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Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes.

Background: The v- abl oncogene of the Abelson murine leukemia virus (A-MuLV) encodes a cytoplasmic tyrosine kinase that can associate with phosphoinositide 3-kinase, Shc and Grb2, and activate the pathway that leads from Ras to mitogen-activated protein (MAP) kinase. Despite the link to these mitogenic signal transducers, v-Abl does not stimulate cell growth in general. Only a subset of NIH3T3 cells, represented by the P-3T3 subclone, can respond to the mitogenic effects of v-Abl, whereas the majority of NIH3T3 cells respond to the growth-inhibitory effects of v-Abl. This restricted mitogenic function suggests that v-Abl may rely on additional signal transducers — which are not required for the physiological mitogenic response – to stimulate DNA synthesis. Results A dominant-negative mutant of the small GTP-binding protein Rac (Rac N17, containing an asparagine residue at position 17) was found to block v-Abl-induced activation of two mitogenic enhancer elements. Activation of the serum response element by v-Abl was inhibited by Rac N17 and Ras N17, whereas activation of the TPA response element was inhibited by Rac N17 but not by Ras N17. The Rac N17 mutant also compromized the ability of v-Abl to activate the MAP-kinase, Erk2, and the stress-activated protein kinase, JNK1. Activation of endogenous Rac was indicated by the ability of v-Abl to stimulate pinocytosis. P-3T3 cells transformed by v- abl could proliferate without serum, but became serum-dependent when Rac N17 was expressed. However, Rac N17 did not inhibit the morphological change or anchorage-independent growth of cells transformed with v-Abl. Conclusion The activation of the mitogenic program by v-Abl tyrosine kinase requires not only Ras but also Rac. Multiple pathways are activated by v-Abl in transformed cells. The availability of some of these pathways may determine whether v-Abl can activate mitogenesis.

In normal cells, the strength and duration of proliferative signaling pathways are tightly regulated. In oncogenic settings, negative regulation is often bypassed to allow constitutive activation of these pathways. In our recent manuscript, we identify a mechanism that allows the v-Abl oncogene to bypass negative regulation by SOCS-1 to constitutively activate Jak-Stat signaling. The mechanism involves post-translational modifications of SOCS-1 that disrupt its interaction with the proteasome, thereby preventing it from targeting activated Jak kinases for degradation. In this review, we discuss the implications of these findings for our understanding of v-Abl oncogenesis and the regulation of SOCS protein function.

The uptake of glucose into mammalian cells, catalysed by members of the GLUT family of glucose transporters, is regulated by a variety of hormones, growth factors and other agents. In adipocytes, skeletal muscle and heart the principal regulator is the hormone insulin, which rapidly stimulates glucose uptake by bringing about the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular compartment to the cell surface. Recent studies have implicated the C-terminal hydrophilic region of this protein as being primarily responsible for its insulin-regulated trafficking. In an attempt to identify the protein machinery involved in this trafficking, we have used glutathione S-transferase fusion proteins bearing hydrophilic domains of various GLUT transporters in affinity purification experiments on detergent-solubilized extracts of 3T3-L1 adipocyte intracellular membranes. The C-terminal region of GLUT4 was found specifically to bind a number of polypeptides in these extracts, which are therefore candidates for components of the trafficking machinery. Although these proteins did not bind to the corresponding region of the more widely-distributed GLUT1 glucose transporter isoform, regulation of this transporter also appears to be of physiological importance in some cell types. To study such regulation we have used as a model system the interleukin-3 (IL-3)-dependent haemopoietic cell line IC.DP. These cells express a temperature-sensitive mutane of the v-abl tyrosine kinase, whose activation at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the kinase were found to stimulate glucose transport by promoting the translocation of GLUT1 to the cell surface. Moreover, inhibition of glucose uptake by addition of transport inhibitors markedly increased the rate of apoptosis, an effect which could be reversed by the provision of alternative energy sources. These observations suggest that the trafficking of GLUT1, regulated by growth factors or oncogenes, may play an important role in the suppression of apoptosis in haemopoietic cells.

A novel cellular protein, Abl-interactor-1 (Abi-1), which specifically interacts with the carboxy-terminal region of Abl oncoproteins, has been identified in a mouse leukemia cell line. The protein exhibits sequence similarity to homeotic genes, contains several polyproline stretches, and includes a src homology 3 (SH3) domain at its very carboxyl terminus that is required for binding to Abl proteins. The abi-1 gene has been mapped to mouse chromosome 2 and is genetically closely linked to the c-abl locus. The gene is widely expressed in the mouse, with highest levels of mRNA found in the bone marrow, spleen, brain, and testes. The Abi-1 protein coimmunoprecipitates with v-Abl and serves as a substrate for kinase activity. When overexpressed in NIH-3T3 cells, abi-1 potently suppresses the transforming activity of Abelson leukemia virus expressing the full-length p160v-abl kinase but does not affect the transforming activity of viruses expressing a truncated p90v-abl or v-src kinases. We suggest that the Abi-1 protein may serve as a regulator of Abl function in transformation or in signal transduction.

The active site substrate specificities of v-Abl and c-Src are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a “dual specificity” with respect to the ability to utilize D- and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response element-binding protein (CREB). We have mapped the specific domains within each of the proteins responsible for this interaction. We have shown that complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor.

A temperature-sensitive mutant of the v-Abl protein has previously been shown to exhibit tyrosine protein kinase activity in Interleukin 3 (IL-3)-dependent IC.DP cells grown at the permissive temperature (32 degrees C) but not at the restrictive temperature (39 degrees C). These IC.DP cells are dependent on IL-3 for suppression of apoptosis at 39 degrees C, but at 32 degrees C cells will survive without added growth factor. Both IL-3 and v-Abl stimulated the tyrosine phosphorylation of SHC and GTPase-activating protein. However, while IL-3 stimulated similar levels of tyrosine phosphorylation in p46shc and p52shc, v-Abl preferentially phosphorylated p52shc, an event that occurred within 1 h of temperature switch. v-Abl also differentially associated with p46shc in a temperature-independent manner. In contrast, only IL-3 stimulated detectable increases in both myelin basic protein kinase and mitogen-activated protein (MAP) kinase kinase in in vitro assays, although in more specific MAP kinase activity assays a very slight increase in the activity of this enzyme was observed after 6 h at the permissive temperature. Time course studies suggest that phosphorylation and association of SHC with v-Abl is insufficient to lead to significant activation of MAP kinase and that activation of the MAP kinase kinase/MAP kinase pathway is not required for apoptotic suppression.

We have found that the platelet-derived growth factor receptor (PDGFR)/Abl signaling pathway is up-regulated as a determinant of the acquisition of resistance to estrogen deprivation in vitro. We aimed to determine its clinical relevance in aromatase inhibitor (AI)-resistant breast cancer. We identified a cohort of 45 patients with estrogen receptor-positive breast cancer who had been treated with an AI, subsequently relapsed and had biopsy material available from both the presentation and post-AI recurrent lesion. PDGFR alpha, PDGFR beta and Abl expression was assessed in formalin-fixed paraffin-embedded sections. Tumor protein expression of PDGFR alpha (1.39-fold, P = 0.0065), PDGFR beta (4.32-fold, P = 0.006) and Abl (1.8-fold, P = 0.001) was increased at the point of relapse. Tumor and stromal expression of PDGFR alpha as well as PDGFR beta was significantly correlated in pre-treatment and relapse samples. High post-treatment tumor and stromal PDGFR beta levels were associated with a short time to treatment failure (TTF). Expression of PDGFR alpha in relapsing tumor specimens was correlated with Abl expression and Ki67 levels. Furthermore, changes in Abl correlated significantly with changes in ER expression. These clinical data support a role for enhanced PDGF/Abl signaling in AI-resistant disease and provide a rationale for targeting the pathway in endocrine-resistant breast cancer.

Background Expression of active c-Abl in adult mouse forebrain neurons in the AblPP/tTA mice resulted in severe neurodegeneration, particularly in the CA1 region of the hippocampus. Neuronal loss was preceded and accompanied by substantial microgliosis and astrocytosis. In contrast, expression of constitutively active Arg (Abl-related gene) in mouse forebrain neurons (ArgPP/tTA mice) caused no detectable neuronal loss or gliosis, although protein expression and kinase activity were at similar levels to those in the AblPP/tTA mice. Methods To begin to elucidate the mechanism of c-Abl-induced neuronal loss and gliosis, gene expression analysis of AblPP/tTA mouse forebrain prior to development of overt pathology was performed. Selected results from gene expression studies were validated with quantitative reverse transcription PCR , immunoblotting and bromodeoxyuridine (BrdU) labeling, and by immunocytochemistry. Results Two of the top pathways upregulated in AblPP/tTA mice with c-Abl expression for 2 weeks were cell cycle and interferon signaling. However, only the expression of interferon signaling pathway genes remained elevated at 4 weeks of c-Abl induction. BrdU incorporation studies confirm that, while the cell cycle pathway is upregulated in AblPP/tTA mice at 2 weeks of c-Abl induction, the anatomical localization of the pathway is not consistent with previous pathology seen in the AblPP/tTA mice. Increased expression and activation of STAT1, a known component of interferon signaling and interferon-induced neuronal excitotoxicity, is an early consequence of c-Abl activation in AblPP/tTA mice and occurs in the CA1 region of the hippocampus, the same region that goes on to develop severe neurodegenerative pathology and neuroinflammation. Interestingly, no upregulation of gene expression of interferons themselves was detected. Conclusions Our data suggest that the interferon signaling pathway may play a role in the pathologic processes caused by c-Abl expression in neurons, and that the AblPP/tTA mouse may be an excellent model for studying sterile inflammation and the effects of interferon signaling in the brain.

We report the characterization of a member of the ras gene family that is overexpressed in cells transformed by abl tyrosine kinase oncogenes. The gene, named kir (for kinase-inducible ras-like), is induced at the transcriptional level. kir mRNA has a rapid turnover and encodes a protein of 33 kDa with guanine nucleotide-binding activity but undetectable intrinsic GTPase activity. kir was cloned by differential screening of genes present in fully malignant versus growth factor-independent cell lines expressing wild-type or mutant forms of BCR/ABL. BCR/ABL and v-Abl induce transcription of the kir gene via specific signaling pathway(s), but kir overexpression alone is not sufficient to mediate transformation.

v-cbl is the transforming gene of a murine retrovirus which induces pre-B cell lymphomas and myelogenous leukaemias. It encodes 40 kDa of a gag fusion protein which is localized in the cytoplasm and nucleus of infected cells. The c-cbl oncogene encodes a 120 kDa cytoplasmic protein and its overexpression is not associated with tumorigenesis. The c-cbl sequence has shown that v-cbl was generated by a truncation that removed 60% of the C-terminus. In this study, we carried out experiments to identify the position within cbl where the transition occurs between non-tumorigenic and tumorigenic forms. These experiments focused attention on a region of 17 amino acids which is deleted from cbl in the 70Z/3 pre-B lymphoma due to a splice acceptor site mutation. This mutation activates cbl's tumorigenic potential and induces its tyrosine phosphorylation. We also show that the expression of the v-abl and bcr-abl oncogenes results in the induction of cbl tyrosine phosphorylation, and that abl and cbl associate in vivo. These findings demonstrate that tyrosine-phosphorylated cbl promotes tumorigenesis and that cbl is a downstream target of the bcr-abl and v-abl kinases.

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. We demonstrate that nontransformed pre-B cells expanded from the mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast with their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, we show that the v-abl viral transforming protein, a tyrosine kinase, blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha. This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement.

Myeloid leukocytes form actin-based plasma membrane protrusions, called podosomes, that are implicated in myeloid cell recruitment into tissues and cell migration within the interstitium. In this study, we show that tyrosine kinases of the Abl family are present in podosomes formed by murine and human macrophages. Silencing of Abl expression in bone marrow-derived macrophages and monocyte-derived macrophages by siRNA or Abl enzymatic inhibition with imatinib resulted in the disassembly of macrophage podosomes and the reduction of their capacity to degrade an extracellular matrix and migrate through matrigel matrices and endothelial cell monolayers. Additionally, macrophages deficient in Src-family kinases, which cross-talk with Abl in regulating macrophage migration, also demonstrated podosome disassembly. These findings suggest that podosome disassembly induced by Abl targeting may inhibit podosome-dependent functions such as leukocyte recruitment into inflammatory sites and osteoclast-dependent bone resorption.

SummaryThe response of the tumour microenvironment to anti-cancer drugs caninfluence treatment efficacy. Current drug-screening methodologies fail todistinguish and quantify simultaneously the concomitant effect of drugs onthe tumour stroma and cancer cells. To overcome this limitation we havedeveloped a fluorescence-based experimental model that employs mCherry-labelled stromal cells (e.g. bone marrow fibroblastic stromal cells) co-cultured in direct contact with enhanced green fluorescent protein-labelledtumour cell lines for accurate assessment of proliferation and viability inboth cell compartments and adhesion of tumour cells. Additionally, we usedfluorescence-based image analysis to determine morphological changes thatcorrelate with cell function (e.g. morphology of the actin cytoskeleton andnuclearity of osteoclasts to predict their bone resorption activity). Using thisplatform we have revealed that dexamethasone induces HS5 fibroblast prolif-eration and contact with multiple myeloma cells via a process involving Src/c-Abl kinases. Osteoclasts also inhibited dexamethasone-induced apoptosisin myeloma cells while retaining their normal morphology and functionalityin bone resorption. Myeloma resistance to dexamethasone mediated by HS5cells and osteoclasts was reversed by treatment with the Src/c-Abl inhibitordasatinib but not with bortezomib. This new experimental platform providesa more precise screening of new therapeutics for improved efficacy oftumour cell killing within the bone marrow microenvironment.Keywords: myeloma, tumour microenvironment, co-culture system, dexa-methasone, drug-resistance.

Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5′-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.

Background The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. Results We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (K XXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. Conclusion Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

Imatinib (IM)-associated myelosuppression is rare in gastrointestinal stromal cell tumors (10%) but common in chronic myeloid leukemia (CML). Selective inhibition of predominantly Philadelphia chromosome (Ph+) driven hematopoiesis may explain myelosuppression in CML. In the absence of clinical methods to quantitate the Ph+/Ph- progenitor ratio, we hypothesized that the pre-IM percentage of BCR-ABL+ cells measured by fluorescence in situ hybridization (FISH) predicts for myelosuppression. FISH analyses performed within 30 days pre-IM 400 mg/day were analyzed in 89 patients with chronic phase CML at three institutions. Patients who developed grade ≥3 cytopenias had a higher percentage of positive FISH (90% vs. 83%; p = 0.02). Cytopenias lasted a median of 16 days, and all patients but one continued IM, achieved complete hematologic and cytogenetic remissions, and did not experience progression, with a follow-up of 61 months. In conclusion, IM-associated cytopenias are associated with a high pre-IM FISH, are reversible, and do not adversely affect outcomes.

Chromosomal rearrangements involving the ABL1 gene, leading to a BCR-ABL1 fusion gene, have been mainly associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (ALL). At present, six other genes have been shown to fuse to ABL1. The kinase domain of ABL1 is retained in all chimeric proteins that are also composed of the N-terminal part of the partner protein that often includes a coiled-coil or a helix-loop-helix domain. These latter domains allow oligomerization of the protein that is required for tyrosine kinase activation, cytoskeletal localization, and neoplastic transformation. Fusion genes that have a break in intron 1 or 2 (BCR-ABL1, ETV6-ABL1, ZMIZ1-ABL1, EML1-ABL1, and NUP214-ABL1) have transforming activity, although NUP214-ABL1 requires amplification to be efficient. The NUP214-ABL1 gene is the second most prevalent fusion gene involving ABL1 in malignant hemopathies, with a frequency of 5% in T-cell ALL. Both fusion genes (SFPQ-ABL1 and RCSD1-ABL1) characterized by a break in intron 4 of ABL1 are associated with B-cell ALL, as the chimeric proteins lacked the SH2 domain of ABL1. Screening for ABL1 chimeric genes could be performed in patients with ALL, more particularly in those with T-cell ALL because ABL1 modulates T-cell development and plays a role in cytoskeletal remodeling processes in T cells.

Cell migration is an essential step in cancer invasion and metastasis. A number of orchestrated cellular events involving tyrosine kinases and signaling receptors enable cancer cells to dislodge from primary tumors and colonize elsewhere in the body. For example, activation of the Src and Abl kinases can mediate events that promote tumor cell migration. Also, activation of the Robo1 receptor can induce tumor cell migration. However, while the importance of Src, Abl, and Robo1 in cell migration have been demonstrated, molecular mechanisms by which they collectively influence cell migration have not been clearly elucidated. In addition, little is known about mechanisms that control Robo1 expression. We report here that Src activates Abl to stabilize Robo1 in order to promote cell migration. Inhibition of Abl kinase activity by siRNA or kinase blockers decreased Robo1 protein levels and suppressed the migration of transformed cells. We also provide evidence that Robo1 utilizes Cdc42 and Rac1 GTPases to induce cell migration. In addition, inhibition of Robo1 signaling can suppress transformed cell migration in the face of robust Src and Abl kinase activity. Therefore, inhibitors of Src, Abl, Robo1 and small GTPases may target a coordinated pathway required for tumor cell migration.

Gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) develops in the chronically inflamed mucosa of patients infected with the bacterial pathogen Helicobacter pylori. Here we use patient material, primary gastric lymphoma cell cultures, and a preclinical model of the disease to examine the role of microRNA (miRNA)-mediated posttranscriptional regulation—focusing in particular on miR-203 and its target ABL1—in gastric MALT lymphomagenesis. Microarray-based miRNA expression profiling revealed a strong downregulation of the putative tumor suppressor miRNA miR-203 in human MALT lymphoma samples, which resulted from extensive promoter hypermethylation of the miR-203 locus and coincided with the dysregulation of the miR-203 target ABL1 in lymphoma biopsies compared with matched adjacent normal material from the same patients. Treatment of lymphoma B cells with demethylating agents led to increased miR-203 expression and the concomitant downregulation of ABL1, confirming the epigenetic regulation of this miRNA. Ectopic reexpression of miR-203 by transfection of a human lymphoma cell line or lentiviral transduction of explanted primary MALT lymphoma cells was sufficient to prevent tumor cell proliferation in vitro. Similarly, the treatment of primary MALT lymphoma cells with the ABL inhibitors imatinib and dasatinib prevented tumor cell growth. Finally, we show that the treatment of tumor-bearing mice with imatinib induces MALT lymphoma regression in a preclinical model of the disease, implicating ABL1 in MALT lymphoma progression. In summary, our results show that the transformation from gastritis to MALT lymphoma is epigenetically regulated by miR-203 promoter methylation and identify ABL1 as a novel target for the treatment of this malignancy. Cancer Res; 71(10); 3616–24. ©2011 AACR.

During or immediately after synthesis in vertebrate cells, the oncogenic protein-tyrosine kinase pp60v-src associates with the approximately 90-kDa heat-shock protein (hsp90). In this complex, pp60v-src is not functional as a kinase. When pp60v-src is subsequently found inserted into the plasma membrane, it is active as a kinase and is no longer associated with hsp90. We have taken advantage of genetic manipulations possible in Saccharomyces cerevisiae to investigate the function and specificity of the association between hsp90 and pp60v-src. Expression of pp60v-src is known to be toxic to S. cerevisiae cells. We find that this toxicity is due to a very specific effect on growth, arrest at a particular point in the cell cycle. In cells expressing v-src, a mutation that lowers the level of hsp90 expression (i) relieves cell cycle arrest and rescues growth, (ii) reduces the level of tyrosine phosphorylation mediated by pp60v-src, (iii) changes the pattern of tyrosine phosphorylation, and (iv) reduces the concentration of pp60v-src. We conclude that hsp90 does not simply suppress pp60v-src kinase activity during transit to the plasma membrane, as previously suggested, but also stabilizes the protein and affects both its activity and specificity. This function of hsp90 is highly selective for pp60v-src: the same hsp90 mutation has no effect on the activity or specificity of the exogenous pp160v-abl tyrosine kinase; similarly, it does not affect the specificity and has only a very small effect on the activity of the exogenous pp60c-src kinase.

Although the biological function of the c-Abl tyrosine kinase remains unsolved, potentially productive avenues towards the elucidation of that function have been identified by recent progress. An F-actin binding and a sequence-specific DNA-binding domain have been discovered in c-Abl, and DNA binding has been shown to be cell-cycle regulated. Deletion of those two domains in the mouse c-Abl results in a loss of biological function despite the production of an active tyrosine kinase. These findings suggest a role for c-Abl in the regulation of processes occurring on F-actin and on specific DNA elements.

The Abl family nonreceptor tyrosine kinases, consisting of closely related Abl and Arg (Abl-related gene), play essential roles in mouse neurulation, but their functions in the subsequent development of CNS are poorly understood. Here, we show that conditional deletion ofAblin precursors of neurons and glia on anArgknock-out background leads to striking cerebellar malformations, including defects in anterior cerebellar morphogenesis, granule cell ectopia, and hypoplasia. Time course analyses reveal that the abnormal anterior cerebellar foliation results from local disruptions of the basement membrane (BM) located between radial glial endfeet and the meninges during embryonic cerebellar development. Granule cell ectopia and hypoplasia are also associated with the breaches in the BM and abnormal Bergmann glial networks during postnatal cerebellar development.In vitroculture experiments indicate that Abl/Arg-deficient granule cells can interact with glial processes and proliferate normally in response to sonic hedgehog compared to cells isolated from control mice. Consistent with these findings, selective ablation of Abl family kinases in cerebellar granule cells alone does not cause any abnormality, suggesting that deletion ofAbl/Argfrom glia is likely required for the mutant phenotype. Together, these results provide compelling evidence that Abl and Arg play key redundant roles in BM maintenance and cortical lamination in the cerebellum.

Chronic myeloid leukemia (CML) is triggered by the BCR-ABL oncogene. Imatinib is the first-line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co-encapsulating anti-BCR-ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti-leukemia activity of the developed formulations co-encapsulating siRNA and imatinib and of the combination of Trf-liposomes carrying siRNA and free imatinib under two different treatment schedules of pre-sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co-encapsulating siRNA and imatinib promote a 3.84-fold reduction on the imatinib IC50 (from 3.49 to 0.91 µM), whereas a 8.71-fold reduction was observed for the pre-sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR-ABL mRNA and Bcr-Abl protein level. Biotechnol. Bioeng. 2010;107: 884–893. © 2010 Wiley Periodicals, Inc.

The ability to resist anoikis is critical for carcinoma cells to metastasize. Although several lung adenocarcinoma cell lines were shown to repress anoikis through the activation of Src, it remains unknown whether Src actually plays a crucial role in anoikis resistance in lung adenocarcinoma tissues. We examined 20 human lung adenocarcinoma tissues with lymphatic permeation and nine cell lines to investigate whether intralymphatic floating carcinoma cells in the tissues, used as an in vivo model of anoikis resistance, actually suppressed anoikis and whether cell lines in suspension culture, an in vitro model of anoikis resistance, survived through Src activation. We observed that the intralymphatic carcinoma cells aggregated tightly to form nests expressing E-cadherin and phosphorylated Src (p-Src). The apoptotic indices of these cells were comparable to those of extracellular matrix adhesive cells in all tissues, indicating that the intralymphatic cells actually evaded anoikis. Next, we found that the nine cell lines in suspension aggregated loosely (five cell lines) or tightly (four cell lines), and all cells resisted anoikis. Upon detachment, four cell lines (LC-KJ, HCC827, H1650, and H1975) formed compact spheroids that expressed E-cadherin and p-Src. The spheroids were similar to intralymphatic tumour nests and were thus considered to be a suitable model of the nests. The spheroids of the four cell lines underwent apoptosis after treatment with the Src/Abl/Kit inhibitor PP1 or Src/Abl inhibitor bosutinib. On the other hand, the Abl/Kit inhibitor imatinib did not affect cell growth or apoptosis in the four types of spheroids. These results indicate that Src, but not Abl or Kit, plays an essential role in the development of anoikis resistance in lung adenocarcinomas.

Dasatinib, (former BMS 354825), is an orally available small-molecule multikinase inhibitor. It potently inhibits BCR-ABL and SRC-family kinases (SRC, LCK, YES, FYN), but also c-KIT, PDGFR-alpha and beta, and ephrin receptor kinase.Dasatinib is about 300 times more potent than imatinib in cells expressing unmutated BCR-ABL in vitro. The drug has demonstrated activity against clinically relevant mutations, including those associated with poor prognosis during ongoing imatinib therapy.Dasatinib is approved for the treatment of patients with BCR-ABL-positive chronic myeloid leukemia (CML), resistant or intolerant to imatinib in chronic, accelerated, and blast phase. It also is approved for the treatment of Philadelphia Chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) resistant or intolerant to imatinib.A single daily dose of 100 mg in chronic phase CML results in high hematologic and molecular remission rates and prolongation of survival. In accelerated and blastic phase as well as in ALL, 70 mg twice daily is recommended. Complete hematologic and cytogenetic remissions (CR) frequently occur even in this patient group with poor prognosis. Remissions however are very short.Side effects of dasatinib are frequent but mostly moderate and manageable and include cytopenias and pleural effusions. The role of dasatinib in other diseases, including solid tumors, has to be identified.

Bosutinib (SKI-606) is a 7-alkoxy-3-quinolinecarbonitrile, which functions as a dual inhibitor of Src and Abl kinases. In biochemical and proliferation assays, the compound was shown to be active against src family kinases and Bcr-Abl at IC50s of 100 and 90 nM, respectively. The bcr-abl fusion gene product, a consecutively activated tyrosine kinase, which is crucial for the development of chronic myeloid leukaemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of the dual src/abl inhibitor are required to ablate Bcr-Abl phosphorylation when compared to first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and in vivo experiments and has demonstrated promising harbouring results in CML patients resistance or intolerance to IM in ongoing phase I/II clinical trials. Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant bcr-abl mutations. A randomised open label phase III clinical study to compare the efficacy of bosutinib and IM in first-line therapy of Ph+ chronic phase (CP) CML has recently been initiated. In a phase I/II clinical study with subjects suffering from advanced stages of solid tumours, long-term responses have also been reported. In conclusion, Bosutinib is a promising novel small molecule inhibitor for targeted therapy of CML and solid tumours.

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src.

The transforming abl proteins p160gag-abl, p185bcr-abl, and p210bcr-abl and the normal protein p140c-abl have identical catalytic sites, but differ in their N-terminal domains. Previous studies have indicated that the transforming abl proteins possess higher tyrosine kinase activity than the normal abl proto-oncogene product. In the present study, we demonstrate that two transforming abl proteins, p210bcr-abl and p160gag-abl, exhibit a higher affinity toward ATP and synthetic tyrosine containing substrates than p140c-abl. Furthermore, protein tyrosine kinase blockers from the tyrphostin family can discriminate between normal abl and transforming abl proteins of both human and mouse origin. These results suggest that the transforming potency of the abl proteins may result from their higher affinities toward intracellular signal transducers and demonstrate for the first time that oncogene products can differ from their homologous proto-oncogene product in substrate specificity. The ability of tyrphostins to discriminate between normal and transforming abl proteins suggests that it may be possible to design specific abl kinase inhibitors to combat abl-associated human leukemias.

We report the design, synthesis, and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well-characterized cancer cell lines. Our study reveals that substitutions in the 3- and 4-positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell-based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.

The FIP1L1-PDGFRalpha fusion gene plays an important role in the pathogenesis of chronic eosinophilic leukemia (CEL) and is a direct therapeutic target of the tyrosine kinase inhibitor imatinib mesylate.In 24 hypereosinophilic syndromes (HES) patients, using reverse transcriptase-polymerase chain reaction (RT-PCR), nested PCR and sequence analysis, we investigated the frequency of FIP1L1-PDGFRalpha and other abnormalities of tyrosine kinase family genes like PDGFRalpha, PDGFRbeta, C-KIT, FGFR1, ABL and FLT3 as well as gene mutation "hotspots", like MPL515 and JAK2V617F, frequently involved in myeloproliferative diseases. Fluorescence in situ hybridization was used to confirm the 4q12 deletion.The FIP1L1-PDGFRalpha fusion transcript was found in 8 (33%) of 24 patients with HES, corresponding to the chromosome 4q12 deletion identified by FISH. The FIP1L1-PDGFRalpha-associated patients diagnosed with CEL, frequently had hepatosplenomegaly, eosinophil-related tissue damage, anemia, thrombocytopenia, myelofibrosis and a short overall survival time. Nevertheless, imatinib mesylate induced rapid and complete hematological responses in treated FIP1L1-PDGFRalpha cases, followed by molecular remission and reversal of myelofibrosis. FIP1L1-PDGFRalpha fusion could co-exist with other mutations of tyrosine kinase family genes, like FLT3 or PDGFRbeta. We also demonstrated that the SNPs of PDGFRbeta were associated with selective splicing of exon 19 in case 20.Correlating the CEL genotype with phenotype, FIP1L1-PDGFRalpha emerges as a relatively homogeneous clinicobiological entity that co-exists with other abnormalities of tyrosine kinase family genes. It reflects the disease progression and there is a good response to imatinib. Detection of the FIP1L1-PDGFRalpha fusion gene is valid for both CEL diagnosis and therapy surveillance.

Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c- abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl -derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.

The axonal localization of the Drosophila abl protein and its genetic interactions with the disabled and fasciclin I genes implicate this cytoplasmic tyrosine kinase in the process of axonal pathfinding. Several changes at the amino terminus of abl permitted proper function and localization of the altered proteins. In contrast, the presence of human c-abl type 1a amino-terminal sequences or the murine c-abl carboxy-terminal domain interfered with function and axonal locallzation. Rescue of phenotypes caused by mutations in abl alone did not require tyrosine kinase activity, indicating a novel kinase-independent function for the properly localizaed abl protein. However, abl kinase activity was required to rescue the mutant phenotypes in genetic backgrounds also mutant for disabled .

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.

c-Cbl functions as a multifunctional adaptor and an E3 ubiquitin ligase. Several studies have shown that c-Cbl is involved in cytoskeleton-mediated events, but the molecular mechanisms linking c-Cbl to cytoskeletal rearrangements remain to be elucidated. Our previous results indicated that c-Cbl facilitates spreading and migration of v-Abl-transformed NIH 3T3 fibroblasts and suggested that small GTPases play important roles in the cytoskeletal effects of c-Cbl in this system. To elucidate the individual contributions of small GTPases to these effects, we assessed the roles of endogenous Rac1, RhoA and Rap1 in the c-Cbl-dependent spreading and migration of v-Abl-transformed fibroblasts overexpressing c-Cbl, using RNAi. Furthermore, since it has been shown that Rap1 can act as an upstream regulator of Rac1 in inducing cell spreading, we analyzed the interplay between Rap1 and Rac1 in the signaling pathways connecting c-Cbl to the cytoskeletal events. Our results indicate that Rac1 is essential for cell migration and spreading, whereas activation of RhoA exerts a negative effect. We have also shown that Rap1 is essential for cell spreading, although not for migration in our experimental system. Furthermore, we provide evidence that Rap1 is located upstream of Rac1 in one of the signaling pathways that regulate c-Cbl-facilitated cell spreading. Overall, our findings are consistent with the model describing the connection of c-Cbl to the cytoskeletal rearrangements via two pathways, one of which is mediated by PI3K and Rac1, and the other, by CrkL/C3G, Rap1 and Rac1.

The cellular RACK1 was shown in association with Abl in BALB/3T3 cells transfected with S-ras(Q(61)K) by immunoprecipitation. An identical finding was demonstrated with cells transfected with the embryonic E-ras, but not in cells without transformation. The Abl-RACK1 of transformed cells as resolvable with Triton X-114 was found with little affinity for FAK, PY(397)-FAK and integrin. Of interests, PY(397)-FAK in the membrane skeleton of transformed cells was shown in significant quantities on the Western blot. However the PY(397)-FAK of transformed cells was not functionally able to react with RACK1 and recruit cytokeratin-1, a substrate of Src, indicating that PY(397)-FAK is not operative to transmit integrin signals. In other words, the Abl-RACK1 of transformed cells cannot replace the Src-RACK1 of cells without transformation to bridge PY(397)-FAK and cytokeratin-1 for integrin signals, and the formation of Abl-RACK1 in transformed cells may block the association of PY(397)-FAK-RACK1. We characterized Abl and RACK1 from transformed cells by chromatography on a HiTrap-PEP(Taxol) affinity column, constructed from a beta-tubulin peptide specific for Taxol binding (PEP(Taxol)). However, the Triton X-100 cannot achieve the same resolution of Abl-RACK1 from plasma membrane as is shown with Triton X-114. A significant fraction of Abl was deposited at the membrane skeleton and was therefore not accessible with Triton X-100. Half of Abl resolved with Triton X-100 was demonstrated to have catalytic activity as shown with positive phosphotyrosine staining on the Western blot and competitive elution with a specific phosphate, such as sodium beta-glycerophosphate, from HiTrap-PEP(Taxol), but this was not associated with RACK1. No significant difference of RACK1 was found in Triton X-100 resolvable membrane preparations from cells with and without transformations. Future studies are planned to differentiate the mechanism operative for RACK1 associated and RACK1 freed Abl in cells transformed with oncogenic ras.

Prostate cancer is one of the most common cancers in men, with more than 500,000 new worldwide cases reported annually, resulting in 200,000 deaths of mainly older men in developed countries. Existing treatments have not proved very effective in managing prostate cancer, and continuing efforts therefore are ongoing to explore novel targets and strategies for future therapies. LAPSER1 has been identified as a candidate tumor suppressor gene in prostate cancer, but its true functions remain unknown. We report here that LAPSER1 colocalizes to the centrosomes and midbodies in mitotic cells with gamma-tubulin, MKLP1, and p80 katanin, and is involved in cytokinesis. Moreover, RNAi-mediated disruption of LAPSER1, which is accompanied by the mislocalization of p80 katanin, results in malformation of the central spindle. Significantly, the enhanced expression of LAPSER1 induces binucleation and renders the cells resistant to oncogenic transformation. In cells transformed by the v-Fps oncogene, overexpressed LAPSER1 induces abortive cytokinesis, followed by mitotic catastrophe in a p80 katanin-dependent manner. Cells that are rescued from this apoptotic pathway with Z-VAD-fmk display karyokinesis. These results suggest that LAPSER1 participates in cytokinesis by interacting with p80 katanin, the disruption of which may potentially cause genetic instability and cancer.

Cellular transformation, the conversion of normal cells into tumorigenic cells in vitro, is characterized by immortalization, anchorage- and serum-independent growth and tumour formation in the nude mouse. Among these, anchorage-independent growth is one of the defining characteristics of transformed cells and tumour cells. Without attachment to the extracellular substrate, most normal cells cannot grow or survive, but tumour cells can proliferate. Many oncogenes and tumour suppressors are involved in regulating this process, among which is Abl tyrosine kinases. Previous work showed that v-Abl, an oncogenic variant of c-Abl kinase, induces anchorage-independent growth in the context of p53 deficiency, and a recent study by our group showed that loss of c-Abl kinase also facilitates anchorage-independent growth. The cellular context, such as a deficiency in both p53 and RB, is critical to induce anchorage independence by loss of c-Abl kinase. In this review, we discuss the mechanisms of cellular transformation by oncogenic and normal Abl kinases.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the presence of the Philadelphia chromosome that expresses the constitutively activated tyrosine kinase Bcr-Abl; this enzyme causes hyperproliferation of the stem cells and the consequent pathology of the disease. Targeted inhibitors of Bcr-Abl have antiproliferative effects on the leukemic cells and induce apoptosis, favouring a regression of the CML chronic phase, but in the successive blast crisis phase cancer cells frequently develop resistance to Bcr-Abl inhibitors. Src is a family of non-receptor tyrosine kinases, fundamental for cell development, growth, replication, adhesion, motility and is overexpressed in a wide number of human cancers. Recently it was demonstrated that Src is increased in hematopoietic cells expressing Bcr-Abl and is involved in the oncogenic pathway that causes CML. For this reason and also for the development of resistance to classical Bcr-Abl inhibitors, various dual Src/Abl inhibitors have been recently synthesized and tested. This mini review will be focused on the latest finding on this matter.

Background: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). Objective: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. Methods: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of gamma H2AX accumulation was studied after ionizing radiation. Results: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. Conclusions: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

To investigate the mechanism of apoptosis of chronic myeloid leukemia (CML) cells induced by the novel p210 bcr/abl inhibitor berbamine.Human Ph+ CML leukemia K562 cells, which express endogenous p210 bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry (FCM) and Annexin-V-Fluos/PI staining kit were used to evaluate the apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure the leukemic cells with activated Caspase-3. Phosphorylation of p210 bcr/abl protein in the leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blotting with p-Tyr (pY99) antibody. The protein levels of p210 bcr/abl, Hsp90 and Hsp70 in the leukemic cells were determined by Western blotting with antibodies to c-abl, Hsp90, and Hsp70 respectively.After treatment with berbamine at 8 microg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43% respectively. IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210 bcr/abl protein in the leukemia cells, and the amount of phosphorylated p210 bcr/abl in the leukemia cells exposed to berbamine at 8 microg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of p210 bcr/abl down-regulated. Significantly, berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in the leukemia cells treated with berbamine at 8 microg/ml for 48 h accounted for 18.37% of that of the untreated leukemia cells. Berbamine at 8 microg/ml had no obvious effect on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis.(1) Berbamine induces caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210 bcr/abl protein and down-regulating its chaperone Hsp90 protein. (2) Unlike Hsp90 inhibitor GA that upregulates Hsp70, berbamine has no obvious effect on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.

Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre–B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

Understanding the mechanisms leading to transformation of early B-lineage precursors is an important step leading to rational design of new treatments for precursor (pre)-B-cell leukemia. We used normal mouse pre-B cells to determine if and how transforming growth factor (TGF)-beta1 affects these precursors to the B-cell lineage and whether transformed pre-B cells respond to TGF-beta1. We found that normal pre-B cells proliferating in the presence of interleukin (IL)-7 enter cell-cycle arrest after exposure to TGF-beta1. However, clonally related IL-7-independent tumors induced by oncogenes abl + myc or raf + myc have reduced sensitivity to TGF-beta1. In contrast, tumor cells induced by myc alone remain sensitive to TGF-beta1 growth suppression. These results suggest that lesions in different molecular signaling pathways can lead to loss of TGF-beta1 sensitivity in a single cell type. The approach of using normal pre-B-cell lines and transformation by overexpression of different oncogenes provides a system to compare and contrast molecular pathways that lead to full malignancy.

Standard antineoplastic treatment for metastatic melanoma is ineffective in the large majority of patients. Therefore, alternative approaches need to be investigated. STI571 is a new antineoplastic compound, which selectively inhibits the tyrosine kinase activity of ABL, c-Kit and platelet-derived growth factor receptor (PDGFR). Melanoma may express all of these proteins. The aim of this study was to investigate whether STI571 inhibits the in-vitro growth of melanoma cells. Nineteen cell lines were obtained from four primary and 15 metastatic melanomas of cutaneous origin. The percentages of positive cells for the putative targets of STI571 were as follows: ABL, 41-100%; c-Kit, 8-97%; PDGFR-alpha, 41-98%; PDGFR-beta, 51-99%. 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium (MTT) and viability assays showed that STI571 clearly inhibits the proliferation of eight of the 19 (42.1%) cell lines. No relationship could be established between the expression of c-Kit, ABL, PDGFR-alpha or PDGFR-beta and the response of cell lines to STI571. Our study shows, for the first time, an antiproliferative effect of STI571 on human melanoma cell lines of cutaneous origin, raising the possibility of the future clinical use of STI571. The identification of the target of STI571 in human cutaneous melanoma cells would allow the selection of patients who could benefit from this treatment.

Imatinib, a potent inhibitor of the oncogenic tyrosine kinase BCR-ABL, has shown remarkable clinical activity in patients with chronic myelogenous leukaemia (CML). However, this drug does not completely eradicate BCR-ABL-expressing cells from the body, and resistance to imatinib emerges. Although BCR-ABL remains an attractive therapeutic target, it is important to identify other components involved in CML pathogenesis to overcome this resistance. What have clinical trials of imatinib and studies using mouse models for BCR-ABL leukaemogenesis taught us about the functions of BCR-ABL beyond its kinase activity, and how these functions contribute to CML pathogenesis?