Cathepsins L and B target HIF1α for oxygen-independent proteolytic cleavage.

The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.

Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome.

Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.

SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma.

Survival rates among patients with high-risk neuroblastoma remain low and novel therapies for recurrent neuroblastomas are required. ALK is commonly mutated in primary and relapsed neuroblastoma tumors and ALK tyrosine kinase inhibitors (TKI) are promising treatments for ALK-driven neuroblastoma; however, innate or adaptive resistance to single-agent ALK-TKIs remain a clinical challenge. Recently, SHP2 inhibitors have been shown to overcome ALK-TKI resistance in lung tumors harboring ALK rearrangements. Here, we have assessed the efficacy of the SHP2 inhibitor TNO155 alone and in combination with the ALK-TKIs crizotinib, ceritinib, or lorlatinib for the treatment of ALK-driven neuroblastoma using in vitro and in vivo models. In comparison to wild-type, ALK-mutant neuroblastoma cell lines were more sensitive to SHP2 inhibition with TNO155. Moreover, treatment with TNO155 and ALK-TKIs synergistically reduced cell growth and promoted inactivation of ALK and MAPK signaling in ALK-mutant neuroblastoma cells. ALK-mutant cells engrafted into larval zebrafish and treated with single agents or dual SHP2/ALK inhibitors showed reduced growth and invasion. In murine ALK-mutant xenografts, tumor growth was likewise reduced or delayed, and survival was prolonged upon combinatorial treatment of TNO155 and lorlatinib. Finally, we show that lorlatinib-resistant ALK-F1174L neuroblastoma cells harbor additional RAS-MAPK pathway alterations and can be resensitized to lorlatinib when combined with TNO155 in vitro and in vivo. Our results report the first evaluation of TNO155 in neuroblastoma and suggest that combinatorial inhibition of ALK and SHP2 could be a novel approach to treating ALK-driven neuroblastoma, potentially including the increasingly common tumors that have developed resistance to ALK-TKIs. SIGNIFICANCE: These findings highlight the translatability between zebrafish and murine models, provide evidence of aberrant RAS-MAPK signaling as an adaptive mechanism of resistance to lorlatinib, and demonstrate the clinical potential for SHP2/ALK inhibitor combinations for the treatment of ALK-mutant neuroblastoma, including those with acquired tolerance or potentially resistance to ALK-TKIs.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Hypoxia-inducible factor underlies von Hippel-Lindau disease stigmata.

von Hippel-Lindau (VHL) disease is a rare hereditary cancer syndrome that causes a predisposition to renal clear-cell carcinoma, hemangioblastoma, pheochromocytoma, and autosomal-recessive familial polycythemia. pVHL is the substrate conferring subunit of an E3 ubiquitin ligase complex that binds to the three hypoxia-inducible factor alpha subunits (HIF1-3α) for polyubiquitylation under conditions of normoxia, targeting them for immediate degradation by the proteasome. Certain mutations in pVHL have been determined to be causative of VHL disease through the disruption of HIFα degradation. However, it remains a focus of investigation and debate whether the disruption of HIFα degradation alone is sufficient to explain the complex genotype-phenotype relationship of VHL disease or whether the other lesser or yet characterized substrates and functions of pVHL impact the development of the VHL disease stigmata; the elucidation of which would have a significant ramification to the direction of research efforts and future management and care of VHL patients and for those manifesting sporadic counterparts of VHL disease. Here, we examine the current literature including the other emergent pseudohypoxic diseases and propose that the VHL disease-phenotypic spectrum could be explained solely by the varied disruption of HIFα signaling upon the loss or mutation in pVHL.

D154Q Mutation does not Alter KRAS Dimerization.

KRAS is one of the most frequently mutated oncogenes in human cancers. Despite nearly 40 years of research, KRAS remains largely undruggable, in part due to an incomplete understanding of its biology. Recently, KRAS dimerization was discovered to play an important role in its signalling function. The KRAS D154Q mutant was described as a dimer-deficient variant that can be used to study the effect of dimerization in KRAS oncogenicity. However, we show here that KRAS D154Q homo- and heterodimerized with KRAS WT using three separate protein-protein interaction assays, and that oncogenic KRAS dimerization was not negatively impacted by the presence of a secondary D154Q mutation. In conclusion, we advise caution in using this variant to study the purpose of dimerization in KRAS oncogenic behaviour.

The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Side population analysis in clear cell renal cell carcinoma.

Cancer stem cells have an important role in tumour biology. While their identity in haematological malignancies is clearly defined, stem cell identity remains elusive in some solid tumours. Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer, but the identity or existence of ccRCC stem cells remains unknown. We aimed to discern their existence using the widely utilised side population approach in ccRCC cell lines. In all cells tested, a well-defined side population was identified, and cell-based assays suggested stem-like properties. However, limiting dilution assays revealed comparable tumour initiating abilities and tumour histology of side and non-side populations, and single cell RNA-sequencing revealed minimal differences between these populations. The results indicate that the side population approach is not sufficient for cancer stem cell discovery in ccRCC.

HIF-1α Hydroxyprolines Modulate Oxygen-Dependent Protein Stability Via Single VHL Interface With Comparable Effect on Ubiquitination Rate.

The basic molecular mechanism underlying mammalian oxygen-dependent regulation of hypoxia-inducible factor (HIF) via the von Hippel-Lindau E3 ubiquitin ligase is well established. The principal step in this critical cellular process is the hydroxylation of either or both of the two conserved proline residues P402 and P564 within the oxygen-dependent degradation domain (ODD) of HIF-1α subunit via prolyl hydroxylases, which is necessary for binding VHL. However, the significance of the two prolines has remained unclear considering that only one hydroxyproline is sufficient for the recruitment of VHL. Here, we show using biophysical analyses that both hydroxyprolines bind to the same interface on VHL with similar affinity; VHL binding affinity to HIF-1α ODD remains relatively unchanged regardless of whether the ODD contains one or two hydroxyprolines; ODD with two hydroxyprolines can accommodate two VHLs; and the rate of in vitro ubiquitination of ODD with one hydroxyproline via VHL E3 ligase is comparable to the rate observed with ODD containing two hydroxyprolines. However, the two hydroxyprolines show distinct contributions to the intracellular stability of HIF-1α ODD. These results demonstrate for the first time that the graduated HIF-1α stability profile observed over a range of oxygen tension is not attributed to the binding of or ubiquitination via VHL per se, but is likely due to the preceding events such as the efficacy of oxygen-dependent prolyl hydroxylase-mediated hydroxylation of HIF-1α.

Hypoxia and viral infectious diseases.

Oxygen-sensing mechanisms allow cells to adapt and respond to changes in cellular oxygen tension, including hypoxic conditions. Hypoxia-inducible factor (HIF) is a central mediator in this fundamental adaptive response, and has critical functions in normal and disease physiology. Viruses have been shown to manipulate HIFs during their life cycle to facilitate replication and invasion. Conversely, HIFs are also implicated in the development of the host immune system and response to viral infections. Here, we highlight the recent revelations of host-pathogen interactions that involve the hypoxic response pathway and the role of HIF in emerging viral infectious diseases, as well as discussing potential antiviral therapeutic strategies targeting the HIF signaling axis.

PD-1 blockade delays tumor growth by inhibiting an intrinsic SHP2/Ras/MAPK signalling in thyroid cancer cells.

BACKGROUND: The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). METHODS: PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. RESULTS: Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. CONCLUSIONS: PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

The long form of pVHL is artifactually modified by serine protease inhibitor AEBSF.

von Hippel-Lindau protein (pVHL) is the tumor suppressor responsible for ubiquitylating the hypoxia-inducible factor (HIF) family of transcription factors for degradation under normoxic conditions. There are two major pVHL isoforms with the shorter isoform (pVHL19 ) lacking the acidic domain present in the N-terminus of the longer isoform (pVHL30 ). Although both isoforms can degrade HIF and suppress tumor formation in experimental systems, previous research suggests that pVHL30 can undergo posttranslational modifications (PTM) and interact with unique proteins. Indeed, pVHL30 has long been observed to migrate as two species on a reducing polyacrylamide gel, indicating the presence of an uncharacterized PTM on the slower-migrating pVHL30 without an identifiable biological consequence. Thus, there has been considerable effort to elucidate the exclusive biological activity of pVHL30 , if any, by first defining the unique features of the slower-migrating species. We show here that the migration of pVHL30 , but not pVHL19 , is retarded by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), an irreversible serine protease inhibitor commonly found in protease inhibitor cocktails.

NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS-MAPK Pathway in Neuroblastoma.

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. SIGNIFICANCE: These findings suggest that conventional therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

A Hypoxia-Inducible HIF1-GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell-Platelet Binding.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

Evolution of metazoan oxygen-sensing involved a conserved divergence of VHL affinity for HIF1α and HIF2α.

Duplication of ancestral hypoxia-inducible factor (HIF)α coincided with the evolution of vertebrate species. Paralogs HIF1α and HIF2α are the most well-known factors for modulating the cellular transcriptional profile following hypoxia. However, how the processes of natural selection acted upon the coding region of these two genes to optimize the cellular response to hypoxia during evolution remains unclear. A key negative regulator of HIFα is von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that evolutionarily-relevant substitutions can modulate a secondary contact between HIF1α Met561 and VHL Phe91. Notably, HIF1α binds more tightly than HIF2α to VHL due to a conserved Met to Thr substitution observed in the vertebrate lineage. Similarly, substitution of VHL Phe91 with Tyr, as seen in invertebrate species, decreases VHL affinity for both HIF1α and HIF2α. We propose that vertebrate evolution involved a more complex hypoxia response with fine-tuned divergence of VHL affinity for HIF1α and HIF2α.

Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation.

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.

Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the pVHL (von Hippel-Lindau Protein) and HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells.

Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.

Translational and HIF-1α-Dependent Metabolic Reprogramming Underpin Metabolic Plasticity and Responses to Kinase Inhibitors and Biguanides.

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

HIF-2α-pVHL complex reveals broad genotype-phenotype correlations in HIF-2α-driven disease.

It is definitively established that mutations in transcription factor HIF-2α are causative of both neuroendocrine tumors (class 1 disease) and polycythemia (class 2 disease). However, the molecular mechanism that underlies this emergent genotype-phenotype relationship has remained unclear. Here, we report the structure of HIF-2α peptide bound to pVHL-elongin B-elongin C (VBC) heterotrimeric complex, which shows topographical demarcation of class 1 and 2 mutations affecting residues predicted, and demonstrated via biophysical analyses, to differentially impact HIF-2α-pVHL interaction interface stability. Concordantly, biochemical experiments showed that class 1 mutations disrupt pVHL affinity to HIF-2α more adversely than class 2 mutations directly or indirectly via impeding PHD2-mediated hydroxylation. These findings suggest that neuroendocrine tumor pathogenesis requires a higher HIF-2α dose than polycythemia, which requires only a mild increase in HIF-2α activity. These biophysical data reveal a structural basis that underlies, and can be used to predict de novo, broad genotype-phenotype correlations in HIF-2α-driven disease.