Benjamin T. Houseman

Arterial-Venous Segregation by Selective Cell Sprouting: An Alternative Mode of Blood Vessel Formation

Making Split Decisions Development of the vertebrate vasculature has been thought to involve just two mechanisms of blood vessel formation. Herbert et al. (p. 294; see the Perspective by Benedito and Adams) identified a third mechanism in zebrafish in which two distinct, unconnected vessels can be derived from a single precursor vessel. Several vascular endothelial growth factors and signaling pathways, including ephrin and notch signaling, coordinated the sorting and segregation of a mixture of arterial and venous-fated precursor cells into distinct arterial and venous vessels. These findings provide a mechanistic framework for how mixed populations of cells can coordinate their behavior to segregate and form distinct blood vessels. An alternative developmental pathway for vertebrate vasculature segregates a precursor vessel into two separate vessels. Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Akt and Autophagy Cooperate to Promote Survival of Drug-Resistant Glioma

Combined inhibition of PI3K, mTOR, and autophagy promotes glioma cell death. Blocking All Escape Routes Many cancers, including glioma, are associated with increased signaling through the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway, which promotes cell growth, proliferation, and survival. This suggests that pharmacological inhibition of key kinases in this pathway could provide an approach to antineoplastic therapy. Disappointingly, however, inhibitors of PI3K, Akt, or mTOR typically block cancer cell growth rather than eliciting the death of malignant cells, limiting their utility as antineoplastic agents. Noting that autophagy, a process of autodigestion that can enable cells to endure periods of stress and nutrient deprivation, could provide a survival mechanism under conditions of decreased PI3K-Akt-mTOR signaling, Fan et al. explored the effects of various combinations of kinase and autophagy inhibitors on glioma cell survival. Inhibition of mTOR complex 1 (mTORC1) with rapamycin induced autophagy; however, cells survived the combination of rapamycin with inhibitors of autophagy by activating Akt signaling. In contrast, the combined inhibition of mTORC1, PI3K, and autophagy, or that of mTORC1, mTORC2, and autophagy, triggered apoptosis—the process of programmed cell death. The authors elicited cell death with combinations of drugs that are either now in use in patients or in clinical trials, raising the hope that this approach could be readily translatable to human therapy. Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5′-triphosphate–competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.

The p110[delta] structure: mechanisms for selectivity and potency of new PI(3)K inhibitors

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in numerous pathologies like cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small molecule and ATP-competitive PI3K inhibitors with a wide range of selectivities have entered clinical development. In order to understand mechanisms underlying isoform selectivity of these inhibitors, we developed a novel expression strategy that enabled us to determine the first crystal structure of the catalytic subunit of the class IA PI3K p110δ. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI3K inhibitors reveal that selectivity towards p110δ can be achieved by exploiting its conformational flexibility and the sequence diversity of active-site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110δ with greatly improved potencies.

Shaping Development of Autophagy Inhibitors with the Structure of the Lipid Kinase Vps34

Lipid Kinase Revealed The lipid kinase, Vps34, makes the key signaling lipid phosphatidylinositol 3-phosphate [PI(3)P] and has essential roles in autophagy, membrane trafficking, and cell signaling. It is a class III PI 3-kinase, a class against which there is currently no specific inhibitor. Miller et al. (p. 1638) now describe the crystal structure of Vps34. Modeling substrate binding and combining structural data with mutagenesis suggests a mechanism in which Vps34 is auto-inhibited in solution, but adopts a catalytically active conformation on membranes. Structures of Vps34 with existing inhibitors might allow for the generation of inhibitors with high affinity and specificity. Structural data might provide a foundation to develop specific inhibitors to this class of phosphoinositide 3-kinases. Phosphoinositide 3-kinases (PI3Ks) are lipid kinases with diverse roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, membrane trafficking, and cell signaling. We solved the crystal structure of Vps34 at 2.9 angstrom resolution, which revealed a constricted adenine-binding pocket, suggesting the reason that specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the carboxyl-terminal helix of Vps34 mediate catalysis on membranes and suppress futile adenosine triphosphatase cycles. Vps34 appears to alternate between a closed cytosolic form and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors reveal the reason that an autophagy inhibitor preferentially inhibits Vps34 and underpin the development of new potent and specific Vps34 inhibitors.

Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages

The class IA isoforms of phosphoinositide 3-kinase (p110α, p110β and p110δ) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110δ selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110δ in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110α, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110δ. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110α also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110δ.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110δ isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110δ-selective PI3K inhibitors may be more effective in inflammation than in cancer.

Discovery of Dual Inhibitors of the Immune Cell PI3Ks p110δ and p110γ: a Prototype for New Anti-inflammatory Drugs

PI3Kdelta and PI3Kgamma regulate immune cell signaling, while the related PI3Kalpha and PI3Kbeta regulate cell survival and metabolism. Selective inhibitors of PI3Kdelta/gamma represent a potential class of anti-inflammatory agents lacking the antiproliferative effects associated with PI3Kalpha/beta inhibition. Here we report the discovery of PI3Kdelta/gamma inhibitors that display up to 1000-fold selectivity over PI3Kalpha/beta and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kdelta is weakly anti-inflammatory, but PI3Kdelta/gamma inhibitors show superior inflammatory marker suppression through suppression of lipopolysaccharide-induced TNFalpha production and T cell activation. Moreover, PI3Kdelta/gamma inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.

Biocompatibility and Reduced Drug Absorption of Sol−Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications

Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices. This study presents evidence that a sol-gel treatment of PDMS that fills the polymer matrix with silica nanoparticles is effective at reducing the absorption of drugs into the material while preserving its biocompatibility, transparency, and oxygen permeability. We show that the absorption of two anticancer drugs, camptothecin and a kinase inhibitor, is reduced to such an extent that on-chip microfluidic cell culture experiments can recapitulate the results obtained off-chip.

Dual blockade of lipid and cyclin-dependent kinases induces synthetic lethality in malignant glioma

Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.

Dose-Dependent Effects of Focal Fractionated Irradiation on Secondary Malignant Neoplasms in Nf1 Mutant Mice

Secondary malignant neoplasms (SMN) are increasingly common complications of cancer therapy that have proven difficult to model in mice. Clinical observations suggest that the development of SMN correlates with radiation dose; however, this relationship has not been investigated systematically. We developed a novel procedure for administering fractionated cranial irradiation (CI) and investigated the incidence and spectrum of cancer in control and heterozygous Nf1 mutant mice irradiated to a moderate (15 Gy) or high dose (30 Gy). Heterozygous Nf1 inactivation cooperated with CI to induce solid tumors and myeloid malignancies, with mice developing many of the most common SMNs found in human patients. CI-induced malignancies segregated according to radiation dose as Nf1(+/-) mice developed predominately hematologic abnormalities after 15 Gy, whereas solid tumors predominated at 30 Gy, suggesting that radiation dose thresholds exist for hematologic and nonhematologic cancers. Genetic and biochemical studies revealed discrete patterns of somatic Nf1 and Trp53 inactivation and we observed hyperactive Ras signaling in many radiation-induced solid tumors. This technique for administering focal fractionated irradiation will facilitate mechanistic and translational studies of SMNs.

PLC-γ and PI3K Link Cytokines to ERK Activation in Hematopoietic Cells with Normal and Oncogenic Kras

Targeting signaling pathways upstream of oncogenic Ras may have therapeutic benefit in the treatment of leukemia. Target Upstream of Oncogenic Ras Members of the K-Ras family of small guanosine triphosphatases mediate signaling by cytokine and growth factor receptors to activate extracellular signal–regulated kinase (ERK), leading to cellular proliferation. Mutant K-Ras molecules, for example, K-RasG12D, accumulate in the active form and are associated with certain leukemias. Through flow cytometric analysis of phosphorylated proteins in mouse bone marrow cells, Diaz-Flores et al. showed that ERK activation downstream of K-RasG12D required cytokine receptor–dependent activation of phospholipase C–γ (PLC-γ) and phosphoinositide 3-kinase (PI3K) signaling. Treatment of mice with a clinically available PI3K inhibitor reduced ERK activation in cells expressing K-RasG12D, suggesting that molecules upstream of oncogenic Ras may provide therapeutic targets against some cancers. Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf–mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C–γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-RasG12D remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.