Malaria, an infectious disease caused by eukaryotic parasites from the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1,358 small molecule kinase inhibitors to evaluate the role of both the human and malaria kinomes in Plasmodium infection of liver cells, the parasites’ obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood stage malaria. Most of the screening hits inhibited both liver and blood stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests several kinases are essential to malaria parasites, including cyclin-dependent kinases, glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.
Plasmodium; malaria; chemical probes; kinase; high-throughput screen
The influence of environmental attitudes on environmental behaviors has long been discussed. However, few studies have addressed the foundation of such attitudes. In the present study, we explored primitive belief underlying environmental attitudes, i.e., connections with nature, and its relationship with pro-environmental behaviors. Specifically, we used scales, a computerized Implicit Association Test, and a situational simulation experiment to examine both explicit and implicit connections with nature, both deliberate and spontaneous environmental behaviors, and to find correlations between environmental connectedness and environmental behaviors. Results showed that explicit connectedness was positively correlated with deliberate environmental behaviors, while implicit connectedness was positively correlated with spontaneous environmental behaviors. Additionally, explicit and implicit connectedness was independent of each other. In conclusion, the current study confirms the positive role played by connections with nature in promoting environmental behavior, and accordingly suggests means to encourage pro-environmental behavior by enhancing people’s connectedness to nature.
Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest.
We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3–7 mice/group). MPS1 expression levels were examined in relation to patient survival.
Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity.
Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.
Chemotherapy-induced peripheral neuropathy (CIPN) characterized by loss of sensory sensitivity and pain in hands and feet is the major dose-limiting toxicity of many chemotherapeutics. At present, there are no FDA-approved treatments for CIPN. The anti-diabetic drug metformin is the most widely used prescription drug in the world and improves glycemic control in diabetes patients. There is some evidence that metformin enhances the efficacy of cancer treatment. The aim of this study was to test the hypothesis that metformin protects against chemotherapy-induced neuropathic pain and sensory deficits. Mice were treated with cisplatin together with metformin or saline. Cisplatin induced increased sensitivity to mechanical stimulation (mechanical allodynia) as measured using the von Frey test. Co-administration of metformin almost completely prevented the cisplatin-induced mechanical allodynia. Co-administration of metformin also prevented paclitaxel-induced mechanical allodynia. The capacity of the mice to detect an adhesive patch on their hind paw was used as a novel indicator of chemotherapy-induced sensory deficits. Co-administration of metformin prevented the cisplatin-induced increase in latency to detect the adhesive patch indicating that metformin prevents sensory deficits as well. Moreover, metformin prevented the reduction in density of intra-epidermal nerve fibers (IENFs) in the paw that develops as a result of cisplatin treatment. We conclude that metformin protects against pain and loss of tactile function in a mouse model of CIPN. The finding that metformin reduces loss of peripheral nerve endings indicates that mechanism underlying the beneficial effects of metformin includes a neuroprotective activity. Because metformin is widely used for treatment of type II diabetes, has a broad safety profile, and is currently being tested as an adjuvant drug in cancer treatment, clinical translation of these findings could be rapidly achieved.
Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway.
Tyrosine kinase; TYK2; STAT1; BCL2; T-ALL
For a subpopulation of acute myeloid leukemia (AML) patients, the constitutively activated tyrosine kinase, mutant FLT3, has emerged as a promising target for therapy. The development of drug resistance, however, is a growing concern for mutant FLT3 inhibitors, such as PKC412. Potential therapeutic benefit can arise from the combination of two structurally diverse inhibitors that target- but bind differently to- the same protein or from two inhibitors with completely different mechanisms of action. Thus, there is a need for identification and development of novel FLT3 inhibitors that have the ability to positively combine with PKC412 or standard chemotherapeutic agents used to treat AML as a way to suppress the development of drug resistance and consequently prolong disease remission. Here, we report the effects of the novel type II ATP competitive inhibitors, HG-7-85-01 and HG-7-86-01, which potently and selectively target mutant FLT3 protein kinase activity, and inhibit the proliferation of cells harboring FLT3-ITD or FLT3 kinase domain point mutants via induction of apoptosis and cell cycle inhibition. Anti-leukemic activity of HG-7-85-01 was demonstrated in vivo to be comparable to that observed with PKC412 in a bioluminescence assay utilizing NCr nude mice harboring Ba/F3-FLT3-ITD-luc+ cells. HG-7-85-01 was also observed to override PKC412 resistance. Finally, HG-7-85-01 and HG-7-86-01 synergized with PKC412 and standard chemotherapeutic agents against mutant PKC412-sensitive and some PKC412-resistant, FLT3-positive cells. Thus, we present a structurally novel class of FLT3 inhibitors that warrants consideration for clinical testing against drug-resistant disease in AML patients.
The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
We describe the discovery of structurally diverse kinase inhibitors to dissect the physiological roles of the NUAK isoforms. We recommend use of an inhibitor-resistant NUAK1[A195T] mutant to verify that the physiological effects of these compounds is indeed mediated through inhibition of NUAKs
AMP-activated protein kinase (AMPK); AMPK-related kinase 5 (ARK5); kinase inhibitor; kinase profiling; liver kinase B1 (LKB1); myosin phosphate-targeting subunit 1(MYPT1); sucrose-non-fermenting protein kinase/AMPKrelated protein kinase (SNARK); ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; BRSK, brain-specific kinase; DMEM, Dulbecco’s modified Eagle’s medium; HA, haemagglutinin; HEK, human embryonic kidney; LKB1, liver kinase B1; MARK, microtubule-affinity-regulating kinase; MEF, mouse embryonic fibroblast; MYPT1, myosin phosphate-targeting subunit 1; NF-κB, nuclear factor κB; PEI, polyethylenimine; PP1, protein phosphatase 1; SIK, salt-induced kinase
In recent years, the roles of chronic stress and depression as an independent risk factor for decreased insulin sensitivity and the development of diabetes have been increasingly recognized. However, an understanding and the mechanisms linking insulin resistance and acute psychological stress are very limited. We hypothesized that acute psychological stress may cause the development of insulin resistance, which may be a risk factor in developing type 2 diabetes. We tested the hypothesis in a well-established mouse model using 180 episodes of inescapable foot shock (IES), followed by a behavioral escape test. In this study, mice that received IES treatment were tested for acute insulin resistance by measuring glucose metabolism and insulin signaling. When compared to normal and sham mice, mice that were exposed to IES resulting in escape failure (defined as IES with behavioral escape failure) displayed elevated blood glucose levels in both glucose tolerance and insulin tolerance tests. Furthermore, mice with IES exposure and behavioral escape failure exhibited impaired hepatic insulin signaling via the insulin-induced insulin receptor/insulin receptor substrate 1/Akt pathway, without affecting similar pathways in skeletal muscle, adipose tissue and brain. Additionally, a rise in murine growth-related oncogene KC/GRO was associated with impaired glucose metabolism in IES mice, suggesting a mechanism by which psychological stress by IES may influence glucose metabolism. The present results indicate that psychological stress induced by IES can acutely alter hepatic responsiveness to insulin and affect whole-body glucose metabolism.
Psychological stress; Inescapable foot shocks; Insulin signaling; Glucose metabolism; Acute insulin resistance
The purpose of this study was to develop a self-aggregated nanoparticulate vehicle using an amphiphilic poly(lactic acid)-grafted-chitosan (PLA-g-CS) copolymer and to evaluate its potential for ocular delivery of amphotericin B.
A PLA-g-CS copolymer was synthesized via a “protection-graft-deprotection” procedure and its structure was confirmed by Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance, and X-ray diffraction spectra. Amphotericin B-loaded nanoparticles based on PLA-g-CS (AmB/PLA-g-CS) were prepared by the dialysis method and characterized for particle size, zeta potential, and encapsulation efficiency. Studies of these AmB/PLA-g-CS nanoparticles, including their mucoadhesive strength, drug release properties, antifungal activity, ocular irritation, ocular pharmacokinetics, and corneal penetration were performed in vitro and in vivo.
Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance, and X-ray diffraction spectra showed that the PLA chains were successfully grafted onto chitosan molecules and that crystallization of chitosan was suppressed. The self-aggregated PLA-g-CS nanoparticles had a core-shell structure with an average particle size of approximately 200 nm and zeta potentials higher than 30 mV. Amphotericin B was incorporated into the hydrophobic core of the nanoparticles with high encapsulation efficiency. Sustained drug release from the nanoparticles was observed in vitro. The ocular irritation study showed no sign of irritation after instillation of the PLA-g-CS nanoparticles into rabbit eyes. The minimal inhibitory concentration of the AmB/PLA-g-CS nanoparticles showed antifungal activity similar to that of free amphotericin B against Candida albicans. The in vivo ocular pharmacokinetic study suggested that the PLA-g-CS nanoparticles have the advantage of prolonging residence time at the ocular surface. The corneal penetration study showed that the PLA-g-CS nanoparticles could penetrate into the cornea.
Our results suggest that this nanoparticulate vehicle based on a PLA-g-CS copolymer might be a promising system for effective ocular delivery of amphotericin B.
chitosan; poly(lactic acid); nanoparticles; amphotericin B
Here we describe the synthesis and characterization of a number of 3-amino-1H-indazol-6-yl-benzamides that were designed to target the “DFG-out” conformation of the kinase activation loop. Several compounds such as 4 and 11 exhibit single-digit nanomolar EC50s against FLT3, c-Kit and the gatekeeper T674M mutant of PDGFRα.
The c-Fes protein-tyrosine kinase modulates cellular signaling pathways governing differentiation, the innate immune response, and vasculogenesis. Here we report the identification of Type I and II kinase inhibitors with potent activity against c-Fes both in vitro and in cell-based assays. One of the most potent inhibitors is the previously described anaplastic lymphoma kinase inhibitor, TAE684. The crystal structure of TAE684 in complex with the c-Fes SH2-kinase domain showed excellent shape complementarity with the ATP-binding pocket and a key role for the gatekeeper methionine in the inhibitory mechanism. TAE684 and two pyrazolopyrimidines with nanomolar potency against c-Fes in vitro were used to establish a novel role for this kinase in osteoclastogenesis, illustrating the value of these inhibitors as tool compounds to probe the diverse biological functions associated with this unique kinase.
The transcription factor FoxO3a is highly expressed in brain, but little is known about the response of FoxO3a to behavioral stress and its impact in the associated behavioral changes.
We tested the response of brain FoxO3a in the learned helplessness (LH) paradigm and tested signaling pathways that mediate the response of FoxO3a.
A single session of inescapable shocks (IES) in mice reduced FoxO3a phosphorylation at the Akt-regulating serine/threonine residues and induced prolonged nuclear accumulation of FoxO3a in the cerebral cortex, both indicate activation of FoxO3a in brain. The response of FoxO3a is accompanied by a transient inactivation of Akt and a prolonged activation of glycogen synthase kinase-3beta (GSK3β). Noticeably, FoxO3a formed a protein complex with GSK3β in the cerebral cortex, and the interaction between the two proteins was stronger in IES-treated mice. Inhibition of GSK3 was able to abolish IES-induced LH behavior, disrupt IES-induced GSK3β-FoxO3a interaction, and reduce nuclear FoxO3a accumulation. In vitro approaches further revealed that the interaction between GSK3β and FoxO3a was strongest when both were active, FoxO3a was phosphorylated by recombinant GSK3β, and GSK3 inhibitors effectively reduced FoxO3a transcriptional activity. Importantly, IES-induced LH behavior was markedly diminished in FoxO3a-deficient mice that have minimal FoxO3a expression and reduced levels of FoxO3a-inducible genes.
FoxO3a is activated in response to IES by interacting with GSK3β, and inhibition of GSK3β or reducing FoxO3a expression promotes resistance to stress-induced behavioral disturbance by disrupting this signaling mechanism.
FoxO3a; GSK3beta; Akt; stress; learned helplessness; signal transduction
Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers of response to targeted agents. To uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines, which represent much of the tissue-type and genetic diversity of human cancers, with 130 drugs under clinical and preclinical investigation. In aggregate, we found mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing’s sarcoma cells harboring the EWS-FLI1 gene translocation to PARP inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.
Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors.
While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.
Squamous cell lung cancer; DDR2; dasatinib; tyrosine kinase inhibitors; lung cancer genomics
Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer. A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M “gatekeeper” resistance mutation relative to wild-type EGFR were discovered by library screening. A representative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-dependent cellular proliferation assays.
The fibroblast growth factor receptor tyrosine kinases (FGFR1, 2, 3, and 4) represent promising therapeutic targets in a number of cancers. We have developed the first potent and selective irreversible inhibitor of FGFR1, 2, 3, and 4 which we named FIIN-1 that forms a covalent bond with cysteine 486 located in the P-loop of the FGFR1 ATP-binding site. We demonstrate that the inhibitor potently inhibits Tel-FGFR1 transformed Ba/F3 cells (EC50 = 14 nM) as well as numerous FGFR-dependent cancer cell lines. A biotin-derivatized version of the inhibitor, FIIN-1-biotin, was shown to covalently label FGFR1 at Cys486. FIIN-1 is a useful probe of FGFR-dependent cellular phenomena and may provide a starting point of the development of therapeutically relevant irreversible inhibitors of wild-type and drug-resistant forms of FGFR kinases.
The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR mutant non-small cell lung cancer (NSCLC) is limited by the development of drug resistance mutations, including the gatekeeper T790M mutation1-3. Strategies aimed at targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild type EGFR4,5. All current EGFR inhibitors possess a structurally related quinazoline based core scaffold and were identified as ATP-competitive inhibitors of wild type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30-100 fold more potent against EGFR T790M, and up to 100 fold less potent against wild type EGFR, than quinazoline based EGFR inhibitors in vitro and are effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant selective kinase inhibitors.
Epidermal growth factor receptor; mutation; drug resistance; kinase inhibitor
To investigate the role of perforin-mediated cell apoptosis in murine models of immune-mediated bone marrow (BM) failure.
We compared C57BL/6J (B6) mice carrying a perforin gene deletion (Prf−/−) with wild type (WT) controls for cellular composition in lymphohematopoietic tissues. Lymph node (LN) cells from Prf−/− mice were co-incubated with BM cells from B10-H2b/LilMcdJ (C.B10) mice in an apoptosis assay in vitro. We then infused Prf−/− and WT B6 LN cells into sublethally-irradiated C.B10 and CByB6F1 recipients with mismatches at the minor- and major-histocompatibility loci, respectively, in order to induce BM failure. Cellular composition was analyzed by flow cytometry.
Prf−/− mice showed normal lymphoid cell composition but Prf−/− LN cells had reduced ability to induce C.B10 BM cell apoptosis in vitro. Infusion of 5–10 × 106 Prf−/− LN cells produced obvious BM failure in C.B10 and CByB6F1 recipients; pancytopenia and BM hypocellularity were only slightly less severe than those caused by infusion of 5 × 106 WT B6 LN cells. Infused Prf−/− LN cells showed less T cell expansion, normal T cell activation, and higher proportions of T cells expressing gamma-interferon, tissue necrosis factor alpha and Fas ligand CD178, in comparison to infused WT B6 LN cells. Fas expression was equally high in residual BM cells in recipient of both Prf−/− and B6 LN cells.
Perforin deficiency alters T cell expansion but up-regulates T cell Fas ligand expression. Perforin-mediated cell death appears to play a minor role in mouse models of immune-mediated BM failure.
Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.