c-Abl Kinase Research Articles

The c-Abl inhibitor in Parkinson disease

Parkinson's disease (PD) is an insidious onset neurodegenerative disease affecting approximately 1% of the population over the age of 65. So far available therapies for PD have only aimed at improving or alleviating symptoms, but not at slowing, preventing, and reversing the course of PD. Recently, some studies have indicated that the levels and activation of Abelson non-receptor tyrosine kinase (c-Abl, Abl1) were up-regulated in the brain tissue of patients with PD and demonstrated that c-Abl inhibitors could improve motor behavior, prevent the loss of dopamine neurons, inhibit phosphorylation of Cdk5, regulate α-synuclein phosphorylation and clearance, inhibit the tyrosine phosphorylation of parkin and decrease parkin substrate, for example, PARIS (zinc finger protein 746), AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein type2), FBP1 (fuse-binding protein 1), and synphilin-1. Therefore, we review the mechanism of the c-Abl inhibitor in PD and conclude that c-Abl inhibitors may be a potential treatment in PD and other neurodegenerative disease.

C-Abl inhibition mitigates diet-induced obesity through improving insulin sensitivity of subcutaneous fat in mice.

High-energy diets are among the main causes of the global epidemic of metabolic disorders, including obesity and type 2 diabetes. The mechanisms of high-energy-diet-induced metabolic disorders are complex and largely unknown. The non-receptor tyrosine kinase c-Abl plays an important role in adipogenesis in vitro but its role in vivo in the regulation of metabolism is still elusive. Hence, we sought to address the role of c-Abl in diet-induced obesity and obesity-associated insulin resistance. The expression of c-Abl in different fat tissues from obese humans or mice fed a high-fat diet (HFD) were first analysed by western blotting and quantitative PCR. We employed conditional deletion of the c-Abl gene (also known as Abl1) in adipose tissue using Fabp4-Cre and 6-week-old mice were fed with either a chow diet (CD) or an HFD. Age-matched wild-type mice were treated with the c-Abl inhibitor nilotinib or with vehicle and exposed to either CD or HFD, followed by analysis of body mass, fat mass, glucose and insulin tolerance. Histological staining, ELISA and biochemical analysis were used to clarify details of changes in physiology and molecular signalling. c-Abl was highly expressed in subcutaneous fat from obese humans and HFD-induced obese mice. Conditional knockout of c-Abl in adipose tissue improved insulin sensitivity and mitigated HFD-induced body mass gain, hyperglycaemia and hyperinsulinaemia. Consistently, treatment with nilotinib significantly reduced fat mass and improved insulin sensitivity in HFD-fed mice. Further biochemical analyses suggested that c-Abl inhibition improved whole-body insulin sensitivity by reducing HFD-triggered insulin resistance and increasing adiponectin in subcutaneous fat. Our findings define a new biological role for c-Abl in the regulation of diet-induced obesity through improving insulin sensitivity of subcutaneous fat. This suggests it may become a novel therapeutic target in the treatment of metabolic disorders.

Tyrosine kinase c-Abl regulates the survival of plasma cells

Tyrosine kinase c-Abl plays an important role in early B cell development. Its deletion leads to reduced pro- and pre-B cell generation in mice. However, its function in B cell terminal differentiation remains unexplored. Here, we used c-Ablf/f Aicdacre/+ mice, in which c-Abl is ablated only in antigen-activated B cells, to study the role of c-Abl in germinal center (GC) B and antibody-secreting plasma cell formation. Upon challenge with a model antigen, we found normal GC and memory B but reduced plasma cells and antigen-specific antibody response in the mutant mice. In-vitro studies revealed that plasma cells lacking c-Abl could be generated but did not accumulate in culture, indicative of survival defect. They also exhibited impaired STAT3 phosphorylation. The plasma cell defects could be rectified by introduction of Bim-deficiency or delivery of colivelin, a STAT3 activator, into c-Ablf/f Aicdacre/+ mice. Hence, c-Abl signalling regulates the survival of plasma cells.

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non-receptor-type tyrosine kinase c-Abl. c-Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N- and C-terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C-terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c-Abl, enhanced the activation of estrogen signaling. In contrast, the c-Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild-type. These results suggest that c-Abl-mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453-1461, 2017. © 2016 Wiley Periodicals, Inc.

Novel Src/Abl tyrosine kinase inhibitor bosutinib suppresses neuroblastoma growth via inhibiting Src/Abl signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Aberrant activation of the non-receptor tyrosine kinases Src and c-Abl contributes to the progression of NB. Thus, targeting these kinases could be a promising strategy for NB therapy. In this paper, we report that the potent dual Src/Abl inhibitor bosutinib exerts anti-tumor effects on NB. Bosutinib inhibited NB cell proliferation in a dose-dependent manner and suppressed colony formation ability of NB cells. Mechanistically, bosutinib effectively decreased the activity of Src/Abl and PI3K/AKT/mTOR, MAPK/ERK, and JAK/STAT3 signaling pathways. In addition, bosutinib enhanced doxorubicin (Dox)- and etoposide (VP-16)-induced cytotoxicity in NB cells. Furthermore, bosutinib demonstrated anti-tumor efficacy in an orthotopic xenograft NB mouse model in a similar mechanism as of that in vitro. In summary, our results reveal that Src and c-Abl are potential therapeutic targets in NB and that the novel Src/Abl inhibitor bosutinib alone or in combination with other chemotherapeutic agents may be a valuable therapeutic option for NB patients.

Autophagy regulates MAVS signaling activation in a phosphorylation-dependent manner in microglia.

Mitochondrial antiviral signaling (MAVS) protein has an important role in antiviral immunity and autoimmunity. However, the pathophysiological role of this signaling pathway, especially in the brain, remains elusive. Here we demonstrated that MAVS signaling existed and mediated poly(I:C)-induced inflammation in the brain. Along with the MAVS signaling activation, there was an induction of autophagic activation. Autophagy negatively regulated the activity of MAVS through direct binding of LC3 to the LIR motif Y(9)xxI(12) of MAVS. We also found that c-Abl kinase phosphorylated MAVS and regulated its interaction with LC3. Interestingly, tyrosine phosphorylation of MAVS was required for downstream signaling activation. Importantly, in vivo data showed that the deficiency of MAVS or c-Abl prevented MPTP-induced microglial activation and dopaminergic neuron loss. Together, our findings reveal the molecular mechanisms underlying the regulation of MAVS-dependent microglial activation in the nervous system, thus providing a potential target for the treatment of microglia-driven inflammatory brain diseases.

Reduction of Blood Amyloid-β Oligomers in Alzheimer's Disease Transgenic Mice by c-Abl Kinase Inhibition.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aβ). The role of the c-Abl tyrosine kinase in Aβ-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aβ in the blood of AD transgenic mice. We found that imatinib reduces Aβ-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of β-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aβ accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.

Mechanism-Based Drug Combinations with the DNA Strand-Breaking Nucleoside Analog CNDAC.

CNDAC (2'-C-cyano-2'-deoxy-1-β-d-arabino-pentofuranosyl-cytosine, DFP10917) and its orally bioavailable prodrug, sapacitabine, are undergoing clinical trials for hematologic malignancies and solid tumors. The unique action mechanism of inducing DNA strand breaks distinguishes CNDAC from other deoxycytidine analogs. To optimize the clinical potentials of CNDAC, we explored multiple strategies combining CNDAC with chemotherapeutic agents targeting distinct DNA damage repair pathways that are currently in clinical use. The ability of each agent to decrease proliferative potential, determined by clonogenic assays, was determined in paired cell lines proficient and deficient in certain DNA repair proteins. Subsequently, each agent was used in combination with CNDAC at fixed concentration ratios. The clonogenicity was quantitated by median effect analysis, and a combination index was calculated. The c-Abl kinase inhibitor imatinib had synergy with CNDAC in HCT116 cells, regardless of p53 status. Inhibitors of PARP1 that interfere with homologous recombination (HR) repair or base excision repair (BER) and agents such as temozolomide that cause DNA damage repaired by the BER pathway were also synergistic with CNDAC. The toxicity of the nitrogen mustards bendamustine and cytoxan, or of platinum compounds, which generate DNA adducts repaired by nucleotide excision repair and HR, was additive with CNDAC. An additive cell killing was also achieved by the combination of CNDAC with taxane mitotic inhibitors (paclitaxel and docetaxel). At concentrations that allow survival of the majority of wild-type cells, the synergistic or additive combination effects were selective in HR-deficient cells. This study provides mechanistic rationales for combining CNDAC with other active drugs. Mol Cancer Ther; 15(10); 2302-13. ©2016 AACR.

Parkin regulates lipopolysaccharide-induced proinflammatory responses in acute lung injury

The acute respiratory distress syndrome (ARDS) is a serious condition resulting from direct or indirect lung injury that is associated with high mortality and morbidity. A key biological event in the pathogenesis of the acute lung injury (ALI) that causes acute respiratory distress syndrome is activation of the lung endothelium cells (ECs), which is triggered by a variety of inflammatory insults leading to barrier disruption and excessive accumulation of neutrophils. Recently, we demonstrated that imatinib protects against lipopolysaccharide (LPS)-induced EC activation by inhibiting c-Abl kinase. In the present study, we explored the role of parkin, a novel c-Abl substrate, in ALI. Parkin is an E3 ubiquitin ligase originally characterized in the pathogenesis of Parkinson disease; however, its potential role in acute inflammatory processes and lung EC function remains largely unknown. Using parkin deficient (PARK2-/-) mice, we now demonstrate that parkin mediates LPS-induced ALI. After LPS, PARK2-/- mice have reduced total protein and cell levels in bronchoalveolar lavage (BAL) compared to wild type. Moreover, in LPS-treated PARK2-/- lungs, the sequestration and activation of neutrophils and release of inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α]) are significantly reduced. The BAL levels of soluble VCAM-1 and ICAM-1 are also decreased in LPS-treated PARK2-/- mice compared to wild type. In cultured human lung endothelial cells, downregulation of parkin by small interfering RNA decreases LPS-induced VCAM-1 expression, IL-8 and IL-6 secretion, and NF-kB phosphorylation. These results suggest a previously unidentified role of parkin in mediating endotoxin-induced endothelial proinflammatory signaling and indicate that it may play a critical role in acute inflammation.

CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families.

CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori. CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori. We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma.

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.

Activation of tyrosine kinase c-Abl contributes to α-synuclein-induced neurodegeneration.

Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies.

C-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution.

Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity.

Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl

The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van ’t Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and 15N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl.

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

Altered S-nitrosothiol homeostasis provides a survival advantage to breast cancer cells in HER2 tumors and reduces their sensitivity to trastuzumab

The monoclonal antibody trastuzumab against HER2/neu, which is overexpressed in 15–20% of breast cancers, has clinical efficacy but many patients do not respond to initial treatment or develop resistance during treatment. Nitric oxide (NO) regulates cell signaling by targeting specific cysteine residues in proteins, forming S-nitrosothiols (SNO) in a process known as S-nitrosylation. We previously reported that molecular characteristics in breast cancer may dictate the tumor response to impaired SNO homeostasis. In the present study, we explored the role of SNO homeostasis in HER2 breast tumors.The antiproliferative action of trastuzumab in HER2-overexpressing BT-474 and SKBR-3 cells was suppressed when S-nitrosoglutathione reductase (GSNOR/ADH5) activity, which plays a key role in SNO homeostasis, was specifically inhibited with the pyrrole derivative compound N6022. Moreover, GSNOR inhibition restored the activation of survival signaling pathways involved in the resistance to anti-HER2 therapies (AKT, Src and c-Abl kinases and TrkA/NRTK1, TrkB/NRTK2, EphA1 and EphA3 receptors) and reduced the apoptotic effect of trastuzumab. Accordingly, GSNOR inhibition augmented the S-nitrosylation of apoptosis-related proteins, including Apaf-1, pSer73/63 c-Jun, calcineurin subunit α and HSF1. In agreement with in vitro data, immunohistochemical analyses of 51 breast tumors showed that HER2 expression was associated with lower expression of GSNOR protein. Moreover, gene expression analysis confirmed that high ADH5/GSNOR gene expression was associated with high patient survival rates in HER2 tumors.In conclusion, our data provide evidence of molecular mechanisms contributing to the progression of HER2+ breast cancers and could facilitate the development of therapeutic options to counteract resistance to anti-HER2 therapies.

C-Abl contributes to glucose-promoted apoptosis via p53 signaling pathway in podocytes

AimTo investigate the role of the non-receptor tyrosine kinase c-Abl in high glucose-induced podocyte injury and its possible signal transduction pathway. MethodsSixteen C57BL/6 mice were randomly assigned to a group with diabetes and a normal control group. Subsequently, differentiated mouse podocytes were exposed to high-glucose conditions, and podocyte apoptosis was then assessed by flow cytometry and Hoechst 33258 staining. Western blot and immunofluorescence assay were used to measure c-Abl expression. Co-immunoprecipitation assay was used and c-Abl siRNA was applied to evaluate the interaction between c-Abl and p53. ResultsHigh glucose promotes podocyte apoptosis. The c-Abl expression in podocytes was increased after exposure to high glucose, stimulating the p53 signaling pathway. Conversely, treatment with c-Abl siRNA restored high glucose-promoted podocyte apoptosis and resulted in the reduction of p53 expression. Conclusionc-Abl contributes to high glucose-induced podocyte apoptosis via p53 signaling pathway.

Hippo/MST1 signaling mediates microglial activation following acute cerebral ischemia–reperfusion injury

Cerebral ischemia–reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1–IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src–MST1–IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

Identification of TKI-Sensitive Point Mutations That Activate c-ABL Kinase Activity and Transformation Potential and Confer in Vitro Resistance to the Allosteric ABL Inhibitor GNF-5

Introduction:Pathologically activated tyrosine kinases represent attractive therapeutic targets, with deregulated ABL kinase representing one of the best-validated examples.While physiologic activation of c-ABL is tightly controlled by intramolecular interactions, ABL fusion proteins are constitutively activated by oligomerization driven by the fusion partner, as well as by loss of kinase autoinhibition due to removal of an autoinhibitory myristate site at the N-terminus of c-ABL (Nagar et al, Cell, 2003). Recent routine genomic tumor sequencing of various human malignancies has identified point mutations of unknown significance in numerous kinases, including c-ABL, and it is expected that the increasingly common practice of tumor genome sequencing will provide new opportunities for tailored targeted therapy. To maximize therapeutic benefit and avoid unnecessary cost and toxicity, it will be critical to distinguish "driver" from "passenger" mutations. We therefore sought to: (i) test the transforming potential of select clinically detected c-ABL mutants, (ii) prospectively identify novel additional activating c-ABL point mutations, and (iii) determine if active mutant c-ABL isoforms retain sensitivity to ABL TKIs.Results:Two of four clinically identified c-ABL mutations tested caused a dramatic increase in ABL kinase activity and transformed Ba/F3 cells to growth factor independence. A mutagenesis screen of c-ABL identified six additional transforming point mutations in the SH3 and kinase domains, including one that has been reported clinically in breast invasive carcinoma (cBioPortal.org). Relative to BCR-ABL, all c-ABL mutants showed heightened sensitivity to imatinib. Allosteric site inhibitors targeting the myristate-binding pocket in the BCR-ABL kinase domain (KD) have been shown to inhibit BCR-ABL kinase activity, purportedly by mimicking binding of the c-ABL myristate group and establishing c-ABL-like autoinhibitory constraints (Hantschel, Haematologica, 2012). While only two c-ABL mutations from the screen were near the allosteric site, all mutants exhibited decreased sensitivity to the allosteric inhibitor GNF-5. Recent studies have shown that derivative compounds of GNF-5 targeting this site in BCR-ABL are subvertable by on-target resistance mutations. We tested whether three BCR-ABL mutations described to confer in vitro, and in one case clinical, resistance to ABL001 (currently undergoing clinical trial evaluation) could impact c-ABL kinase activity presumably by perturbing this autoinhibitory pocket. In the context of c-ABL, all three ABL001-resistant mutations activated ABL kinase, transformed Ba/F3 cells, and conferred substantial resistance to allosteric inhibition relative to native BCR-ABL. These findings raise the possibility that c-ABL mutations may play a role in resistance to emerging allosteric ABL inhibitors. Importantly, previous work suggested that the genomic breakpoint on chromosome 9 in one third of CML cases is located 5' to ABL exon 1b, which would be predicted to result in simultaneous KD mutation expression in BCR-ABL and c-ABL (Uphoff et al, Leuk Res, 1999). To definitively demonstrate that co-expression of KD mutations in c-ABL and BCR-ABL can occur in primary patient samples, we analyzed RNA from five CML patients harboring a dominant BCR-ABL T315I mutation, and identified a heterozygous c-ABL 1b T315I mutation in one case.Conclusions:We have identified eight novel activating point mutations in c-ABL (three found in clinical isolates) that are potential therapeutic targets of ABL TKIs. Activating c-ABL mutants, including T315I, confer substantial resistance to the allosteric ABL inhibitor GNF-5 regardless of proximity to the inhibitor-binding site, implying resistance mechanisms beyond mere steric hindrance. Thus, mutations may induce distal conformational changes by disrupting a kinase conformation required for allosteric compound inhibition. In a subset of CML cases, c-ABL point mutants are co-expressed from the same allele as BCR-ABL, suggesting that the location of the chromosome 9 breakpoint may negatively impact clinical responsiveness to allosteric ABL inhibitors. Sequence analysis of c-ABL in patients with resistance to allosteric ABL inhibitors is required to validate c-ABL point mutants as a clinically important mechanism of resistance to this emerging class of targeted therapeutics. DisclosuresOff Label Use: the use of ABL TKIs for malignancies associated with point mutations in c-ABL. Shah:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Plexxikon Inc.: Research Funding.

Neuronal gene repression in Niemann–Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway

BackgroundNiemann–Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. ResultsOur data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1−/− mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-β-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1−/− mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. ConclusionOur data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.