Abl Kinase Activity Research Articles

Advances in the Treatment of Chronic Myeloid Leukemia

Background: The treatment of chronic myeloid leukemia has progressed in recent decades, becoming a model for a disease whose pathogenesis is primarily based on a genetic mutation and has led to survivals comparable to those of the general population. Objective: This review aims to present recent therapeutic advances in this area. Methods: A mini-review was achieved using the articles published in Web of Science and Pub- Med between January 2021 - May 2022, and new patents were made in this field. Results: The three generations of tyrosine kinase inhibitors have transformed chronic myeloid leukemia into a manageable disorder and greatly improved the treatment results of the chronic phase, the prognosis, survival, and quality of life of patients. The therapeutic goals today include achieving a deep and lasting molecular response as soon as possible, successful treatment-free remission, and discovering and applying new therapeutic strategies to act on impaired immune modulation and dormant leukemic stem cells. The allosteric inhibitor asciminib targets the ABL myristoyl pocket, reduces Abl kinase activity, and is effective against most cells that have mutations in the ABL1 kinase domain. Progress and recommendations for achieving long-term treatment- free remission are set out. Nearly 50% of the patients who received first-line tyrosine kinase inhibitors required a change of treatment by 10 years due to intolerance or resistance to treatment. Their main side effects are presented. Conclusion: Obtaining a deep and persistent molecular response contributes to achieving longterm treatment-free remission.

Mechanism of Late Recurrences of Chronic Myeloid Leukemia after Discontinuation of TKI Therapy: BCR::ABL1 Ins35bp Loss-of-Function Splicing Mutation and Regulatory T Cells

Introduction: Treatment-free remission (TFR) as one of the goals of treatment of BCR::ABL-positive chronic phase chronic myeloid leukemia (CML) has been proposed. Actually, approximately 40-60% of CML patients maintain TFR without recurrence after discontinuation of tyrosine kinase inhibitors (TKIs). In some of such patients, there are fluctuated cases in which BCR::ABL positive clones continue to be detected for long periods at levels of international scale (IS) BCR::ABL < 0.1%. Such fluctuated cases show no apparent molecular recurrence and maintain TFR for a long time, but they have also been shown to be more likely to eventually develop late recurrence (Haematologica 2022, Blood adv. 2020). We have reported that a higher percentage of PD-1 positive CD8 + T cells before and after TKI discontinuation is associated with molecular recurrence, and together with the possibility of assessing host immune response capacity after TKI discontinuation by using regulatory T (Treg) cells (Fujioka, et al. Cancers 2021). On the other hands, mutations in BCR::ABL caused by the alternative splicing, such as BCR::ABL Ins35bp, are known to be one of the TKI resistance in CML. Loss of BCR::ABL kinase activity is thought to result in loss of TKI sensitivity and thus failure to achieve deep molecular remission (Yuda, et al. Cancer Science 2017). In this study, we analyzed the mutations that cause splicing variants such as BCR::ABL Ins35bp and host immune response in CML patients to elucidate molecular mechanisms in the long-term maintenance of TFR and late recurrence. Methods: Twenty-four CML patients who discontinued TKI treatment for any reason at Akita University Hospital were included in the study. The trends of IS level after TKI discontinuation were monitored, and immune profile analysis by multi-color flow cytometry (FCM) was performed in all patients. Mutation analysis of the BCR::ABL region including splicing variants was performed in selected patients. Mutation analysis was performed on samples with sufficient cell count and IS > 0.0032% (MR 4.5), and the BCR::ABL region of these samples was amplified by long nested PCR and then analyzed by the next-generation sequencer (NGS). Results: First, BCR::ABL Ins35bp was evaluated by mutation analysis. Among CML patients who underwent mutation analysis, BCR::ABL Ins35bp was detected in all cases in which the samples could perform analysis at newly diagnosis (ND). Even after TKI discontinuation, a certain percentage of BCR::ABL Ins35bp was detected in all patients, and the percentage of BCR::ABL Ins35bp increased at the time of recurrence regardless of whether early or late. These results suggest that function-dead BCR::ABL Ins35bp clones may remain in CML patients with detectable BCR::ABL under and after TKI treatment, and eventually leading to recurrence with transcriptional promotion of native BCR::ABL. On the other hand, there were cases in which the BCR::ABL Ins35bp was lost at the time of recurrence. There is a possibility of immunological involvement in such cases. CML patients with detectable BCR::ABL in the TFR for a long time had a decreased immunosuppressive Treg cells, while analysis using late recurrence increased Treg cells prior to recurrence (figure). These suggest the presence of cases in which early recurrence is suppressed by host immune responses. Conclusion: Although the loss of function mutations such as BCR::ABL Ins35bp was previously thought to occur after TKI administration as a cause of TKI resistance, this analysis reveals that the clone is included from the ND before TKI treatment. Examining the BCR::ABL Ins35bp clone at ND may be possible to predict the possibility of recurrence after TFR. Host immune capacity, as assessed by the kinetics of Treg after TKI discontinuation, may contribute to prolonged TFR. Thus Treg monitoring may be important in predicting molecular recurrence, especially in BCR::ABL Ins35bp cases.

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl−) cells through genome editing and compared HCV production between Abl− cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

Hypolipemiant besides Antileukemic Effect of Imatinib Mesylate (Case Report).

Introduction. The tyrosine kinase inhibitor imatinib mesylate (IM) specifically inhibits the kinase activity of ABL, platelet-derived growth factor receptor beta (PDGFR[]), and c-KIT tyrosine kinases and has impressive clinical efficacy in chronic myeloid leukemia (CML)[1]. IM prevented also the development of atherosclerotic lesions and diabetes-induced inflammatory cytokine overexpression [2,3].Case report. A 36 years old male patient with a history of acute myocardial infarction and double coronarian by-pass eight years ago was admitted for the first time on December 2005 complaining of wheigt loss, intermittent discomfort in upper left abdomenum, polydipsia.and polyuria. By the examination of the peripheral blood and of the bone marrow the diagnosis of chronic phase CML-Ph(+) has been established. Simultaneously, type II diabetes mellitus and dyslipidemia (Table 1) were discovered. After a short course of Hydrea, associated with diet and oral antidiabetic therapy, IM 400 mg/d has been started on April 2006. The hematologic remission and a concomitant reduction of the serum levels of cholesterol, and of low-density lipoproteins (LDL) has been obtained (Table 1).Table 1The dynamics of the peripheral blood counts and lipidic profile during IM therapyParametersAprilMayJuneJullyHemoglobin (g/dl)9,611,11212Leukocyte count (×109/l)5.24.67.57.8Platelet count (×109/l)168287210215Serum total cholesterol (mg/dl)310189157152Serum LD lipoproteins (mg/dl)2191288587Serum HD lipoproteins(mg/dL)49535250Comments and conclusions. The effects observed in this reported case and in other similar patients [4] indicates that the treatment with IM induces a favourable control not only on cell growth and replication but also on the homeostasis of lipoproteins and glucose. A possible mechanism of action could be the inhibition by IM of PDGFR-dependent phosphorylation of the LDL-receptor related protein. IM appears to be a novel therapeutic option to retard the development of atherosclerosis, specifically in the context of diabetes and the treatment of choice for patients suffering of CML and dyslipidemia. Further prolonged observation of this and other similar patients is necessary for more pertinent data.

Molecular modeling piloted analysis for semicarbazone derivative of curcumin as a potent Abl-kinase inhibitor targeting colon cancer

The human Abl kinases comprise a family of proteins that are known to be key stimulus drivers in the signaling pathways modulating cell growth, cell survival, cell adhesion, and apoptosis. Recent collative studies have indicated the role of activation of Abl and Abl-related genes in solid tumors; further terming the Abl kinases as molecular switches which promote proliferation, tumorigenesis, and metastasis. The up-regulated Abl-kinase expression in colorectal cancer (CRC) and the role of Abl tyrosine kinase activity in the Matrigel invasion of CRC cells have cemented its significance in CRC advancement. Therefore, the requisite of identifying small molecules which serve as Abl selective inhibitors and designing anti-Abl therapies, particularly for CRC tumors, has driven this study. Curcumin has been touted as an effective inhibitor of cancer cells; however, it is limited by its physicochemical inadequacies. Hence, we have studied the behavior of heterocyclic derivatives of curcumin via computational tools such as pharmacophore-based virtual screening, molecular docking, free-energy binding, and ADME profiling. The most actively docked molecule, 3,5-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-carboxamide, was comparatively evaluated against Curcumin via molecular dynamics simulation using Desmond, Schrödinger. The study exhibited the improved stability of the derivative as compared to Curcumin in the tested protein pocket and displayed the interaction bonds with the contacted key amino acids. To further establish the claim, the derivatives were synthesized via the mechanism of cyclization of Curcumin and screened in vitro using SRB assay against human CRC cell line, HCT 116. The active derivative indicated an IC50 value of 5.85 µM, which was sevenfold lower as compared to Curcumin’s IC50 of 35.40 µM. Hence, the results base the potential role of the curcumin derivative in modulating Abl-kinase activity and in turn may have potential therapeutic value as a lead for CRC therapy.

Effects of imatinib on vascular insulin sensitivity and free fatty acid transport in early weight gain.

Vascular endothelial dysfunction is an essential part of the pathophysiology of type 2 diabetes and its complications. In type 2 diabetes, endothelial dysfunction is characterized by reduced insulin signaling and increased transendothelial transport of fatty acids (FA). As the Abl kinase inhibitor imatinib was previously shown to reverse type 2 diabetes and to inhibit VEGF signaling via Abl kinases, we studied the effect of imatinib on vascular insulin sensitivity and fatty acid transport in vivo and in vitro. C57/BL6J mice were fed a chow diet or Western diet (WD), and received daily imatinib injections for two weeks. Insulin-mediated vasoreactivity of resistance arteries was studied using intravital microscopy, and metabolic insulin sensitivity using the hyperinsulinemic-euglycemic clamp. The effect of imatinib on triglyceride content in skeletal muscle and heart in vivo was also determined. In vitro, the effect of imatinib on fatty acid transport was studied in human umbilical vein endothelial cells (HUVECs) by evaluating the effect of imatinib on fluorescently labeled FA uptake both under basal and VEGF-B-stimulated conditions. Imatinib prevented the WD-induced weight gain in mice, independently from food intake. In line with this, imatinib enhanced insulin-mediated vasoreactivity of resistance arteries in the WD-fed mice. However, imatinib did not affect triglyceride content in muscle. In cultured endothelial cells, VEGF-B stimulation resulted in a time-dependent uptake of fatty acids in parallel with increased phosphorylation of the Abl kinase substrate Crk-like protein (CrkL) at Tyr207. Although imatinib effectively prevented VEGF-B-mediated Abl kinase activation, it had no effect on VEGF-B mediated endothelial FA uptake. Imatinib prevents weight gain and preserves insulin-mediated vasodilation in WD-fed mice, but does not affect endothelial FA transport despite inhibiting VEGF-B signaling. The beneficial effect of imatinib on insulin-mediated vasodilation may contribute to the anti-diabetic effects of imatinib.

Molecular insights on ABL kinase activation using tree-based machine learning models and molecular docking.

Abelson kinase (c-Abl) is a non-receptor tyrosine kinase involved in several biological processes essential for cell differentiation, migration, proliferation, and survival. This enzyme's activation might be an alternative strategy for treating diseases such as neutropenia induced by chemotherapy, prostate, and breast cancer. Recently, a series of compounds that promote the activation of c-Abl has been identified, opening a promising ground for c-Abl drug development. Structure-based drug design (SBDD) and ligand-based drug design (LBDD) methodologies have significantly impacted recent drug development initiatives. Here, we combined SBDD and LBDD approaches to characterize critical chemical properties and interactions of identified c-Abl's activators. We used molecular docking simulations combined with tree-based machine learning models—decision tree, AdaBoost, and random forest to understand the c-Abl activators' structural features required for binding to myristoyl pocket, and consequently, to promote enzyme and cellular activation. We obtained predictive and robust models with Matthews correlation coefficient values higher than 0.4 for all endpoints and identified characteristics that led to constructing a structure–activity relationship model (SAR).Graphic abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-021-10261-z.

Cortactin Is Required for Efficient FAK, Src and Abl Tyrosine Kinase Activation and Phosphorylation of Helicobacter pylori CagA

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin’s phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]5, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR]x (x = 6-9) and hybrid cyclic-linear peptides, [R6K]W6 and [R5K]W7, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]9 was found to be the most potent tyrosine kinase inhibitor. [WR]9 showed higher inhibitory activity (IC50 = 0.21 μM) than [WR]5, [WR]6, [WR]7, and [WR]8 with IC50 values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-33P]ATP. Consistent with the result above, [WR]9 inhibited other protein kinases such as Abl kinase activity with an IC50 value of 0.35 μM, showing 2.2-fold higher inhibition than [WR]5 (IC50 = 0.79 μM). [WR]9 also inhibited PKCa kinase activity with an IC50 value of 2.86 μM, approximately threefold higher inhibition than [WR]5 (IC50 = 8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]9 exhibited IC50 values of <0.25 μM against Akt1, Alk, and Btk. These data suggest that [WR]9 is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R6K]W6 and [R5K]W7 against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR](5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]5 or [WR]9 affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]9.

Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR-ABL positive cells.

Chronic myeloid leukemia (CML) is characterized by constitutive BCR-ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR-ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%-30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR-ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR-ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L-NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1μM) of imatinib. Mechanistically, imatinib or BCR-ABL siRNA following deglutathionylation of NF-κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase-3 however associated with increased caspase-3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington’s disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer’s disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αβ neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αβ neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

Diversity of Long-Lived Intermediates along the Binding Pathway of Imatinib to Abl Kinase Revealed by MD Simulations.

Imatinib, a drug used for the treatment of chronic myeloid leukemia and other cancers, works by blocking the catalytic site of pathological constitutively active Abl kinase. While the binding pose is known from X-ray crystallography, the different steps leading to the formation of the complex are not well understood. The results from extensive molecular dynamics simulations show that imatinib can primarily exit the known crystallographic binding pose through the cleft of the binding site or by sliding under the αC helix. Once displaced from the crystallographic binding pose, imatinib becomes trapped in intermediate states. These intermediates are characterized by a high diversity of ligand orientations and conformations, and relaxation timescales within this region may exceed 3-4 ms. Analysis indicates that the metastable intermediate states should be spectroscopically indistinguishable from the crystallographic binding pose, in agreement with tryptophan stopped-flow fluorescence experiments.

Abelson Kinases Mediate the Depression of Spontaneous Synaptic Activity Induced by Amyloid Beta 1-42 Peptides.

Amyloid beta (Aβ) peptides represent one of the most studied etiological factors of Alzheimer's disease. Nevertheless, the effects elicited by different molecular forms of amyloid beta peptides widely vary between the studies, mostly depending on experimental conditions. Despite the enormous amount of accumulated evidences concerning the pathological effects of amyloid beta peptides, the exact identity of the amyloid beta species is still controversial, and even less is clear as regards to the downstream effectors that mediate the devastating impact of these peptides on synapses in the central nervous system. Recent publications indicate that some of the neurotoxic effects of amyloid beta peptides may be mediated via the activation of proteins belonging to the Abelson non-receptor tyrosine kinase (Abl) family, that are known to regulate actin cytoskeleton structure as well as phosphorylate microtubule-associated tau protein, a hallmark of Alzheimer's disease. By performing series of miniature excitatory postsynaptic currents (mEPSC) recordings in cultured hippocampal cells, we demonstrate that activation of Abl kinases by acute application of 42 amino acid-length monomeric amyloid beta (Aβ1-42) peptides reduces spontaneous synaptic release, while this effect can be rescued by pharmacologic inhibition of Abl kinase activity, or by reduction of Abl expression with small interfering RNAs. Our electrophysiological data are further reinforced by a subsequent biochemical analysis, showing enhanced phosphorylation of Abl kinase substrate CT10 Regulator of Kinase-homolog-Like (Crkl) upon treatment of hippocampal neurons with Aβ peptides. Thus, we conclude that Abl kinase activation may be involved in Aβ-induced weakening of synaptic transmission.

Research Progress on Pathogenic Genes of Ph-like Acute Lymphoblastic Leukemia--Review

Abstract Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL or BCR-ABL1-like ALL) is a kind of acute leukemia which has the similar gene expression profiles and manifests the biological behavior same to Ph-positive ALL, but lacks the BCR-ABL1 fusion gene. Ph-like ALL was involved in multiple abnormal changes of genomes, activating kinase and cytokine receptor signaling. This review focuses on the progress of classical genetic abnormalities of PH-like ALL in the JAK-STAT signaling, ABL kinase activation, TKI resistance in Ph-like ALL, SH2B3 gene inactivating mutation and IKZF1 gene abnormality. Besides, also summarizes the frontier progress of novel gene mutation (ATF7IP exon 9 fused with PDGFRB exon 11, PDGFRBC843G mutation caused by fusion of exon 11-23 of PDGFRB with exon 1-6 of AGGF1 gene) in recent years.

Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases.

Many commonly employed strategies to map kinase activities in live cells require expression of genetically encoded proteins (e.g. FRET sensors). In this work, we describe the development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We further demonstrate probe multiplexing and pixel-by-pixel quantification to estimate the relative proportion of modified probe, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular dynamics of multiple kinases concurrently in live cells.

Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood–brain barrier

Exposure of the brain to high levels of glucocorticoids during ischemia–reperfusion induces neuronal cell death. Oxidative stress alters blood–brain barrier (BBB) function during ischemia–reperfusion, and so we hypothesized that it might impair P-glycoprotein (P-gp)-mediated efflux transport of glucocorticoids at the BBB. Therefore, the purpose of this study was to clarify the molecular mechanism of this putative decrease of P-gp-mediated efflux function. First, we established that H2O2 treatment of a human in vitro BBB model (hCMEC/D3) reduced both P-gp efflux transport activity and protein expression on the plasma membrane within 20 min. These results suggested that the rapid decrease of efflux function might be due to internalization of P-gp. Furthermore, H2O2 treatment markedly increased tyrosine-14-phosphorylated caveolin-1, which is involved in P-gp internalization. A brain perfusion study in rats showed that cortisol efflux at the BBB was markedly decreased by H2O2 administration, and inhibitors of Abl kinase and Src kinase, which phosphorylate tyrosine-14 in caveolin-1, suppressed this decrease. Overall, these findings support the idea that oxidative stress-induced activation of Abl kinase and Src kinase induces internalization of P-gp via the phosphorylation of tyrosine-14 in caveolin-1, leading to a rapid decrease of P-gp-mediated cortisol efflux at the BBB.

Assays for tyrosine phosphorylation in human cells.

Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors.

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step that is required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes, depending on the cell or tissue type. The virus spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abelson (Abl) kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titres and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which makes experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titres by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying the host cell pathways required for coronavirus infection. This will provide an insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.