Search

Objectives Leukemia is one of the severe cancer types all around the globe. Even though some chemotherapeutic drugs are available for treating leukemia, they have various side effects. As an alternative approach, herbal drugs are focused on current research to overcome leukemia. The present work was conducted to investigate the antileukemic mechanism of active phytochemical vitexin, which was isolated from ethno-medicine (Prosopis cineraria leaf) used by traditional healers of West Bengal, India. Methods Antiproliferative mechanisms of selected phyto-compound against K-562 cells were evaluated using cellular uptake, morphological changes, DNA fragmentation, mitochondrial membrane potential and signaling pathways analysis. Key findings Vitexin exhibited cytotoxicity by reducing mitochondrial membrane potential (32.40%) and causing DNA fragmentation (84.15%). The western blotting study indicated inhibition of cell survival proteins (BCR, ABL, H-RAS, N-RAS, K-RAS and RAF) and expression of apoptotic proteins (p38, BAX and caspase-9) in leukemia cells upon treatment with vitexin. Conclusions Based on the results, presently investigated phyto-compound vitexin could be considered for developing safe and natural drugs to treat leukemia after conducting suitable preclinical and clinical trials.

Imatinib is a drug for the treatment of Chronic Myeloid Leukemia (CML) and other cancers that 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.

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib’s high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib’s half-maximal inhibitory concentration (IC(50)). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.

SUMMARY Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease., Graphical Abstract, In brief Metabolic reprogramming in tumors is an adaptation that generates vulnerabilities that can be exploited for developing new therapies. Here Luttman et al. identify synergism between ABL allosteric inhibitors and lipophilic statins to impair metastatic lung cancer cell outgrowth and colonization, leading to increased survival in mouse models of advanced disease.

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

Key Points Question Are new-generation tyrosine kinase inhibitors associated with a higher risk of hepatotoxicity than imatinib in patients with chronic myeloid leukemia? Findings In this meta-analysis of 9 studies including 3475 patients, bosutinib, nilotinib, and ponatinib were associated with an increased risk of hepatotoxicity compared with imatinib. No significant difference was found with dasatinib. Meaning These findings suggest that hepatic function should be monitored frequently in patients treated with bosutinib, nilotinib, and ponatinib., This systematic review and meta-analysis examines the relative risk of hepatotoxicity of new-generation BCR-ABL fusion oncoprotein tyrosine kinase inhibitors (TKIs) vs imatinib among patients with chronic myeloid leukemia., Importance Although BCR-ABL fusion oncoprotein tyrosine kinase inhibitors (BCR-ABL TKIs) can substantially improve the survival rate of chronic myeloid leukemia (CML), they are clinically accompanied by severe hepatotoxicity. Objective To compare the relative risk (RR) of hepatotoxicity of new-generation BCR-ABL TKIs with that of imatinib, and to provide an overall assessment of the clinical benefit. Data Sources PubMed, Embase, Cochrane library databases, and ClinicalTrials.gov were searched for clinical trials published between January 2000 and April 2020. Study Selection Study selection was conducted independently by 2 investigators according to the inclusion and exclusion criteria published previously in the protocol: only randomized phase 2 or phase 3 clinical trials that compared bosutinib, dasatinib, nilotinib, or ponatinib with imatinib were included. Among the 2666 records identified, 9 studies finally fulfilled the established criteria. Data Extraction and Synthesis Two investigators extracted study characteristics and data independently using a standardized data extraction form. Data were extracted according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines. When substantial heterogeneity was observed, pooled estimates were calculated based on the random-effect model; otherwise, the fixed-effect model was used. Main Outcomes and Measures Data extracted included study characteristics, baseline patient information, interventions and data on all-grade and high-grade (grades 3 and 4) elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, overall survival, and major molecular response (MMR). The RRs and 95% CIs were calculated using the inverse variance method. Results Nine trials involving 3475 patients were analyzed; the median (range) age was 49 (18-91) years; 2059 (59.2%) were male patients. Increased risks were observed for each new-generation TKI except for dasatinib. Patients receiving new-generation TKIs were more likely to experience all grades of ALT elevation (pooled RR, 2.89; 95% CI, 1.78-4.69; P

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.

Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.

Background: Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. Materials and Methods: The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. Results: The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC50 values of 2.6 and 77.6 μg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WT1protein expressions by 75 and 49.5%, respectively when compared to vehicle control. Conclusion: The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.

Our patient was diagnosed with chronic myeloid leukemia (CML) in chronic phase (CP) when he was 40 years old. Although dasatinib (DAS) was prescribed during his clinical course, he was poorly compliant with the treatment. In November 20XX, at 65 years of age, he visited our hospital with leukocytosis. He was diagnosed with CML in CP and recommenced DAS at 50 mg/day, achieving a complete hematological response after 2 months. However, DAS was increased to 100 mg/day because only minimum cytogenetic response was evident even after 9 months, but CML progressed to the accelerated phase after 18 months. The ABL kinase domain mutations T315I and F317L were detected. Ponatinib (PON) was not yet approved, and he declined allogeneic stem cell transplantation therapy. He commenced interferon-α (IFN-α) in addition to DAS, and the F317L mutation (only) disappeared after 7 months; the patient achieved a major cytogenetic response. In January 20XX＋4, he commenced PON monotherapy (the drug was approved by this time) and achieved a major molecular response after 8 months. The T315I mutation disappeared during PON therapy. Although IFN-α is rarely used in the treatment of CML, this case suggests that IFN-α should be re-considered in patients with CML who exhibit tyrosine kinase inhibitor resistance.

Summary Cyclophilin A (CypA) is over-expressed in a number of human cancer types, but the mechanisms by which CypA promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds to and prevents the CrkII adaptor protein from switching to the inhibited state. CrkII is involved in cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII, by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binging site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical, and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly up-regulated.

Background The mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis. Results In the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ. Conclusions Our findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed. Electronic supplementary material The online version of this article (10.1186/s40246-018-0160-8) contains supplementary material, which is available to authorized users.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Unlike most other cancers, the incidence of melanoma is continuing to increase, particularly in young women, and the disease remains incurable for many due to its aggressive, metastatic nature, and high rate of resistance to conventional, targeted, and immunological agents. Cathepsin proteases are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave/degrade proteins that restrict cancer progression, while extracellular cathepsins directly cleave extracellular matrix and activate pro-invasive proteases in the tumor microenvironment. Cathepsin secretion is dramatically increased in cancer cells; however, the signaling pathways have not been extensively studied. Here, we describe new pathways leading to cathepsin secretion in melanoma cells. We show that Abl and Arg non-receptor tyrosine kinases (Abl/Arg) promote cathepsin B/L secretion by activating transcription factors with key roles in epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance (Ets1, Sp1, NF-κB/p65). In some melanoma cells, Abl/Arg drive an Ets1/p65->cathepsin pathway via a kinase independent mechanism, whereas, in others, Abl/Arg promote kinase-dependent Sp1/Ets1/p65->cathepsin L and Sp1/p65->cathepsin B pathways. The signaling pathways we identify are functionally and clinically relevant, as Abl/Arg, Sp1, Ets1, and cathepsin mRNAs are correlated in primary melanomas, and Abl/Argdriven invasion and metastasis requires cathepsin secretion. These data are significant as they indicate that drugs targeting Abl kinases, many of which are FDA-approved, might serve as cathepsin secretion inhibitors for melanomas expressing activated Abl kinases. The data also have important translational relevance given the renewed interest in targeting extracellular cathepsins as a therapeutic strategy for aggressive cancers.

Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.

Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

International audience; Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation and apoptosis. Previously we have shown that Gα13 is essential for both G protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK)-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.

ETV6–ABL1 fusion is a rare but recurrent oncogenic lesion found in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), without an established chromosomal abnormality, and is associated with poor outcome. In ETV6–ABL1-positive cases, an in-frame fusion produced by a complex rearrangement results in constitutive chimeric tyrosine kinase activity. Monosomy 7 is also a rare and unfavorable chromosomal abnormality in childhood BCP-ALL. Here, we report a 14-year-old female BCP-ALL patient with ETV6–ABL1 fusion combined with monosomy 7. She was admitted to our hospital because of persistent fever. Bone marrow nuclear cell count on admission was 855,000/µL with 90.0% blastic cells of lymphoid morphology. Blasts were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c, had a karyotype of 45, XX, − 7 [18/20] and a split signal for ABL1 FISH probe (92.7%), and were sensitive to tyrosine kinase inhibitors, imatinib and dasatinib, in vitro. ETV6–ABL1 fusion transcript was identified by whole transcriptome sequencing and confirmed by RT-PCR. She was treated with the high-risk protocol based on ALL-BFM 95, achieved complete remission (CR) after induction chemotherapy, and maintained CR for 4 months. To our knowledge, this is the first report of ETV6–ABL1 fusion combined with monosomy 7 in childhood BCP-ALL.

Background MicroRNAs (miRNAs) have emerged as crucial factors that regulate proliferation and apoptosis of cardiac c-kit+ cells. Although much is known about their role in maintaining cardiac c-kit+ cell pluripotency, the mechanisms by which they affect cell fate decisions that are an essential part of the repair of heart failure remain poorly understood. Methods Cardiac c-kit+ cells were obtained from Balb/c mice and cultured in vitro. Lentiviral vectors of miR199a-3p, its corresponding anti-miRNA, or short hairpin RNA against Cables1 were transfected into cells. The proliferation of cardiac c-kit+ cells was evaluated using EdU and flow cytometry. Furthermore, we examined cell apoptosis by flow cytometry under treatment with 200nM angiotensin II for 48 h. The levels of miR199a-3p and Cables1 mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to examine the expression of Cables1 and P53 proteins. Results We demonstrated a significantly decreased expression of miR199a-3p in heart failure samples compared with healthy donors. Meanwhile, we identified miR199a-3p as a proliferation- and apoptosis-associated regulator impacted through Cdk5 and Abl enzyme substrate 1 (CABLES1) targeting, and also attributed their repression to P53 protein expression. We further demonstrated that P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. Conclusion Collectively, our findings uncover one new mechanism by which P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. Therefore, miR199a-3p and P53 are coupled through CABLES1 and comprise a novel negative feedback loop that likely contributes to cardiac c-kit+ cell proliferation and apoptosis.

Background Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC) models. Methods CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive. Results We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression. Conclusion Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.

Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.

Several Rho GDP/GTP exchange factors include a Sec14 domain and spectrin repeats. The Sec14 domain of Kalirin 7 is a determinant of spine length. Together the noncatalytic Sec14 domain and spectrin repeats of Kalirin 7 support spine formation, localize to the postsynaptic density, and attract presynaptic endings., Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis.

The mammalian ABL1 gene encodes the ubiquitously expressed nonreceptor tyrosine kinase ABL. In response to growth factors, cytokines, cell adhesion, DNA damage, oxidative stress, and other signals, ABL is activated to stimulate cell proliferation or differentiation, survival or death, retraction, or migration. ABL also regulates specialized functions such as antigen receptor signaling in lymphocytes, synapse formation in neurons, and bacterial adhesion to intestinal epithelial cells. Although discovered as the proto-oncogene from which the Abelson leukemia virus derived its Gag-v-Abl oncogene, recent results have linked ABL kinase activation to neuronal degeneration. This body of knowledge on ABL seems confusing because it does not fit the one-gene-one-function paradigm. Without question, ABL capabilities are encoded by its gene sequence and that molecular blueprint designs this kinase to be regulated by subcellular location-dependent interactions with inhibitors and substrate activators. Furthermore, ABL shuttles between the nucleus and the cytoplasm where it binds DNA and actin--two biopolymers with fundamental roles in almost all biological processes. Taken together, the cumulated results from analyses of ABL structure-function, ABL mutant mouse phenotypes, and ABL substrates suggest that this tyrosine kinase does not have its own agenda but that, instead, it has evolved to serve a variety of tissue-specific and context-dependent biological functions.

The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.

Background & objectives: Chronic myeloid leukaemia is (CML) characterized by the presence of a hallmark chromosomal translocation, the Philadelphia chromosome. Although there are many reports available regarding the different variants of BCR-ABL in CML, we studied the co-expression of e13a2 and e14a2 transcripts and a few polymorphisms in CML patients. Methods: Molecular genetics approach was adapted to screen for polymorphisms, mutation and translocation in BCR, ABL kinase domain and BCR-ABL breakpoint region in 73 CML patients. Results: All eight patients with dual transcripts were found to harbour an exonic polymorphism (c.2700 T>C) and an intronic polymorphism (g.109366A>G) that were earlier reported to be associated with co-expression of both the transcripts. We also observed c.763G>A mutation in ABL kinase domain and two polymorphisms, c.2387 A>G and c.2736A>G in the BCR gene. Interpretation & conclusions: Though our data support the previous findings that co-expression of BCR-ABL transcripts is due to the occurrence of exonic and intronic polymorphisms in the BCR gene, it also shows that the intronic polymorphism can arise without the linked exonic polymorphism. The occurrence of ABL kinase domain mutation is less frequent in Indian population.

Summary Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends., Graphical Abstract Image 1, Highlights • PAXX-deficient pro-B cells support normal V(D)J recombination • PAXX and XLF are mutually redundant in repairing RAG-DNA breaks • PAXX function in V(D)J recombination depends on its interaction with Ku • Unlike XLF, PAXX is not redundant with ATM and the RAG complex in repairing DNA breaks, Developing lymphocytes rely on nonhomologous end joining (NHEJ) to repair programmed DNA double-strand breaks generated during antigen receptor gene assembly. Lescale et al. show that PAXX—a component of the NHEJ machinery—has a key role in V(D)J recombination that is masked by functional redundancy with its paralog XLF.

Depression is a common symptom among gastric cancer (GC) patients and serves as a potential indication of poor prognosis and advanced cancer clinical stage. However, the molecular mechanism of depression‑associated poor prognoses of GC patients remains unclear. Recent studies have revealed that GC patients with depression are under high levels of oxidative stress (OS) status that is accompanied by the dysfunction of numerous proto‑oncogenes, including the ABL proto‑oncogene 1 (ABL1), which is a non‑receptor tyrosine kinase. Recent evidence indicates that ABL1 was dysregulated in both major depressive disorder (MDD) and cancer patients with depression, and high levels of reactive oxygen species (ROS) can lead to the activation of ABL1 in response to OS and that activated ABL1 subsequently contributes to development of GC via interactions with the downstream targets and corresponding signaling pathways. In this review, we examine the evidence to illuminate the molecular mechanism of ABL1 in the progression of GC patients with depression and identify out new and effective methods for the initial and long‑term treatment of GC.

Understanding the conformational propensities of proteins is key to solving many problems in structural biology and biophysics. The co‐variation of pairs of mutations contained in multiple sequence alignments of protein families can be used to build a Potts Hamiltonian model of the sequence patterns which accurately predicts structural contacts. This observation paves the way to develop deeper connections between evolutionary fitness landscapes of entire protein families and the corresponding free energy landscapes which determine the conformational propensities of individual proteins. Using statistical energies determined from the Potts model and an alignment of 2896 PDB structures, we predict the propensity for particular kinase family proteins to assume a “DFG‐out” conformation implicated in the susceptibility of some kinases to type‐II inhibitors, and validate the predictions by comparison with the observed structural propensities of the corresponding proteins and experimental binding affinity data. We decompose the statistical energies to investigate which interactions contribute the most to the conformational preference for particular sequences and the corresponding proteins. We find that interactions involving the activation loop and the C‐helix and HRD motif are primarily responsible for stabilizing the DFG‐in state. This work illustrates how structural free energy landscapes and fitness landscapes of proteins can be used in an integrated way, and in the context of kinase family proteins, can potentially impact therapeutic design strategies.

BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist. We investigated, at transcriptional, translational and post-translational level, the mechanisms involved in BCR/Abl suppression in K562 and KCL22 CML cells. BCR/abl mRNA steady-state analysis and ChIP-qPCR on BCR promoter revealed that BCR/abl transcriptional activity is reduced in K562 cells under oxygen shortage. The SUnSET assay showed an overall reduction of protein synthesis under oxygen/glucose shortage in both cell lines. However, only low oxygen decreased polysome-associated BCR/abl mRNA significantly in KCL22 cells, suggesting a decreased BCR/Abl translation. The proteasome inhibitor MG132 or the pan-caspase inhibitor z-VAD-fmk extended BCR/Abl expression under oxygen/glucose shortage in K562 cells. Glucose shortage induced autophagy-dependent BCR/Abl protein degradation in KCL22 cells. Overall, our results showed that energy restriction induces different cell-specific BCR/Abl protein suppression patterns, which represent a converging route to TKi-resistance of CML cells. Thus, the interference with BCR/Abl expression in environment-adapted CML cells may become a useful implement to current therapy.

// Carolina Simioni 1, * , Simona Ultimo 1, * , Alberto M. Martelli 2 , Giorgio Zauli 1 , Daniela Milani 1 , James A. McCubrey 3 , Silvano Capitani 1, 4 , Luca M. Neri 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 3 Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA 4 LTTA Center, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Luca M. Neri, email: luca.neri@unife.it Silvano Capitani, email: silvano.capitani@unife.it Keywords: NUP214-ABL1, PI3K/Akt/mTOR signaling, T-Acute Lymphoblastic Leukemia, targeted therapies, autophagy Received: August 04, 2016 Accepted: October 19, 2016 Published: November 03, 2016 ABSTRACT Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis. We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr412 (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G0/G1 to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.

Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cδ (PKCδ) is crucial for fractionated-radiation-induced expansion of glioma-initiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCδ activity, expanded the CD133-positive (CD133+) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCδ expression blocked fractionated-radiation-induced CD133+ cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133+ cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCδ activation, suggesting that Abl acts upstream of PKCδ. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl–PKCδ signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors.

Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.

In the present study, we have examined the effect of perforin (pfp) deficiency in 4 models of mouse B-cell lymphomagenesis. We have examined pfp loss on the background of either Mlh1 tumor suppressor allele loss or oncogene expression [Ig heavy chain (Eμ)-v-Abl, Eμ-myc, and vav-bcl2]. Pfp was shown to act as a suppressor of B-cell malignancies characteristically driven by v-Abl or bcl-2, whereas Mlh loss cooperated in accelerating spontaneous B-cell lymphomas characteristic of pfp loss. No protective role for pfp was observed in the more aggressive Eμ-myc model of B-cell lymphoma. These transgenic models have allowed us to distinguish the role of pfp in surveillance of B-cell lymphomagenesis, as opposed to its loss simply driving the onset of a spontaneous lymphoma characteristic of pfp deficiency.

Conformational change is a common molecular mechanism for the regulation of kinase activities. Small molecule modulators of protein conformations, including allosteric kinase inhibitors, are highly wanted as tools for the interrogation of kinase biology and as selective therapeutic agents. However, straightforward cellular assays monitoring kinase conformations in a manner conducive to high-throughput screening (HTS) are not readily available. Here we describe such an HTS-compatible conformational sensor assay for Abl based on a split luciferase construct. The Abl sensor responds to intramolecular structural rearrangements associated with intracellular Abl deactivation and small molecule inhibition. The intact regulatory CAP–SH3–SH2 domain is required for the full functionality of the sensor. Moreover, a T334I Abl mutant (T315I in Abl1a) was found to be particularly well suited for HTS purposes and mechanistic intracellular studies of T334I mutant inhibitors. We expect that the split luciferase-based conformational sensor approach might be more broadly useful to probe the intracellular activation of other kinases and enzymes in general.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs and widespread vasculopathy. Current therapies for SSc focus on treating specific symptoms, but disease-modifying agents targeting the underlying pathogenesis are lacking. The pathogenesis of SSc involves activation of profibrotic pathways, with over-expression of the cytokines transforming growth factor (TGF)-β and platelet derived growth factor (PDGF). A recent report showed that SSc patients have autoantibodies against the PDGF receptor, which stimulate the production of reactive oxygen species and type I collagen expression(1). PDGF receptors are upregulated in the skin and bronchoalveolar lavage fluid of patients with SSc, and when activated, lead to fibroblast and myofibroblast proliferation(2, 3). PDGF participates in smooth muscle cell recruitment and mitogenic signaling that underlie the vasculopathy associated with pulmonary arterial hypertension (PAH), a complication of SSc associated with high mortality(4). In addition, stimulation of the TGF-β profibrotic pathway involves activation of c-Abl(2). Thus, the PDGF and TGF-β pathways are thought to contribute to the fibrotic and vascular complications in SSc. Imatinib mesylate (Gleevec, Novartis, East Hanover, New Jersey) is a small molecule that antagonizes specific tyrosine kinases that mediate fibrotic pathways, including c-Abl, a downstream mediator of TGF-β(2) and PDGF receptors(5). Imatinib has been shown to inhibit lung and dermal fibrosis in bleomycin-induced mouse models(6, 7), and the proliferation of synovial fibroblasts derived from patients with rheumatoid arthritis(8). Imatinib has also been reported to provide benefit in the treatment of refractory idiopathic PAH through its effects on vascular remodeling(9). We now describe two patients with early diffuse SSc who experienced clinical improvement in response to imatinib therapy and provide evidence that both c-Abl and PDGFR are targets of imatinib in scleroderma skin. Finally, we show that an imatinib-responsive gene signature is present in most cases of diffuse SSc.

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome, which results from a reciprocal translocation between the long arms of the chromosomes 9 and 22 t(9;22)(q34;q11). This translocation creates two new genes, BCR-ABL on the 22q- (Ph chromosome) and the reciprocal ABL-BCR on 9q-. The BCR-ABL gene encodes for a 210-kD protein with deregulated tyrosine kinase (TK) activity, which is crucial for malignant transformation in CML. The recognition of the BCR-ABL gene and corresponding protein led to the synthesis of small-molecule drugs, designed to interfere with BCR-ABL tyrosine kinase activation by competitive binding at the ATP-binding site. The first tyrosine kinase inhibitor (TKI), introduced into clinical practice in 1998, was imatinib mesylate. Imatinib became the first choice drug in chronic phase CML, because of its high efficacy, low toxicity and ability to maintain durable hematological and cytogenetic responses. However, approximately 20-25% of patients initially treated with imatinib will need alternative therapy, due to drug resistance, which is often caused by the appearance of clones expressing mutant forms of BCR-ABL. Second-generation TKIs have provided new therapeutic option for the patients resistant to imatinib. Dasatinib is the first, second-generation TKI, approved in the US and European Union for the treatment of CML patients with imatinib resistance or intolerance. This drug is a dual SRC-ABL kinase inhibitor, active in most clinically relevant BCR-ABL mutations, except highly resistant T315I. Other second-generation TKIs include nilotinib, bosutinib and INNO 406. Apart from TKIs, the promising group of molecules is inhibitors of Aurora family of serine-threonine kinases. One of these molecules, MK0457, has entered clinical trials, and initial reports indicate that this compound could be active in disease associated with T315I mutation. Thus, wide spectrum of new agents, with different mode of action, is currently in clinical development for CML. It is likely that combination therapy will be the best therapeutic strategy in the future.

Dasatinib (BMS-354825) is a Src/ABL tyrosine kinase inhibitor currently approved for the treatment of chronic myeloid leukemia. Dasatinib has increased potency against ABL compared to the current therapy imatinib, and is effective in many cases where disease is resistant to imatinib. Dasatinib also inhibits many Src-family tyrosine kinases. We have demonstrated in this study that dasatinib is able to block the function of normal human T-lymphocytes in vitro at clinically relevant concentrations. T-cell functions including proliferation, activation and cytokine production were all uniformly inhibited in the presence of dasatinib. We also demonstrated inhibition of TCR signalling through Src-family kinase LCK, and predicted that inhibition of LCK and other kinases involved in T-cell signalling by dasatinib is responsible for the suppression of T-cell function. These findings raise the concern about potential T-cell inhibition in patients taking dasatinib, and suggest a possible application for the treatment of T-cell mediated immune disorders.

The precise mechanisms by which Abl oncogenes transform hematopoietic cells are unknown. We have examined the role of Pim kinases in v-Abl–mediated transformation. In v-Abl transformants, expression of Pim-1 and Pim-2, but not Pim-3, is dependent on Abl kinase activity. Transformation assays demonstrate that v-Abl cannot efficiently transform bone marrow cells derived from Pim-1−/−/Pim-2−/− mice. Ectopic expression of either Pim-1 or Pim-2 in Pim-1−/−/Pim-2−/− cells restores transformation by v-Abl, strongly suggesting that either Pim-1 or Pim-2 is required for v-Abl–mediated tumorigenesis. Interestingly, the combined deficiency of Pim-1, Pim-2, and Suppressor of Cytokine Signalling (SOCS)-1 resulted in partial restoration of v-Abl transformation efficiency. In addition, Pim kinases are involved in modification of SOCS-1 and in regulating SOCS-1 protein levels in v-Abl–transformed cells. Furthermore, Pim kinases regulate the proapoptotic proteins Bcl-XS and BAD. Pim kinases inhibit the expression of Bcl-XS. Pim deficiency decreases the phosphorylation levels of BAD, whereas ectopic expression of Pim-1 increases the amount of phospho-BAD. This correlates with an increased protection from apoptosis in Abl transformants expressing Pim kinases. Together, these data suggest that Pim kinases play a key role in the v-Abl transformation, possibly via participating in modulation of SOCS-1 and via regulating the apoptotic signaling.

We have investigated two patients with acquired chromosomal rearrangements, a male presenting with a t(1;9)(p34;q34) and B cell progenitor acute lymphoid leukemia and a female presenting with a t(8;12)(p11;q15) and the 8p11 myeloproliferative syndrome. We determined that the t(1;9) fused ABL to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing factor. The t(8;12) fused CPSF6, a cleavage and polyadenylation specificity factor, to FGFR1. The fusions were confirmed by amplification of the genomic breakpoints and RT-PCR. The predicted oncogenic products of these fusions, SFPQ-ABL and CPSF6-FGFR1, are in-frame and encode the N-terminal domain of the partner protein and the entire tyrosine kinase domain and C-terminal sequences of ABL and FGFR1. SFPQ interacts with two FGFR1 fusion partners, ZNF198 and CPSF6, that are functionally related to the recurrent PDGFRalpha partner FIP1L1. Our findings thus identify a group of proteins that are important for pre-mRNA processing as fusion partners for tyrosine kinases in hematological malignancies.

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.

The molecular mechanisms that control the proliferation and differentiation of specific cell types remain poorly understood. Positive ETS factors play important roles in mediating proliferative responses to Ras/MAPK signaling in many cell types following mitogenic stimulation. PE-1/METS, a member of the ETS-domain family transcription factors that functions as a transcriptional repressor, can block mitogenic responses mediated by positively acting Ets factors. The anti-proliferative functions of PE-1/METS require its interaction with DP103, a multifunctional DEAD-box protein that mediates interactions with corepressor proteins and acts in a cooperative manner with Rb family members and to repress cell cycle control genes. ETS-2 repressor factor (ERF) is structurally related to and also functions as a transcriptional repressor, but endogenous target genes and mechanisms of repression remain unknown. Here, we demonstrate that like PE-1/METS, ERF-mediated repression also requires DP103, and that ERF negatively regulates the c-myc and cdc2 genes. In contrast to PE-1/METS, however, ERF-mediated repression of these genes is inactivated by MAPK signaling through phosphorylation sites that are ERF-specific. Furthermore, constitutive activation of the Ras/MAPK pathway in RAW 264.7 cells transformed by the v-Abelson leukemia virus is associated with constitutive inactivation of ERF in this cell type. We propose that ERF and PE-1/METS function to impose 'repression checkpoints' on a subset of cell cycle control genes that are differentially regulated by growth factor signaling pathways that control proliferation and differentiation and that ERF is targeted for inactivation by transforming oncogenes such as vAbl.

Tyrosine kinase inhibitors (TKIs) are now standard up-front therapy for chronic myeloid leukemia (CML). Patients with newly diagnosed chronic-phase CML treated with the TKI imatinib mesylate typically experience a complete cytogenetic remission. Outcomes for patients with advanced-phase disease are distinctly worse. Unfortunately, a small proportion of chronic-phase patients experience relapse during this therapy, and most with advanced-phase disease develop resistance to imatinib mesylate after months of therapy. Hematopoietic cell transplantation remains the only curative approach for CML and can salvage patients with advanced-phase disease. Therefore, physicians must carefully monitor patients with chronic-phase CML treated with TKIs so that they can undergo hematopoietic cell transplant (or treatment with another TKI or experimental therapy) before frank progression occurs. Fortunately, monitoring CML using cytogenetic and molecular methods (i.e., quantitative polymerase chain reaction) effectively defines end points that correlate highly with outcome.

The multi-functional protein c-Cbl is an important modulator of actin cytoskeletal dynamics in diverse biological systems. We had previously reported that c-Cbl facilitates cell spreading and adhesion and suppresses anchorage-independent growth of v-Abl-transformed fibroblasts. To assess the importance of membrane localization of c-Cbl for the observed effects of c-Cbl in v-Abl-3T3 cells, we first mapped the membrane interactive domain(s) of c-Cbl. Our studies indicate that localization of c-Cbl to the membrane is likely to be mediated by the tyrosine kinase binding (TKB) domain and the proline-rich region of c-Cbl, whereas C-terminal tyrosine phosphorylation does not play a role. The association of v-Cbl, which encompasses the TKB domain, with the membrane was unusual as it was not entirely dependent on SH2-phosphotyrosine interactions. Our studies further demonstrate that Src-like adaptor protein (SLAP), which binds to v-Cbl in a tyrosine phosphorylation-independent manner, facilitates membrane association of Cbl. The interaction between c-Cbl and SLAP in v-Abl-3T3 cells positively influenced c-Cbl-mediated spreading and adhesion of these cells. SLAP appears to exert its effects not simply by increasing the amount of c-Cbl in the membrane but by facilitating binding of p85-phosphatidylinositol-3-kinase (PI3K) with membrane-associated c-Cbl.

Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of

Acute lymphocytic leukaemia (ALL) is a heterogeneous group of disorders that result from the clonal proliferation and expansion of malignant lymphoid cells in the bone marrow, blood and other organs. Distinct clinicopathological ALL entities have been identified, resulting in the adoption of risk-oriented treatment approaches. Advances in ALL therapy have led to long-term survival rates of >80% in children. However, only approximate, approximately 30-40% of adults achieve long-term disease-free survival. Contemporary ALL treatment programmes include induction, intensified consolidation, maintenance phases and CNS prophylaxis. The optimal treatment of Philadelphia chromosome-positive patients requires the addition of BCR-ABL tyrosine kinase inhibitors, such as imatinib, whereas allogeneic stem-cell transplantation remains the preferred approach for high-risk patients in first remission. Since only approximate, approximately 38% of adult ALL patients are free of disease 5 years after diagnosis and the outcome of salvage chemotherapy is very poor (complete remission rates of 20-30%, median survival of 3-6 months), novel agents are desperately required. Of those currently in clinical studies, the outlook for sphingosomal vincristine, pegylated asparaginase (pegaspargase), liposomal annamycin, ABT-751, pemetrexed, talotrexin, nelarabine and the novel BCR-ABL kinase inhibitors is discussed.