Inhibition Of c-Abl Research Articles

C-Abl-dependent Molecular Circuitry Involving Smad5 and Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2-induced Osteogenesis

Skeletal remodeling consists of timely formation and resorption of bone by osteoblasts and osteoclasts in a quantitative manner. Patients with chronic myeloid leukemia receiving inhibitors of c-Abl tyrosine kinase often show reduced bone remodeling due to impaired osteoblast and osteoclast function. BMP-2 plays a significant role in bone generation and resorption by contributing to the formation of mature osteoblasts and osteoclasts. The effects of c-Abl on BMP-2-induced bone remodeling and the underlying mechanisms are not well studied. Using a pharmacological inhibitor and expression of a dominant negative mutant of c-Abl, we show an essential role of this tyrosine kinase in the development of bone nodules containing mature osteoblasts and formation of multinucleated osteoclasts in response to BMP-2. Calvarial osteoblasts prepared from c-Abl null mice showed the absolute requirement of this tyrosine kinase in maturation of osteoblasts and osteoclasts. Activation of phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling by BMP-2 leads to osteoblast differentiation. Remarkably, inhibition of c-Abl significantly suppressed BMP-2-stimulated PI 3-kinase activity and its downstream Akt phosphorylation. Interestingly, c-Abl regulated BMP-2-induced osteoclastogenic CSF-1 expression. More importantly, we identified the requirements of c-Abl in BMP-2 autoregulation and the expressions of alkaline phosphatase and osterix that are necessary for osteoblast differentiation. c-Abl contributed to BMP receptor-specific Smad-dependent transcription of CSF-1, osterix, and BMP-2. Finally, c-Abl associates with BMP receptor IA and regulates phosphorylation of Smad in response to BMP-2. We propose that activation of c-Abl is an important step, which induces into two signaling pathways involving noncanonical PI 3-kinase and canonical Smads to integrate BMP-2-induced osteogenesis.

The Hippo pathway kinase Lats2 prevents DNA damage-induced apoptosis through inhibition of the tyrosine kinase c-Abl

The Hippo pathway is an evolutionarily conserved pathway that controls cell proliferation, organ size, tissue regeneration and stem cell self-renewal. Here we show that it also regulates the DNA damage response. At high cell density, when the Hippo pathway is active, DNA damage-induced apoptosis and the activation of the tyrosine kinase c-Abl were suppressed. At low cell density, overexpression of the Hippo pathway kinase large tumor suppressor 2 (Lats2) inhibited c-Abl activity. This led to reduced phosphorylation of downstream c-Abl substrates, the transcription coactivator Yes-associated protein (Yap) and the tumor suppressor p73. Inhibition of c-Abl by Lats2 was mediated through Lats2 interaction with and phosphorylation of c-Abl. Lats2 knockdown, or expression of c-Abl mutants that escape inhibition by Lats2, enabled DNA damage-induced apoptosis of densely plated cells, while Lats2 overexpression inhibited apoptosis in sparse cells. These findings explain a long-standing enigma of why densely plated cells are radioresistant. Furthermore, they demonstrate that the Hippo pathway regulates cell fate decisions in response to DNA damage.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5′-phosphatase interaction with c-Abl

It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Claudin-1 induces epithelial–mesenchymal transition through activation of the c-Abl-ERK signaling pathway in human liver cells

Claudins (CLDNs) are a family of integral membrane proteins central to the formation of tight junctions, structures that are involved in paracellular transport and cellular growth and differentiation, and are critical for the maintenance of cellular polarity. Recent studies have provided evidence that CLDNs are aberrantly expressed in diverse types of human cancers, including hepatocellular carcinomas (HCCs). However, little is known about how CLDN expression is involved in cancer progression. In this study, we show that CLDN1 has a causal role in the epithelial-mesenchymal transition (EMT) in human liver cells, and that the c-Abl-Ras-Raf-1-ERK1/2 signaling axis is critical for the induction of malignant progression by CLDN1. Overexpression of CLDN1 induced expression of the EMT-regulating transcription factors Slug and Zeb1, and thereby led to repression of E-cadherin, β-catenin expression, enhanced expression of N-cadherin and Vimentin, a loss of cell adhesion, and increased cell motility in normal liver cells and HCC cells. In line with these findings, inhibition of either c-Abl or ERK clearly attenuated CLDN1-induced EMT, as evidenced by a reversal of N-cadherin and E-cadherin expression patterns, and restored normal motility. Collectively, these results indicate that CLDN1 is necessary for the induction of EMT in human liver cells, and that activation of the c-Abl-Ras-Raf-1-ERK1/2 signaling pathway is required for CLDN1-induced acquisition of the malignant phenotype. The present observations suggest that CLDN1 could be exploited as a biomarker for liver cancer metastasis and might provide a pivotal point for therapeutic intervention in HCC.

α6 Integrin and CD44 Enrich for a Primary Keratinocyte Population That Displays Resistance to UV-Induced Apoptosis

Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrinhigh+/CD44+ sub-population of basal keratinocytes. These α6 integrinhigh+/CD44+ keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrinhigh+/CD44+ cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrinhigh+/CD44+ cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrinhigh+/CD44+ cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents.

C-Abl inhibition delays motor neuron degeneration in the G93A mouse, an animal model of amyotrophic lateral sclerosis.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive death of motor neurons. Although the pathogenesis of ALS remains unclear, several cellular processes are known to be involved, including apoptosis. A previous study revealed the apoptosis-related gene c-Abl to be upregulated in sporadic ALS motor neurons.Methodology/FindingsWe investigated the possibility that c-Abl activation is involved in the progression of ALS and that c-Abl inhibition is potentially a therapeutic strategy for ALS. Using a mouse motor neuron cell line, we found that mutation of Cu/Zn-superoxide dismutase-1 (SOD1), which is one of the causative genes of familial ALS, induced the upregulation of c-Abl and decreased cell viability, and that the c-Abl inhibitor dasatinib inhibited cytotoxicity. Activation of c-Abl with a concomitant increase in activated caspase-3 was observed in the lumbar spine of G93A-SOD1 transgenic mice (G93A mice), a widely used model of ALS. The survival of G93A mice was improved by oral administration of dasatinib, which also decreased c-Abl phosphorylation, inactivated caspase-3, and improved the innervation status of neuromuscular junctions. In addition, c-Abl expression in postmortem spinal cord tissues from sporadic ALS patients was increased by 3-fold compared with non-ALS patients.Conclusions/SignificanceThe present results suggest that c-Abl is a potential therapeutic target for ALS and that the c-Abl inhibitor dasatinib has neuroprotective properties in vitro and in vivo.

Combined Inhibition of c-Abl and PDGF Receptors for Prevention and Treatment of Murine Sclerodermatous Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic bone marrow transplantation, and has a major effect on the long-term prognosis. The molecular mechanisms underlying cGvHD have been only partially revealed, and molecular targeted therapies have not yet been established for clinical use. We examined the effects of the combined inhibition of the Abelson kinase (c-Abl) and platelet-derived growth factor receptors (PDGFR) in experimental sclerodermatous cGvHD. Treatment using imatinib or nilotinib abolished the aberrant activation of c-Abl and PDGFR and protected against experimental cGvHD. Preventive therapy using imatinib or nilotinib inhibited the development of sclerodermatous cGvHD. Clinical features such as weight loss, alopecia, and skin ulcers, and histologic features with dermal thickening and accumulation of collagen were significantly reduced in mice that received imatinib or nilotinib therapy, but not in mice that received prednisone therapy. Of note, imatinib and nilotinib were also effective for treatment of experimental cGvHD that had already been clinically manifested. In summary, the combined inhibition of c-Abl and PDGFR is effective for prevention and treatment of experimental sclerodermatous cGvHD. Considering the high morbidity associated with cGvHD, the lack of efficient molecular therapies for clinical use, and first positive signals from uncontrolled studies of imatinib, combined inhibition of c-Abl and PDGFR might be a promising future strategy for treatment of sclerodermatous cGvHD.

Mechanistic Investigation of Imatinib-Induced Cardiac Toxicity and the Involvement of c-Abl Kinase

The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.

Clinical or Sub-Clinical Pancreatitis (PA) Associated with Nilotinib (Nb) As Frontline for Chronic Myelogenous Leukemia (CML)

Abstract 4443 Background:Nilotinib is now standard therapy in chronic phase (CP) CML, both as initial therapy and after imatinib failure. One of the adverse effects of nilotinib is the increase in serum amylase and/or lipase levels with or without clinical pancreatitis.The mechanism by which nilotinib increases serum pancreatic enzymes is not clear. Nilotinib is able to inhibit the non-receptor tyrosine kinase c-abl with high affinity. It is possible that c-abl inhibition might interfere with the molecular mechanisms regulating pancreatic cell death, inducing pancreatic damage. Nilotinib may release calcium from the intracellular acinar stores which regulate exocrine pancreatic secretion, or it may promote the accumulation of fatty acid inside the pancreatic acinar cells which disturbs exocytose. (JOP. J Pancreas (Online) 2010 May 5; 291–293). Purpose:To understand the frequency, risk factors and management strategy for patients who develop pancreatitis (clinical or subclinical) during nilotininb frontline therapy for CML. Method:We reviewed the records of 105 CML patients treated at our institution on a clinical trial from 7/2005 to 7/2011 with nilotinib 400 mg twice daily as initial therapy for CML in chronic phase. Results:The median age for the total population was 52 yrs (range, 17 to 89), and 63 were males (60%). Thirteen (12%) pts experienced pancreatitis at least once, most frequently asymptomatic (ie, grade 1). Pancreatitis episodes occurred 28 times (2 episodes were grade 3, 2 episodes were grade 4, 10 episodes were grade 2, and 14 episodes were grade 1). Thirteen episodes (47%) occurred at a dose of 200 mg daily, 4 (14%) at 400 mg daily, and 11 (39%) at 800 mg daily. In 3 instances total bilirubin was also elevated (2 grade 2 and 1 grade 4). One patient experienced recurrent elevations (16 episodes) of lipase and amylase despite dose reduction to 200mg once daily. Concurrent use of hydrochlorothiazide and moderate alcoholic intake were identified as risk factors associated with pancreatitis in the patient who experienced recurrent episodes. Diabetes was identified as a risk factor for pancreatitis in other 4 pts. Twenty four episodes (86%) were characterized by an elevation of lipase and amylase without any clinical symptoms. In four instances, patients experienced a moderate abdominal pain without other symptoms of pancreatitis. In 3 instances, imaging (ultrasound and/or CT scan) demonstrated normal pancreases. Management of pancreatitis included transient nilotinib interruption in most instances. In most patients, lipase and amylase values returned to normal within 14 days of stopping nilotinib. Three patients eventually required change from nilotinib to other tyrosine kinase inhibitors (TKI) (imatinib 2, dasatinib 1) without recurrence of pancreatitis. One of these pts switched to imatinib because of recurrent pancreatitis and the other 2 were switched because of nilotinib toxicities (liver and cardiac toxicity). At the time of pancreatitis episodes, 54% had achieved complete cytogenetic response (CCyR), and 8% complete molecular response (CMR). Rates of (CCyR) were 92% and 94% in patients who experienced and who did not experience pancreatitis, respectively. Rates of major molecular response (MMR) were 100%and 87% in patients who experienced and who did not experience pancreatitis, respectively. Rates of complete hematologic response (CHR) were 100% and 98% in patients who experienced and who did not experience pancreatitis, respectively. The 3-year event-free survival (EFS), treatment- free survival (TFS), and overall survival (OS) rates were 100% in patients who experienced and 93%, 97%, and 100% in patients who did not experience pancreatitis, respectively. Conclusion:Pancreatitis associated with nilotinib is most frequently grade 1 and 2 (asymptomatic enzyme elevation). Close monitoring and timely intervention may allow patients to continue therapy and achieve the desired clinical benefit. Rarely, pancreatitis may require treatment discontinuation. Disclosures:Cortes:BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Deciphera: Research Funding.

Dasatinib as Salvage Therapy for Steroid Refractory and Imatinib Resistant or Intolerant Sclerotic Chronic Graft-versus-Host Disease

Sclerotic chronic graft-versus-host disease (scGVHD) is a severe form of this disease that resembles systemic sclerosis and has limited and disappointing treatment options. Tyrosine kinase inhibitors (TKI) targeting up-regulated profibrotic pathways, such as imatinib mesylate, have been proposed as a potential therapeutic approach for patients with scGVHD. Dasatinib, a second-generation TKI with a well-established safety and efficacy profile in chronic myeloid leukemia patients, who are refractory or intolerant to imatinib, has also shown potent antifibrotic effects. We present here the first direct clinical evidence, from 3 patients treated in a small single-center series, suggesting that dasatinib can be a therapeutic option for patients with severe scGVHD resistant or intolerant to imatinib. All patients achieved partial response, with improvement in scGHVD target organs severity, joint mobility, lung impairment, and deep fibrotic lesions. This clinical response has remained stable or continued to improve after a median of 22 months (20-25) on dasatinib treatment, with very good tolerance. In addition, corticosteroids could be discontinued or significantly reduced in all patients. This clinical evidence suggests that dasatinib could be a safe and effective alternative for scGVHD patients refractory to corticosteroids and resistant or intolerant to imatinib. Based on these preliminary findings, and in order to address appropriate patient selection, time of intervention, and choice of drug, future larger studies should more formally establish the efficacy and safety of second-generation TKI for the treatment of scGVHD.

The c-Abl-MST1 Signaling Pathway Mediates Oxidative Stress-Induced Neuronal Cell Death

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. The protein kinase MST1 (mammalian Ste20-like kinase 1) plays a major role in oxidative stress-induced cell death in primary mammalian neurons. However, the mechanisms that regulate MST1 in oxidative stress responses remain largely unknown. In the present study, we demonstrate that the protein kinase c-Abl phosphorylates MST1 at Y433, which triggers the stabilization and activation of MST1. Inhibition of c-Abl promotes the degradation of MST1 through C terminus of Hsc70-interacting protein (CHIP)-mediated ubiquitination, and thereby attenuates cell death. Oxidative stress induces the c-Abl-dependent tyrosine phosphorylation of MST1 and increases the interaction between MST1 and FOXO3 (Forkhead box O3), thereby activating the MST1-FOXO signaling pathway, leading to cell death in both primary culture neurons and rat hippocampal neurons. The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST1 suggests that the c-Abl-MST1 signaling cascade plays an important role in cellular responses to oxidative stress.

High glucose induces apoptosis in embryonic neural progenitor cells by a pathway involving protein PKCδ

Diabetic-induced neural tube defects in embryos are caused by apoptosis of neural progenitor cells (NPCs); however, the underlying mechanisms are poorly understood. The present study is aimed to investigate the specific cellular proteins that may be involved in apoptosis of NPCs. We show here that hyperglycemia-induced apoptosis of NPCs was through a PKCδ-dependent mechanism. Tyrosine phosphorylation of PKCδ was required for PKCδ binding to c-Abl in the cytoplasm, and inhibition of c-Abl by STI571 or knock-down of c-Abl by RNAi decreased the phosphorylation of PKCδ. Moreover, translocation of PKCδ and c-Abl complex from the cytoplasm to the nucleus, was blocked by down-regulation of PKCδ or c-Abl. Furthermore, we found that interaction of PKCδ and c-Abl played a crucial role in p53 accumulation in the nucleus, which was linked to the apoptosis of NPCs in response to high glucose.

C-Abl regulates Mcl-1 gene expression in chronic lymphocytic leukemia cells

AbstractChronic lymphocytic leukemia (CLL) is a malignancy characterized by clonal expansion of mature B cells that are resistant to apoptosis. This resistance to apoptosis partly results from Mcl-1 expression because high levels of this protein in CLL cells correlate with poor disease prognosis and resistance to chemotherapy. Thus, understanding the mechanism(s) regulating Mcl-1 expression in CLL cells may be useful in the development of new therapies for this incurable disease. In the present study, we show a strong relationship between c-Abl and Mcl-1 expression in CLL cells. We show that treatment of CLL cells with Abl-specific siRNA or with imatinib, to inhibit c-Abl activity, results in the down-regulation of Mcl-1 protein and mRNA. A major regulator of Mcl-1 gene expression is STAT3. Our data show that CLL cells expressing high levels of c-Abl also show elevated levels of phospho-STAT3, and that STAT3 phosphorylation in CLL cells is dependent on c-Abl activity. However, STAT3 phosphorylation by c-Abl requires activation of nuclear factor-κB, secretion of autocrine interleukin-6, and active protein kinase C. Taken together, our data demonstrate the mechanism involved in c-Abl regulation of Mcl-1 expression in CLL cells, and suggest that c-Abl inhibition has therapeutic application in the treatment of this disease.

The LAR protein tyrosine phosphatase enables PDGF β-receptor activation through attenuation of the c-Abl kinase activity

The receptor tyrosine phosphatase (RPTP) LAR negatively regulates the activity of several receptor tyrosine kinases. To investigate if LAR affects the platelet-derived growth factor (PDGF) receptor signaling, mouse embryonic fibroblasts (MEFs) from mice where the LAR phosphatase domains were deleted (LARΔP), and wt littermates, were stimulated with 20 ng/ml PDGF-BB. In LAR phosphatase deficient MEFs, the phosphorylation of the PDGF β-receptor was surprisingly reduced, an event that was rescued by re-expression of wt LAR. The decreased phosphorylation of the PDGF β-receptor was observed independent of ligand concentration and occurred on all tyrosine residues, as determined by immunoblotting analysis using site-selective phosphotyrosine antibodies. This suggests that LAR is required for full PDGF β-receptor kinase activation. Downstream of receptor activation, phosphorylation of Akt and PLCγ were decreased in LARΔP MEFs, whereas Src and Erk MAP kinase pathways were less affected. The proliferation of LARΔP MEFs in response to PDGF-BB was also reduced. The inhibitory effect on the PDGF β-receptor in LARΔP cells was exerted via increased basal activity of c-Abl, since inhibition of c-Abl, by AG957 or siRNA, restored PDGF β-receptor phosphorylation. These observations suggest that LAR reduces the basal c-Abl activity thereby allowing for PDGF β-receptor kinase activation.

Dual inhibition of the homologous recombinational repair and the nonhomologous end-joining repair pathways in chronic lymphocytic leukemia therapy

Resistance to chlorambucil in chronic lymphocytic leukemia (CLL) has been associated with increased DNA repair. Specifically, inhibition of either c-abl, which modulates Rad51 directed homologous recombination or DNA-PK dependent nonhomologous end joining has been shown to sensitize primary CLL lymphocytes to chlorambucil. Here we report that inhibition of c-abl can result in a compensatory increase in DNA-PK and thus inhibition of both c-abl and DNA-PK optimally sensitizes CLL lymphocytes to chlorambucil. In this paper we report a drug-induced compensatory change between two DNA repair pathways with potential therapeutic implications in CLL therapy.

Role of c-Abl in L-selectin shedding from the neutrophil surface

L-selectin is a key molecule that participates in neutrophil tethering and subsequent rolling. It is cleaved from the surface of neutrophils activated in the presence of lipopolysaccharides, N-formyl-methionine-leucine-phenylalanine (fMLP), or Interleukin-8 (IL-8). We previously showed that L-selectin is also shed from the neutrophil surface during rolling on sialyl Lewis-x coated surfaces in a force-, ADAM-17 sheddase-, and p38 MAP kinase-dependent manner under flow. c-Abl tyrosine kinase is phosphorylated when L-selectin on the surface of neutrophils is cross-linked with anti-L-selectin antibodies. Here, we study the effect of c-Abl inhibition on L-selectin shedding from primary human neutrophils in static conditions following exposure to fMLP, IL-8, and hypotonic buffer and under flow through sialyl Lewis-x coated microtubes. Results indicate that c-Abl inhibition by STI571 significantly affects neutrophil adhesion via L-selectin, by decreasing the average rolling velocity and increasing the flux of rolling cells. The change in surface receptor expression was verified by flow cytometry. Interestingly, other forms of L-selectin shedding induced by fMLP, IL-8 or osmotic swelling were unaffected by STI571 treatment. These findings implicate the c-Abl signaling molecule in regulating L-selectin mechanical shedding in response to shear stress, setting this type of signaling apart from those triggered by the presence of a hypotonic environment, fMLP, or IL-8. This study sheds light on the role of c-Abl in neutrophil adhesion not previously reported in the literature.

Novel Regulation of Parkin Function through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.

C-Abl Regulates Mcl-1 Gene Expression In Chronic Lymphocytic Leukaemia Cells

AbstractAbstract 3579Chronic lymphocytic leukaemia (CLL) is a prevalent malignancy characterised by the clonal expansion of mature B cells that are resistant to apoptosis. This resistance to apoptosis will partly be due to Mcl-1 expression because high levels of this protein in CLL cells correlate with poor disease prognosis and resistance to chemotherapy. Thus, understanding the mechanism(s) regulating Mcl-1 expression in CLL cells may be useful in the development of new therapies for this incurable disease. In the present study we show a strong relationship between c-Abl and Mcl-1 expression in CLL cells. We show that treatment of CLL cells with Abl-specific siRNA or with imatinib, to inhibit c-Abl activity, results in the downregulation of Mcl-1 protein and mRNA. A major regulator of Mcl-1 gene expression is STAT3. Our data show that CLL cells expressing high levels of c-Abl also show elevated levels of phospho-STAT3, and that STAT3 phosphorylation in CLL cells is dependent on c-Abl activity. However, STAT3 phosphorylation by c-Abl is not direct, and requires activation of NFkB, secretion of autocrine IL6 and active PKC. Taken together, our results provide clearer definition of the pathobiological role of c-Abl in CLL cells. Given that high NFkB activation, plasma IL6 levels and Mcl-1 all correlate with poor disease prognosis in CLL, our work potentially connects several features of CLL cells that are important to their pathophysiology by suggesting a central role of c-Abl in their regulation. Since CLL cells expressing high levels of c-Abl and Mcl-1 belong to the unmutated, poor prognostic group of CLL, c-Abl inhibition may have therapeutic application in the treatment of this disease, especially for those patients who are resistant to conventional chemotherapeutic agents. Disclosures:No relevant conflicts of interest to declare.

Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP(+)) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571's protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD.

A Phase II Study of Imatinib in Patients with Advanced Anaplastic Thyroid Cancer

Currently, there is no standard treatment for metastatic anaplastic thyroid cancer (ATC). DNA microarray analysis has shown platelet-dervived growth factor receptor (PDGFR) overexpression in ATC relative to well-differentiated thyroid cancer. In p53-mutated/deficient ATC cell lines, cABL is overexpressed, and selective inhibition of cABL results in a cytostatic effect. Imatinib inhibits tyrosine kinase activity of Bcr-ABL and PDGF. We hypothesize that patients with ATC that over-expresses PDGF receptors or cABL will respond to imatinib. Patients with histologically confirmed ATC who had measurable disease and whose disease expressed PDGF receptors by immunohistochemistry were eligible for study. Imatinib was administered at 400 mg orally twice daily without drug holiday. Response to treatment was assessed every 8 weeks. Patients with complete response, partial responses, or stable disease were treated until disease progression. The study was terminated early due to poor accrual. From February 2004 to May 2007, 11 patients were enrolled and were started on imatinib. At baseline, 4/11 had locoregional disease, 5/11 had distant metastases, and 2/11 had both. Nine of 11 had prior chemoradiation, and 7/11 had thyroidectomy. Eight of 11 were evaluable for response; 4 were excluded for lack of follow-up with radiologic evaluation. The overall response rates at 8 weeks were complete response 0/8, partial response 2/8, and stable disease 4/8. The median time to follow-up was 26 months (ranges 23-30 months). The rate of 6-month progression-free survival was 36% (95% confidence interval, 9%-65%). The rate of 6-month overall survival was 45% (95% confidence interval, 16%-70%). The most common grade 3 toxicity was edema in 25%; other grade 3 toxicities included fatigue and hyponatremia (12.5% each). There were no grade 4 toxicities or treatment related deaths. Imatinib appears to have activity in advanced ATC and is well tolerated. Due to difficulty of accruing patients with a rare malignancy at a single institution, further investigation of imatinib in ATC may be warranted in a multi-institutional setting.