Mandy Kwong

APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA.

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-γ. The induction of cytokine production by NK cells was preceded by activation of NF-κB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.

An ARL3–UNC119–RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium

The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic β sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice‐site mutation in PDE6D, encoding a prenyl‐binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild‐type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl‐dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP‐bound ARL3, which acts as a cargo‐release factor for PDE6D‐bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl‐binding‐dependent trafficking in ciliopathies.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma

Significance Mutation of isocitrate dehydrogenase 1 (IDH1) is believed to be the initiating event for the majority of secondary glioblastomas and lower-grade diffuse gliomas; however, the basis for tissue specificity of oncogenesis initiated by IDH1 mutation has not been apparent. We report evidence to suggest that specialization of human neocortex for glutaminergic neurotransmission provides a metabolic niche particularly suited for growth of IDH1R132H glioma. Our findings reveal that IDH1-mutant enzyme challenges growth of murine glioma progenitor cells but that these cells thrive if they are engineered to express the hominoid-specific brain enzyme GLUD2, a mitochondrial enzyme that converts glutamate to alpha-ketoglutarate in human cortex. The current findings raise the possibility that evolutionary changes contributing to human cognitive abilities may have conferred vulnerability to brain tumors driven by IDH1 mutation. Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1R132H exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1R132H exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

Cutting edge : Novel human dendritic cell- and monocyte-attracting chemokine-like protein identified by fold recognition methods

Chemokines play an important role in the immune system by regulating cell trafficking in homeostasis and inflammation. In this study, we report the identification and characterization of a novel cytokine-like protein, DMC (dendritic cell and monocyte chemokine-like protein), which attracts dendritic cells and monocytes. The key to the identification of this putative new chemokine was the application of threading techniques to its uncharacterized sequence. Based on our studies, DMC is predicted to have an IL-8-like chemokine fold and to be structurally and functionally related to CXCL8 and CXCL14. Consistent with our predictions, DMC induces migration of monocytes and immature dendritic cells. Expression studies show that DMC is constitutively expressed in lung, suggesting a potential role for DMC in recruiting monocytes and dendritic cells from blood into lung parenchyma.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Structural Basis for Resistance to Diverse Classes of NAMPT Inhibitors.

Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

Structural basis of the broadly neutralizing anti-interferon-α antibody rontalizumab

Interferons‐alpha (IFN‐α) are the expressed gene products comprising thirteen type I interferons with protein pairwise sequence similarities in the 77–96% range. Three other widely expressed human type I interferons, IFN‐β, IFN‐κ and IFN‐ω have sequences 29–33%, 29–32% and 56–60% similar to the IFN‐αs, respectively. Type I interferons act on immune cells by producing subtly different immune‐modulatory effects upon binding to the extracellular domains of a heterodimeric cell‐surface receptor composed of IFNAR1 and IFNAR2, most notably anti‐viral effects. IFN‐α has been used to treat infection by hepatitis‐virus type C (HCV) and a correlation between hyperactivity of IFN‐α‐induced signaling and systemic lupus erythematosis (SLE), or lupus, has been noted. Anti‐IFN‐α antibodies including rontalizumab have been under clinical study for the treatment of lupus. To better understand the rontalizumab mechanism of action and specificity, we determined the X‐ray crystal structure of the Fab fragment of rontalizumab bound to human IFN‐α2 at 3Å resolution and find substantial overlap of the antibody and IFNA2 epitopes on IFN‐α2.

Abstract 5002: Abstract Submission

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Protein tyrosine kinase 7 (PTK7), a catalytically inactive receptor tyrosine kinase (RTK) that is highly expressed by T-lineage cells during intrathymic development, is a novel marker for human CD4+ recent thymic emigrants (RTEs), and is also highly expressed on some T-lineage thymomas, e.g., Jurkat cells as well as inprimary T-Acute Lymphoblastic leukemia. The function of PTK7 in normal human T-cell development and in oncogenesis remains unclear. Here, using RNAi-mediated gene silencing in T-lineage tumor cells, primary human peripheral T-cells, and thymocytes, we found that targeting PTK7 consistently decreased cell survival by augmenting caspase-3 activation of apoptosis. The PTK7 knockdown also decreased AKT phosphorylation and PI3 kinase activity, suggesting an essential role for PTK7 in survival of RTEs and developing thymocytes involving the PI3K/AKT pathway. Using mass spectrometry we identified insulin-like growth factor-1 (IGF-1) receptor as an active kinase partner of PTK7. This interaction was biologically relevant in that PTK7 downregulation also reduced IGF-1R-dependent survival signals in T-lineage cells. As enhanced IGF-1-dependent signaling is a frequent event in oncogenesis, the intersection of PTK7 with the IGF-1 signaling pathway suggests the potential of PTK7-directed therapy of T-lineage tumors. Citation Format: Swati Acharya, Kira Y.D Petersen, Vladislav Golubkov, Mandy Kwong, Christopher M. Adams, Peter K. Jackson, David B. Lewis. Abstract Submission. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2015-5002