Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

Coordinated Regulation of Metabolic Transporters and Migration/Invasion by Carbonic Anhydrase IX

Hypoxia is a prominent feature of the tumor microenvironment (TME) and cancer cells must dynamically adapt their metabolism to survive in these conditions. A major consequence of metabolic rewiring by cancer cells in hypoxia is the accumulation of acidic metabolites, leading to the perturbation of intracellular pH (pHi) homeostasis and increased acidosis in the TME. To mitigate the potentially detrimental consequences of an increasingly hypoxic and acidic TME, cancer cells employ a network of enzymes and transporters to regulate pH, particularly the extracellular facing carbonic anhydrase IX (CAIX) and CAXII. In addition to the role that these CAs play in the regulation of pH, recent proteome-wide analyses have revealed the presence of a complex CAIX interactome in cancer cells with roles in metabolite transport, tumor cell migration and invasion. Here, we explore the potential contributions of these interactions to the metabolic landscape of tumor cells in hypoxia and discuss the role of CAIX as a hub for the coordinated regulation of metabolic, migratory and invasive processes by cancer cells. We also discuss recent work targeting CAIX activity using highly selective small molecule inhibitors and briefly discuss ongoing clinical trials involving SLC-0111, a lead candidate small molecule inhibitor of CAIX/CAXII

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion

Conversion of the cellular prion protein PrPC into its pathogenic isoform PrPSc is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti‐prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis‐folding events

The crystal structure of an octapeptide repeat of the Prion protein in complex with a fab fragment of the POM2 antibody

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein, PrP(c) , into an insoluble, protease-resistant, aggregated isoform termed PrP(sc) . In native conditions, PrP(c) has a structured C-terminal domain and a highly flexible N-terminal domain. A part of this N-terminal domain consists of 4-5 repeats of an unusual glycine rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this paper, we successfully report the first crystal structure of an octapeptide repeat of PrP(c) bound to the POM2 Fab antibody fragment. The structure was solved at a resolution of 2.3 Å by molecular replacement. While several studies have previously predicted a β-turn like structure of the unbound octapeptide repeats, our structure shows an extended conformation of the octapeptide repeat when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the octapeptide repeats. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo

Structural basis of prion inhibition by phenothiazine compounds

Conformational transitions of the cellular form of the prion protein, PrP(C), into an infectious isoform, PrP(Sc), are considered to be central events in the progression of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Tricyclic phenothiazine compounds exhibit antiprion activity; however, the underlying molecular mechanism of PrP(Sc) inhibition remains elusive. We report the molecular structures of two phenothiazine compounds, promazine and chlorpromazine bound to a binding pocket formed at the intersection of the structured and the unstructured domains of the mouse prion protein. Promazine binding induces structural rearrangement of the unstructured region proximal to β1, through the formation of a "hydrophobic anchor." We demonstrate that these molecules, promazine in particular, allosterically stabilize the misfolding initiator-motifs such as the C terminus of α2, the α2-α3 loop, as well as the polymorphic β2-α2 loop. Hence, the stabilization effects of the phenothiazine derivatives on initiator-motifs induce a PrP(C) isoform that potentially resists oligomerization

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides