Daniel Sherman

VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism

Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.

Persephin, a Novel Neurotrophic Factor Related to GDNF and Neurturin

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two structurally related, potent survival factors for sympathetic, sensory and central nervous system neurons. GDNF mediates its actions through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-α) and the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by which the NTN signal is transmitted is not well understood. Here we describe the identification and tissue distribution of a GPI-linked protein (designated NTNR-α) that is structurally related to GDNFR-α. We further demonstrate that NTNR-α binds NTN (Kd ∼ 10 pM) but not GDNF with high affinity; that GDNFR-α binds to GDNF but not NTN with high affinity; and that cellular responses to NTN require the presence of NTNR-α. Finally, we show that NTN, in the presence of NTNR-α, induces tyrosine-phosphorylation of Ret, and that NTN, NTNR-α and Ret form a physical complex on the cell surface. These findings identify Ret and NTNR-α as signalling and ligand-binding components, respectively, of a receptor for NTN and define a novel family of receptors for neurotrophic and differentiation factors composed of a shared transmembrane protein tyrosine kinase and a ligand-specific GPI-linked protein.

ANGPTL3 Stimulates Endothelial Cell Adhesion and Migration via Integrin αvβ3 and Induces Blood Vessel Formation in Vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to αvβ3 and induced integrin αvβ3-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin αvβ3 and suggest a possible role in the regulation of angiogenesis.

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

Correction: A GPI-linked protein that interacts with Retto form a candidate neurturin receptor

This corrects the article DOI: 10.1038/42722

Abstract 4065: Modulation of cancer metabolism with novel PKM2 activators exerts anti-tumor activity

Cancer cells adapt altered metabolism to enable efficient conversion of glucose into biomass needed for massive cell growth and proliferation. The metabolic switch takes place even when oxygen is abundant, a phenomenon known as the Warburg effect. A key mediator of this effect is pyruvate kinase type M2 (PKM2), a rate-limiting enzyme, which catalyzes the last step in glycolysis, converting PEP and ADP to pyruvate and ATP. PKM2 switches between a highly active tetrameric form and low activity monomeric or dimeric forms, following binding to its natural allosteric activator FBP (an upstream glycolytic intermediate). Our goal was to identify novel small molecule PKM2 activators that can be used as potential anti-cancer therapeutics. Using our proprietary structure-based screening tools, we identified several novel, potent, allosteric PKM2 activators, with AC50s as low as 50nM and 150% activation levels, similar to the natural activator FBP (AC50=116nM, 150% activation). Kinetics studies showed that these compounds significantly improve the rate and efficiency of the enzymatic reaction to the same level as FBP (a 4-fold decrease in KM, and a 2-fold increase in VMAX). Furthermore, a greater increase in VMAX (3-fold) is observed when combining these compounds with FBP. These small-molecule activators induce the formation of the (active) tetrameric form of PKM2, increasing the PKM2 tetramer:monomer ratio by more than 10-fold, quantitatively similar to the action of FBP. In cellular assays, these compounds show significant reduction in the proliferation rate of cancer cell lines, including colorectal, lung and hepatic carcinomas, in a dose- and time-dependent manner (up to 80% proliferation inhibition). In line with the ability of these novel compounds to re-direct cellular metabolism from aerobic glycolysis to oxidative phosphorylation, we found that inhibition of oxidative phosphorylation with oligomycin extends the anti proliferative effect of our compounds. When used in combination with commonly used chemotherapeutic agents, such as 5FU in colorectal cancer cells, they show an additive effect and yielded close to 100% inhibition of cell growth. Our compounds demonstrate excellent in vitro and in vivo DMPK profiles, including metabolic stability, excellent permeability with no efflux (CaCO-2 PApp=20.5 106/cm·s-1), and in vivo mouse pharmacokinetics with 20% oral bioavailability, good half life PO and volume of distribution. Several animal efficacy studies, as mono-therapy and in combination with chemotherapy or specific kinase inhibitors, are ongoing. Taken together, we present a new class of potent PKM2 activators that modulate the metabolism within cancer cells and show a promising anti cancer therapeutics potential. The favorable DMPK profile of these compounds suggests that they can be further developed either as mono-therapy or in combination with targeted therapeutics or chemo-therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2011-4065