, orchestrating the cellular response to low O(2) levels, is the transcription factor, termed hypoxia-inducible factor (HIF). The alpha subunits of HIF are regulated by 2-oxoglutarate-dependent oxygenases that, in the presence of O(2), hydroxylate specific prolyl and asparaginyl residues of HIF-alpha, inducing its proteasome-dependent degradation and repression of transcriptional activity, respectively. Hypoxia inhibits oxygenases, stabilized HIF-alpha translocates to the nucleus, dimerizes with HIF-beta, recruits the coactivators p300/CBP, and induces expression of its transcriptional targets via binding to hypoxia-responsive elements (HREs). HREs are composite regulatory elements, comprising a conserved HIF-binding sequence and a highly variable flanking sequence that modulates the transcriptional response. In summary, the transcriptional response of a cell is the end product of two major functions. The first (trans-acting) is the level of activation of the HIF pathway that depends on regulation of stability and transcriptional activity of the HIF-alpha. The second (cis-acting) comprises the characteristics of endogenous HREs that are determined by the availability of transcription factors cooperating with HIF and/or individual HIF-alpha isoforms.