Kinase GSK3β Research Articles

Abstract 3989: IQGAP2 inhibits cell proliferation and migration in hepatocellular carcinoma

Abstract While hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer worldwide, the signaling pathways involved are not fully understood and treatment of advanced disease still represents an area of high unmet medical need. We previously reported that IQ-motif-containing GTPase-activating-like proteins IQGAP1 and IQGAP2 play opposing roles in hepatic carcinogenesis. IQGAP2 was identified as a novel tumor suppressor linked to the Wnt/β-catenin signaling pathway in HCC. In this study, mouse embryonic fibroblasts (MEFs) isolated from Iqgap2−/- mice displayed higher proliferation and migration rates compared to wild-type MEFs as evident by MTT proliferation assay and wound healing assay. In contrast to HepG2, the SK-Hep1 and SNU387 HCC cell lines are characterized by low expression of IQGAP2 and relatively high levels of IQGAP1. Migration assay using blind well chambers with polycarbonate filters revealed 2-fold increased motility for both SK-Hep1 and SNU387 compared to HepG2 (p < 0.05). HepG2 cells stably expressing IQGAP2-specific shRNA showed an increase in their motility. Conversely, overexpression of IQGAP2 in HEK293 resulted in up to 50% inhibition of their migration rate. qRT-PCR of naïve SK-Hep1 and SNU387 cells and HepG2 cells overexpressing IQGAP2 shRNA confirmed upregulation of genes encoding MET, MMP1, MMP2, MTA2 and TWIST1, reinforcing a role of IQGAP2 in cell migration. Akt kinase, one of the key regulators of cell migration, co-immunoprecipitated with IQGAP2 in both mouse liver lysates and HEK293 cells transiently expressing human IQGAP2. IQGAP2, but not IQGAP1, also co-immunoprecipitated with GSK3β kinase in mouse liver lysates. Furthermore, to address whether IQGAP2 affects Akt function in liver, insulin stimulation experiments were performed with wild-type, Iqgap2−/- and Iqgap1−/−/Iqgap2−/- mice. It was shown that upon stimulation with a single intraperitoneal injection of 5 U/kg insulin, levels of the phosphorylated (at Ser9) form of GSK3β were elevated in both Iqgap2−/- and Iqgap1−/−/Iqgap2−/- livers compared to wild-type, suggesting that in IQGAP2-deficiency, GSK3β kinase activity is inhibited by, perhaps, Akt activation. Additionally, the strength of the GSK3β - β-catenin interaction was found to be dependent on the presence of both IQGAP1 and IQGAP2, with Iqgap2−/- livers showing weaker interaction and Iqgap1−/−/Iqgap2−/- livers the weakest compared to wild-type. Using IQGAP2 deletional mutants, it was determined that IQ domains may be primarily responsible for IQGAP2 interactions with both Akt and GSK3β. While it remains unclear whether IQGAP2 binds Akt and GSK3β directly or as part of a larger protein complex, these findings suggest that tumor suppressive function of IQGAP2 in the liver may be realized through inhibition of cell proliferation and migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3989. doi:1538-7445.AM2012-3989

Abstract 828: Src plays a role in chemoresistance and migration in chondrosarcoma cell lines

Abstract Chondrosarcomas (CS) are malignant cartilage-forming tumors of bone. High grade chondrosarcomas show metastasis formation in 71% of cases, for which currently no treatment strategies exist. Kinome profiling revealed high GSK3β, AKT, and Src pathway activity. These kinases play a role in either cell survival, migration, or both. Our aim was to identify the roles of these pathways in chemoresistance and migration and investigate downstream effects while pinpointing the most important kinase in chondrosarcoma. We used 5 conventional chondrosarcoma cell lines and 3 dedifferentiated chondrosarcoma cell lines for the experimental procedures. Kinase inhibition was performed with enzastaurin (GSK3β and AKT inhibition) and dasatinib (Src inhibition). WST and Live cell imaging with AnnexinV staining (BD Pathway 855 Bioimager) were performed to investigate cell viability and apoptosis formation. Proliferation and migration assays were performed with the RTCA xCelligence System (Roche). Tissue microarrays (TMAs) were constructed containing 8 EC, 92 central CS (grade I n=42, grade II n=35, grade III n=14), 11 OC and 45 peripheral CS (grade I n=31, grade II n=11, grade III n=3). Overexpression of Src family members can lead to excess Nrf2 translocation from the nucleus. Immunohistochemistry was performed for the Src family members and GPX3, the target of the transcription factor Nrf2. Western blot was performed for Src family members, Nrf2, and GPX3. Inhibition of GSK3β and Akt with enzastaurin in chondrosarcoma cell lines was ineffective, also when combined with Src inhibition (dasatinib) or doxorubicin. Combination treatment of dasatinib with doxorubicin, however, showed synergistic loss in cell viability and apoptosis formation, with cleaved caspase3 activation, which was not observed after dasatinib single treatment. Migration assays in cell lines (n=6) revealed that at low concentrations of dasatinib (200nM), migration was successfully inhibited. TMA staining revealed sporadic expression of Yes and Lck (5%), expression of Src (57%) and abundant expression of Fyn (78%) throughout our panel of CS tissues. Fyn is described to be associated with metastasis formation and is a good candidate to play a role in chondrosarcoma progression. Overall pSrc (active Src) approached 100% in all grades. Western blot analysis showed nuclear localization of Nrf2 and restoration of GPX3 transcription after dasatinib treatment of cell lines. We show that Src family kinases rather than GSK3β and Akt kinases contribute to chemoresistance in chondrosarcoma. Src kinase inhibition successfully prepared cells for chemotherapy and acts synergistically with doxorubicin, and moreover was able to completely inhibit chondrosarcoma cell migration and restore GPX3 transcription. These results strongly indicate Src family kinases, and in particular Fyn, to be a potential target for the treatment of inoperable and metastatic chondrosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 828. doi:1538-7445.AM2012-828

APC and Smad7 link TGFβ type I receptors to the microtubule system to promote cell migration

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3β (GSK-3β). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-β (TGFβ) and is known to inhibit various TGFβ-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFβ stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3β kinases to facilitate local TGFβ/p38-dependent inactivation of GSK-3β, accumulation of β-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFβ type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFβ.

O‐GlcNAc, A Novel Paradigm for Regulating Stress‐Induced Signal Transduction Pathways

The modification of nucleocytoplasmic proteins by O‐linked β‐Nacetylglucosamine (O‐GlcNAc) is a novel post‐translational modification that regulates the cell's ability to sense and signal cellular stress. The mechanisms by which O‐GlcNAc regulates protein function, promoting cell survival, are unknown. To determine if O‐GlcNAc regulates stress‐induced signaling pathways, promoting cell survival, we have defined the basal and stress‐induced phosphoproteome of the cytoplasmic and nuclear fractions of O‐GlcNAc transferase (OGT) wild‐type and null cells. More than 700 phosphorylation sites were identified in the nuclear fraction, of which 65% showed altered phosphorylation when O‐GlcNAc levels were lowered. Analysis of these data suggests that O‐GlcNAc regulates signaling downstream of the ATM and GSK3β kinases, which both play key roles in cell survival decisions. Using orthologous approaches we have demonstrated that O‐GlcNAc appears to regulate both the expression and autophosphorylation of ATM kinase, such that lowering O‐GlcNAc levels appears to enhance phosphorylation of ATM substrates (p53, H2AX, Chk2). These data suggest that one mechanism by which O‐GlcNAc protects cells is by regulating the ATM kinase. Grant support: American Heart Association (NEZ, SDG #0930162N), NIH (NEZ, R21 #HL108003), NIH (NEZ, P01 #HL107153‐01), a PURA award (SS), ASBMB UAN award (SS).

Curcumin Protects Against Regional Myocardial Ischemia/Reperfusion Injury Through Activation of RISK/GSK-3β and Inhibition of p38 MAPK and JNK

Curcumin, the active ingredient of turmeric (Curcuma longa), is known to have anti-inflammatory and antioxidative properties. The present study was aimed to determine the effect of curcumin in regional myocardial ischemia/reperfusion (I/R) injury and its underlying mechanisms involving the role of prosurvival kinases and apoptotic kinases. Sprague-Dawley rats (n = 109) subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion followed by reperfusion were assigned to receive saline (control), curcumin (100 mg/kg), wortmannin (inhibitor of phosphatidylinositol-3-OH kinase [PI3K]-Akt), wortmannin + curcumin, U0126 (inhibitor of extracellular signal-regulated kinase [ERK1/2]), U0126 + curcumin, SB216763 (inhibitor of glycogen synthase kinase [GSK-3β]), and SB216763 + curcumin 20 minutes before LAD occlusion. Infarct size was measured after 2 hours of reperfusion by triphenyl tetrazolium chloride staining. The phosphorylation of Akt, ERK1/2, GSK-3β, p38, and c-Jun N-terminal kinases (JNK) was determined by immunoblotting after 10 minutes of reperfusion. Curcumin significantly reduced the infarct size compared with the control (33.1% ± 6.2% vs 50.1% ± 3.9%; P < .05). Wortmannin or U0126 alone did not affect the infarct size but abolished the curcumin-induced cardioprotective effect. Curcumin significantly enhanced the phosphorylation of Akt, ERK1/2, and GSK-3β, while it reduced that of p38 and JNK. Wortmannin or U0126 abolished enhanced phosphorylation of GSK-3β induced by curcumin. SB216763 alone or combined with curcumin reduced the infarct size and enhanced phosphorylation of GSK-3β compared with the control. Preconditioning by curcumin effectively protects against regional myocardial I/R injury through the activation of prosurvival kinases involving PI3K-Akt, ERK1/2, and GSK-3β, and attenuation of p38 and JNK.

Prolyl Isomerase Pin1 Promotes Amyloid Precursor Protein (APP) Turnover by Inhibiting Glycogen Synthase Kinase-3β (GSK3β) Activity: NOVEL MECHANISM FOR Pin1 TO PROTECT AGAINST ALZHEIMER DISEASE

Alzheimer disease (AD) is characterized by the presence of senile plaques of amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) and neurofibrillary tangles made of hyperphosphorylated Tau. Increasing APP gene dosage or expression has been shown to cause familial early-onset AD. However, whether and how protein stability of APP is regulated is unclear. The prolyl isomerase Pin1 and glycogen synthase kinase-3β (GSK3β) have been shown to have the opposite effects on APP processing and Tau hyperphosphorylation, relevant to the pathogenesis of AD. However, nothing is known about their relationship. In this study, we found that Pin1 binds to the pT330-P motif in GSK3β to inhibit its kinase activity. Furthermore, Pin1 promotes protein turnover of APP by inhibiting GSK3β activity. A point mutation either at Thr-330, the Pin1-binding site in GSK3β, or at Thr-668, the GSK3β phosphorylation site in APP, abolished the regulation of GSK3β activity, Thr-668 phosphorylation, and APP stability by Pin1, resulting in reduced non-amyloidogenic APP processing and increased APP levels. These results uncover a novel role of Pin1 in inhibiting GSK3β kinase activity to reduce APP protein levels, providing a previously unrecognized mechanism by which Pin1 protects against Alzheimer disease.

Voluntary running in mice beneficially modulates myocardial ischemic tolerance, signaling kinases, and gene expression patterns

Exercise triggers hormesis, conditioning hearts against damaging consequences of subsequent ischemia-reperfusion (I/R). We test whether "low-stress" voluntary activity modifies I/R tolerance and molecular determinants of cardiac survival. Male C57BL/6 mice were provided 7-day access to locked (7SED) or rotating (7EX) running-wheels before analysis of cardiac prosurvival (Akt, ERK 1/2) and prodeath (GSK3β) kinases, transcriptomic adaptations, and functional tolerance of isolated hearts to 25-min ischemia/45-min reperfusion. Over 7 days, 7EX mice increased running from 2.1 ± 0.2 to 5.3 ± 0.3 km/day (mean speed 38 ± 2 m/min), with activity improving myocardial I/R tolerance: 7SED hearts recovered 43 ± 3% of ventricular force with diastolic contracture of 33 ± 3 mmHg, whereas 7EX hearts recovered 63 ± 5% of force with diastolic dysfunction reduced to 23 ± 2 mmHg (P < 0.05). Cytosolic expression (total protein) of Akt and GSK3β was unaltered, while ERK 1/2 increased 30% in 7EX vs. 7SED hearts. Phosphorylation of Akt and ERK 1/2 was unaltered, whereas GSK3β phosphorylation increased ∼90%. Microarray interrogation identified significant changes (≥1.3-fold expression change, ≤5% FDR) in 142 known genes, the majority (92%) repressed. Significantly modified paths/networks related to inflammatory/immune function (particularly interferon-dependent), together with cell movement, growth, and death. Of only 14 induced transcripts, 3 encoded interrelated sarcomeric proteins titin, α-actinin, and myomesin-2, while transcripts for protective actin-stabilizing ND1-L and activator of mitochondrial biogenesis ALAS1 were also induced. There was no transcriptional evidence of oxidative heat-shock or other canonical "stress" responses. These data demonstrate that relatively brief voluntary activity substantially improves cardiac ischemic tolerance, an effect independent of shifts in Akt, but associated with increased total ERK 1/2 and phospho-inhibition of GSK3β. Transcriptomic data implicate inflammatory/immune and sarcomeric modulation in activity-dependent protection.

Brain Deletion of Insulin Receptor Substrate 2 Disrupts Hippocampal Synaptic Plasticity and Metaplasticity

ObjectiveDiabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2).Research Design and MethodsTo study neuronal function and synaptic plasticity in the absence of confounding factors such as hyperglycaemia, we used a mouse model with a central nervous system- (CNS)-restricted deletion of IRS-2 (NesCreIrs2KO).ResultsWe report a deficit in NMDA receptor-dependent synaptic plasticity in the hippocampus of NesCreIrs2KO mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β.ConclusionsThese findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling.

γ-Aminobutyric Acid Type A (GABAA) Receptor Activation Modulates Tau Phosphorylation

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.

Protein kinase GSK3β regulates tumor suppressor pdcd4 expression in lung cancer cells

Protein kinase GSK3β regulates tumor suppressor pdcd4 expression in lung cancer cells

Chemotherapy-Associated Angiogenesis in Neuroblastoma Tumors

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvβ3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cβ and Akt. Pharmacological inhibition of protein kinase Cβ or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.

RACK1 Suppresses Gastric Tumorigenesis by Stabilizing the β-Catenin Destruction Complex

Dysregulation of Wnt signaling has been involved in gastric tumorigenesis by mechanisms that are not fully understood. The receptor for activated protein kinase C (RACK1, GNB2L1) is involved in development of different tumor types, but its expression and function have not been investigated in gastric tumors. We analyzed expression of RACK1 in gastric tumor samples and their matched normal tissues from 116 patients using immunohistochemistry. Effects of knockdown with small interfering RNAs or overexpression of RACK1 in gastric cancer cell lines were evaluated in cell growth and tumor xenograft. RACK1 signaling pathways were investigated in cells and zebrafish embryos using immunoblot, immunoprecipitation, microinjection, and in situ hybridization assays. Expression of RACK1 was reduced in gastric tumor samples and correlated with depth of tumor infiltration and poor differentiation. Knockdown of RACK1 in gastric cancer cells accelerated their anchorage-independent proliferation in soft agar, whereas overexpression of RACK1 reduced their tumorigenicity in nude mice. RACK1 formed a complex with glycogen synthase kinase Gsk3β and Axin to promote the interaction between Gsk3β and β-catenin and thereby stabilized the β-catenin destruction complex. On stimulation of Wnt3a, RACK1 repressed Wnt signaling by inhibiting recruitment of Axin by Dishevelled 2 (Dvl2). Moreover, there was an inverse correlation between expression of RACK1 and localization of β-catenin to the cytoplasm/nucleus in human gastric tumor samples. RACK1 negatively regulates Wnt signaling pathway by stabilizing the β-catenin destruction complex and act as a tumor suppressor in gastric cancer cells.

Novel aspects in structure–activity relationships of profiled 1-aza-9-oxafluorenes as inhibitors of Alzheimer's disease-relevant kinases cdk1, cdk5 and gsk3β

A novel series of derivatized 1-aza-9-oxafluorenes has been synthesized. Structure-directed substituents have been introduced and their effect on the inhibition of Alzheimer's disease-relevant kinases has been determined in in vitro assays. Novel structure–activity relationships were found depending on the varying substituents. Nanomolar inhibitors have been identified as promising candidates for the treatment of Alzheimer's disease.

Electrochemical investigations into Tau protein phosphorylations

Hyperphosphorylation of Tau, a protein that stabilizes microtubules, leads to the breakdown of the microtubular structure and ultimately to the formation of neurofibrillar tangles within neurons. Here, we report monitoring of Tau phosphorylations electrochemically, using Tau protein films chemically linked to gold surfaces and 5'-γ-ferrocenyl (Fc) adenosine triphosphate (Fc-ATP) as a co-substrate. Fc-phosphorylation reactions of Tau are explored using the three protein kinases, glycogen synthase kinase (GSK-3β), sarcoma (Src)-related kinase, and protein kinase A (PKA), which catalyze Fc-phosphorylation of different residues and regions within Tau. The kinetic parameters of the biochemical process (K(M) and V(max)) were determined.

Discovery and structural insight of a highly selective protein kinase inhibitor hit through click chemistry

Novel bisaryl maleimide derivatives to mimic natural kinase inhibitors were prepared through click chemistry. A highly selective hit was discovered in a 124-kinase-assay, and docking studies revealed a π-π stacking interaction with the Phe67 at the P-loop of GSK-3β kinase.

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.

NPM-ALK Regulated GSK3β Phosphorylation Results in Mcl-1 and CDC25A Accumulation and Enhanced Oncogenic Potential

Abstract 586Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. ALCLs are frequently characterized by translocations involving the anaplastic lymphoma kinase (ALK) gene. In 70–80% of these cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of ALK with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK tyrosine kinase. NPM-ALK is an oncogenic tyrosine kinase which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules in ALCLs have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β, as a potential signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the phosphorylation of GSK3β at serine-9 (pS9-GSK3β) in a time and dose-dependent manner. Expression of NPM-ALK in 293T cells led to increased phosphorylation of GSK3β at serine-9 compared to kinase-defective K210R mutant NPM-ALK but did not affect total GSK3β levels. Inducible knockdown of ALK in SU-DHL-1 cells also led to decreased phosphorylation of GSK3β at serine-9 without affecting total GSK3β levels. Selective inhibition of PI3Kδ by CAL-101 resulted in decreased phosphorylation of GSK3β at serine-9 in a dose dependent manner without affecting total GSK3β or NPM-ALK activity. Ablation of ALK kinase activity resulted in proteasomal degradation of GSK3β substrates Mcl-1 and CDC25A. This degradation was recovered upon chemical inhibition of GSK3β (GSK3 IX). To address the role of GSK3β in ALCL proliferation, WST-1 assay revealed that ALK inhibition resulted in a decrease in cell viability which was rescued by GSK3β inhibition. ALK inhibition resulted in significant cellular apoptosis as detected by propidium iodide and Annexin V staining followed by flow cytometry. This apoptotic response was rescued by GSK3β inhibition. The effect of GSK3β on cellular oncogenic potential was addressed by colony formation assay using ALCL-derived cell lines. While ALK inhibition resulted in decreased colony numbers, simultaneous ALK and GSK3β inhibition recovered colony numbers compared to ALK inhibition alone. Additionally, stable GSK3β knockdown conferred resistance to growth inhibitory effect of ALK inhibition as determined by colony formation assay. Furthermore, pS9-GSK3β and its known substrate, CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies and showed a significant correlation (p<0.001). Conversely, ALK-ALCL tissue biopsies showed no significant correlation of pS9-GSK3β and CDC25A expression (p<0.2). Our results demonstrate that NPM-ALK signals through PI3K/AKT to phosphorylate GSK3β at serine-9. This phosphorylation inhibits GSK3β kinase activity which results in the accumulation of CDC25A and Mcl-1 thus providing growth advantage and protection from apoptosis. These findings provide support for the role of GSK3β as a novel mediator of NPM-ALK oncogenesis. Disclosures:No relevant conflicts of interest to declare.

GSK3β Is Involved in the Relief of Mitochondria Pausing in a Tau-Dependent Manner

Mitochondrial trafficking deficits have been implicated in the pathogenesis of several neurological diseases, including Alzheimer's disease (AD). The Ser/Thre kinase GSK3β is believed to play a fundamental role in AD pathogenesis. Given that GSK3β substrates include Tau protein, here we studied the impact of GSK3β on mitochondrial trafficking and its dependence on Tau protein. Overexpression of GSK3β in neurons resulted in an increase in motile mitochondria, whereas a decrease in the activity of this kinase produced an increase in mitochondria pausing. These effects were dependent on Tau proteins, as Tau (−/−) neurons did not respond to distinct GSK3β levels. Furthermore, differences in GSK3β expression did not affect other parameters like mitochondria velocity or mitochondria run length. We conclude that GSK3B activity regulates mitochondrial axonal trafficking largely in a Tau-dependent manner.

Design and combinatorial synthesis of a novel kinase-focused library using click chemistry-based fragment assembly

Fragment-based lead discovery is a new approach for lead generation that has emerged in the past decade. Because the initial fragments identified in the fragment screening typically show weak binding affinity, an intensive medicinal chemistry effort would be required to grow initial fragments into a potential lead compound. Here we demonstrate a kinase focused evolved fragment (KFEF) library, constructed by click chemistry-based fragment assembly, that is a valuable source of kinase inhibitors. This combinatorial assembly of two fragments, kinase-privileged alkyne fragments and diversified azide fragments, by two cycloaddition reactions shows a unique potential for the one-step synthesis of structurally diverse evolved fragments. The screening of this triazole-based KFEF library allowed the rapid identification of potent lead candidates for FLT3 and GSK3β kinase.

Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition

Systems biology approaches that combine experimental data and theoretical modelling to understand cellular signalling network dynamics offer a useful platform to investigate the mechanisms of resistance to drug interventions and to identify combination drug treatments. Extending our work on modelling the PI3K/PTEN/AKT signalling network (SN), we analyse the sensitivity of the SN output signal, phospho-AKT, to inhibition of HER2 receptor. We model typical aberrations in this SN identified in cancer development and drug resistance: loss of PTEN activity, PI3K and AKT mutations, HER2 overexpression, and overproduction of GSK3β and CK2 kinases controlling PTEN phosphorylation. We show that HER2 inhibition by the monoclonal antibody pertuzumab increases SN sensitivity, both to external signals and to changes in kinetic parameters of the proteins and their expression levels induced by mutations in the SN. This increase in sensitivity arises from the transition of SN functioning from saturation to non-saturation mode in response to HER2 inhibition. PTEN loss or PIK3CA mutation causes resistance to anti-HER2 inhibitor and leads to the restoration of saturation mode in SN functioning with a consequent decrease in SN sensitivity. We suggest that a drug-induced increase in SN sensitivity to internal perturbations, and specifically mutations, causes SN fragility. In particular, the SN is vulnerable to mutations that compensate for drug action and this may result in a sensitivity-to-resistance transition. The combination of HER2 and PI3K inhibition does not sensitise the SN to internal perturbations (mutations) in the PI3K/PTEN/AKT pathway: this combination treatment provides both synergetic inhibition and may prevent the SN from acquired mutations causing drug resistance. Through combination inhibition treatments, we studied the impact of upstream and downstream interventions to suppress resistance to the HER2 inhibitor in the SN with PTEN loss. Comparison of experimental results of PI3K inhibition in the PTEN upstream pathway with PDK1 inhibition in the PTEN downstream pathway shows that upstream inhibition abrogates resistance to pertuzumab more effectively than downstream inhibition. This difference in inhibition effect arises from the compensatory mechanism of an activation loop induced in the downstream pathway by PTEN loss. We highlight that drug target identification for combination anti-cancer therapy needs to account for the mutation effects on the upstream and downstream pathways.