Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

Abl family tyrosine kinases are essential for basement membrane integrity and cortical lamination in the cerebellum.

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 of Abl in precursors of neurons and glia on an Arg knock-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 vitro culture 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 of Abl/Arg from 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.

Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl.

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 (KXXK--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.

c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl.

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.

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.

Abl tyrosine kinase in signal transduction and cell-cycle regulation.

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.

LAPSER1 is a putative cytokinetic tumor suppressor that shows the same centrosome and midbody subcellular localization pattern as p80 katanin.

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.

The suppression of drug-induced apoptosis by activation of v-ABL protein tyrosine kinase.

Cells with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP) were treated with the anticancer drugs melphalan or hydroxyurea. At the restrictive temperature for v-ABL protein tyrosine kinase activity, drug-treated IC.DP cells died by apoptosis. In contrast, apoptotic cell death induced by either drug was suppressed when v-ABL was active. However, melphalan-induced accumulation of cells in the S and G2-M phases of the cell cycle was unaffected by v-ABL activation. Moreover, the continuous presence of v-ABL activity was necessary to suppress apoptosis. This suggested that melphalan had interacted with DNA and that v-ABL activity prevented the coupling of drug-induced damage to the apoptotic pathway. IC.DP cells exhibited similar levels and subcellular localization of the BCL-2 protein irrespective of v-ABL activation status, thus v-ABL-mediated cell survival appeared to be independent of BCL-2.

Inhibition of v-Abl transformation in 3T3 cells overexpressing different forms of the Abelson interactor protein Abi-1.

The abi-1 gene encodes a protein that binds and is phosphorylated by the Abelson protein tyrosine kinase. Constructs expressing a full-length abi-1 cDNA, and a smaller cDNA arising from an alternatively spliced form, were generated and tested for their effect on transformation of NIH3T3 cells by the Abelson murine leukemia virus. Overexpression of both forms of the protein strongly inhibited transformation by the wild-type P160 strain of the virus, but not by the non-interacting mutant P90A strain. The inhibition required the SH3 domain of Abi-1, suggesting that a direct interaction was required for the effect. Rare breakthrough P160 transformants of the Abi-1 overexpressing lines were found to have downregulated Abi-1 protein levels by a post-transcriptional mechanism.

Endogenous growth factor patterns modulate hemopoietic lineage development.

Hemopoietic growth factors play an important role during stem cell differentiation, and multipotent hemopoietic cells expressing abl oncogenes can cause stem cell diseases in mice. To further elucidate the mechanism of disease development, we examined the initial changes of normal and abl-transduced progenitor cells early in culture, including the endogenous production of growth factors. From 2-3 days methylcellulose cultures, we isolated colonies of early cells and subjected them to RT-PCR analysis. They were found to produce endogenous IL-1 alpha and IL-4. Treatment of these early cells with the antisense oligonucleotides directed against the mRNA of the growth factors did not prevent colony formation. However, the percentage of pure macrophage colonies in cultures containing the antisense IL-1 alpha was increased from 26-60%. This effect was not observed when the antisense oligomers were added 2 days after initiation of culture. We also transduced the progenitor cells with retrovirus vectors carrying either a neor gene (N2) or a v-abl oncogene (A-MuLV or NSabl). After their culture in methylcellulose, we examined the types of colonies developed, growth factor expression by the early cells and their proliferation rate. While the ratio of erythroid to non-erythroid hemopoietic colonies in uninfected culture and N2-infected culture was 0.8, the ratio in A-MuLV-infected culture was 6.0, and in NSabl-infected culture, 3.1. RT-PCR analysis on colonies of abl-transduced cells from 2-3 day cultures indicated a reduction of IL-4 and IL-1 alpha in these cells, and they entered cell division sooner. Our data suggest that hematopoietic lineage development may be a function of the pattern of endogenous as well as exogenous growth factors, and alteration of this pattern either through antisense treatment or v-abl transduction affects the hemopoietic differentiation program.

Bcr-Abl has a greater intrinsic capacity than v-Abl to induce the neoplastic expansion of myeloid cells.

The Bcr-Abl/p210 fusion protein plays a primary role in the pathogenesis of chronic myelogenous leukemia (CML). Abelson murine leukemia virus, which encodes v-Abl/p160, induces a pre-B cell leukemia/lymphoma in mice. It has been unclear whether the apparent specificity of these two abl oncogenes for myeloid versus lymphoid neoplasms is due to specific intrinsic properties of these Abl oncoproteins, or due to the properties of the target cells expressing them. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder in mice resembling human CML. In this study, we compared Bcr-Abl/p210 and v-Abl/p160 in this mouse CML model. We found that early in the course of disease, both Bcr-Abl/p210 and v-Abl/p160 expanded early immature hematopoietic cells. Later Bcr-Abl/p210 selectively expanded myeloid cells while v-Abl/p160 primarily induced the rapid in vivo expansion of B lymphoblastic cells, along with a minor population of myeloid cells. In vitro, Bcr-Abl/p210 induced more growth of myeloid colonies from 5-fluorouracil treated bone marrow than v-Abl/p160. These results, obtained under equal bone marrow transduction/transplantation conditions, indicate that Bcr-Abl/p210 has a greater intrinsic capacity than v-Abl/p160 to induce the neoplastic growth of myeloid cells. In addition, we found that cultured cells expressing Bcr-Abl/p210 had more activated STAT5 than cells that expressed v-Abl/p160. This suggests that activation of STAT5 might be one part of the mechanism of abl oncogene disease specificity.

Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity.

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.

v-Abl utilizes multiple mechanisms to drive G1/S progression in fibroblasts.

Transformation of 3T3 fibroblasts by the v-Abl tyrosine kinase replaces mitogenic and adhesion signals normally required for cell cycle progression. A 3T3 cell line conditionally transformed with v-Abl has been used to study v-Abl's effects on cell cycle in the context of either serum depletion or absence of adhesion signals. We show that E2F-dependent mRNAs, encoding proteins required for cell cycle progression, are induced by v-Abl. In addition, we identify two previously unknown targets of v-Abl signaling: (1) cyclin D1 and D2 mRNAs are induced upon v-Abl activation; and (2) the CDK inhibitor p27 is decreased upon v-Abl activation.

Biological consequences of p160v-abl protein tyrosine kinase activity in a primitive, multipotent haemopoietic cell line.

A temperature sensitive abl protein tyrosine kinase gene was transferred into a multipotent haemopoietic stem cell line, and the primary biological effects of expression of the gene were examined at the permissive and non-permissive temperatures. Unlike previous studies in factor-dependent cell lines, we found that expression of the functional abl protein tyrosine kinase did not lead to growth autonomy. Furthermore, the cells were still able to undergo terminal myeloid differentiation. However, expression of the functional gene did lead to a delay in maturation with a concomitant increase in cell production, had a modest effect in terms of delayed apoptosis particularly when the cells were maintained at a high cell density, and slightly increased the response to sub-optimal concentrations of IL-3. In many respects, therefore, the effects of abl protein tyrosine kinase in these cells mimics the effect of bcr/abl in primary haemopoietic cells where growth factor independence and an aberrant differentiation profile are relatively late events in clonal evolution and are not intermediate consequences of activation of the abl gene.

Mechanisms of Bcr-Abl-mediated NF-kappaB/Rel activation.

Bcr-Abl constitutes a deregulated tyrosine kinase involved in the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Although activation of the transcription factor NF-kappaB/Rel has been demonstrated, mechanisms of NF-kappaB/Rel activation by Bcr-Abl remain obscure. In this paper we demonstrate activation of NF-kappaB/Rel by Bcr-Abl and for the first time by v-Abl. Furthermore, we investigated mechanisms of NF-kappaB/Rel induction by Bcr-Abl and v-Abl. Both Bcr-Abl and v-Abl induced NF-kappaB/Rel DNA binding in Ba/F3 cells. DNA binding was a result of nuclear translocation of p65/RelA, whereas p65/RelA expression was unaffected. Nuclear translocation of p65/RelA is at least partially due to increased IkappaBalpha degradation, which is independent of IkappaB kinase (IKK) activity. IKK activity is not deregulated by Bcr-Abl and v-Abl. NF-kappaB/Rel transactivation was dependent on abl kinase activity but independent of Grb2 and Grb10 binding tobcr sequences. In addition, NF-kappaB/Rel activation was dependent on Ras activity. Primary CML blasts showed constitutive p65/RelA NF-kappaB/Rel DNA binding activity. Thus NF-kappaB/Rel represents a potential target for molecular therapies in CML.

Phosphorylation of the catalytic subunit of type-1 protein phosphatase by the v-abl tyrosine kinase.

The catalytic subunit of type-1 protein phosphatase (PP1) was phosphorylated by the tyrosine kinase v-abl as follows: (i) cytosolic PP1 was phosphorylated more (0.73 mol/mol) than PP1 obtained from the glycogen particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PP1 was phosphorylated even less and catalytic subunit complexed with inhibitor-2 was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PP1 and 3 h incubation at 30 degrees C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C-terminal region of PP1 which is lost during proteolysis; (iv) phosphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitor-2 of a PP1 form that in its dephospho-form did not convert.

Genetic analysis of transcription factors implicated in B lymphocyte development.

The transcription factors Oct-2, NF-kappa B and PU.1 have been implicated in regulating the development of B lymphocytes. Genetic approaches have been used to analyze the developmental functions of these regulatory proteins. Using gene targeting in murine embryonic stem cells, PU.1 is shown to be required for the development of progenitor B cells. Strikingly, PU.1 is also essential for the development of T lymphoid, granulocytic and monocytic progenitors. Transcription factors of the NF-kappa B/Rel family, which appear to regulate immunoglobulin kappa gene expression, are shown to be a target of the viral transforming protein (v-abl) which arrests B lineage development at the precursor B stage. This suggests a mechanism by which v-abl blocks precursor B cell differentiation. The Oct-2 transcription factor was considered to represent a development regulator of immunoglobulin gene expression. Using gene targeting in a murine B cell, Oct-2 is shown to be dispensable for immunoglobulin gene expression. This suggests the existence of an alternate pathway, involving the ubiquitous related protein, Oct-1, in immunoglobulin gene regulation.

Antagonistic effects of ABL and BCRABL proteins on proliferation and the response to genotoxic stress in normal and leukemic myeloid cells.

Following the discovery of the p210bcrabl protein product of the bcrabl chimeric fusion gene generated by the Philadelphia chromosome translocation in chronic myelogenous leukemia (CML), structure function studies quickly identified which parts of this molecule were playing a role in the generation of the phenotypes of growth factor independent growth, anchorage independent growth, and genetic instability which are associated with this disease. These latter changes result in abnormally high levels of mature myeloid elements circulating in the systemic circulation of CML patients. In addition, the genetic instability which is associated with the presence of the Philadelphia chromosome drives the evolution of the disease from an indolent chronic non life-threatening leukemia, to a fulminant acute leukemic syndrome which results in the death of patients from bleeding and infection. Multiple sites of contact between the p210bcrabl and its substrates have already been identified which are relevant to the phenotypic changes characteristic of CML cells and define their response to therapy. In this review, we will discuss what is known about the relationships between the structural domains of the p210bcrabl protein and the characteristics of the disease process which it causes. We will also discuss how this information may be applied to the establishment of new directions in therapy.

Transforming oncogenes regulate glucose transport by increasing transporter affinity for glucose: contrasting effects of oncogenes and heat stress in a murine marrow-derived cell line.

Transforming oncogenes often overcome the growth factor requirements of cells by activating growth factor signal transduction pathways. Increased energy utilization by transformed cells is a well known phenomenon, but whether glucose uptake is regulated at the level of the glucose transporter has not been clearly established. To determine whether cell transformation by specific oncogenes like, v-H-ras and v-abl affects the activation state of glucose transporters, bone marrow-derived IL-3-dependent 32D (clone3) cells transfected with temperature-sensitive ras and abl oncogenes were used to compare proliferative responses and glucose transporting ability of these cells with the parental cell line at permissive (32 degrees C) and non-permissive (40 degrees C) temperatures. Transformed cells showed elevated incorporation of [3H]thymidine and enhanced tyrosine kinase activity, both of which were abrogated in temperature-sensitive mutants maintained at the non-permissive temperature. Compared with control cells, 2-deoxy-D-[1-(3)H]glucose (2-DOG) uptake was not significantly different in transformed cells at the permissive temperature. However, transformation was associated with a 2-2.5-fold greater affinity of glucose transporters for glucose (Km) and this was reversed following treatment with tyrosine kinase inhibitor, genistein. Maximum velocity of glucose transport (Vmax) and membrane expression of transporters were reduced in oncogene-transformed cells. At the non-permissive temperature, glucose uptake was elevated in both control and oncogene-transformed cells. This increase in glucose transport was not associated with a change in transporter affinity for glucose, but increased Glut-1 expression was observed indicating a 'heat stress' effect that overrode the effects attributable to oncogene loss. The 'heat stress' effect was inhibited by protein synthesis inhibitor cycloheximide. These results provide evidence for intrinsic activation of glucose transporters by the transforming oncogenes ras and abl, and indicate that oncogenes and 'heat stress' regulate glucose transport by different mechanisms.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility.

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.