BCR Domain Research Articles

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms — TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.

Enhanced and Selective Killing of Chronic Myelogenous Leukemia Cells with an Engineered BCR-ABL Binding Protein and Imatinib

The oncoprotein Bcr-Abl stimulates prosurvival pathways and suppresses apoptosis from its exclusively cytoplasmic locale, but when targeted to the mitochondrial compartment of leukemia cells, Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria. The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE)) contains an EGFP tag and two previously characterized motifs: (1) an optimized Bcr-Abl binding motif that interacts with the coiled-coil domain of Bcr (ccmut3; 72 residues), and (2) a cryptic mitochondrial targeting signal (cMTS; 51 residues) that selectively targets the mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive leukemic cells) via phosphorylation at a key residue (T193) by protein kinase C. While the iCE colocalized with Bcr-Abl, it did not relocalize to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts and 1471.1 murine breast cancer cells. The toxicity of the iCE to leukemic cells was equivalent to 10 μM imatinib at 48 h and the iCE combined with imatinib potentiated cell death beyond imatinib or the iCE alone. Substitution of either the ccmut3 or the cMTS with another Bcr-Abl binding domain (derived from Ras/Rab interaction protein 1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE. Additionally, a phosphorylation null mutant of the iCE also abolished the killing effect. The mitochondrial toxicity of Bcr-Abl and the iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation and cell viability were assessed by microscopy. Subcellular localization of constructs was determined using confocal microscopy (including statistical colocalization analysis). Overall, the iCE was highly active against K562 leukemia cells and the killing effect was dependent upon both the ccmut3 and functional cMTS domains.

Another tool in the BCR–ABL kit?

This Cell paper shows that an interaction between the SH2 and kinase domains of BCR–ABL is necessary for kinase activation; inhibition of this interface prevents leukaemogenesis in mice and can restore sensitivity to tyrosine kinase inhibitors.

Two alternatively spliced 5′BCR/3′JAK2 fusion transcripts in a myeloproliferative neoplasm with a three-way t(9;18;22)(p23;p11.3;q11.2) translocation

Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) are known to harbor alterations of the tyrosine kinase JAK2 (9p24), resulting in the constitutive autoactivation of the encoded protein. Here, we report an unclassifiable MPN case, BCR/ABL1-negative, showing a three-way t(9;18;22)(p23;p11.3;q11.2) translocation, which generates a 5'BCR/3'JAK2 gene by fusing BCR at intron 1 to JAK2 at intron 14 on the derivative chromosome 22. The fusion gene produced two alternatively spliced 5'BCR/3'JAK2 transcripts, fusing in-frame BCR exon 1 to JAK2 exon 15 and exon 17. This is the first report of the simultaneous occurrence of two BCR/JAK2 fusion transcripts in the same sample and of the longer transcript isoform (BCR exon 1 fused to JAK2 exon 15). Notably, both BCR/JAK2 encoded fusion proteins are predicted to juxtapose the coiled-coil dimerization domain of BCR to the catalytically inactive pseudokinase domain (JH2), entirely or partially deprived of the inhibitory region 1 (IR1). Interestingly, IR1 is involved in the auto-inhibitory interaction with the JAK2 kinase domain (JH1), which may result in deregulation of JAK2 activity.

Transglutaminase 2 Regulates the GTPase-activating Activity of Bcr

Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPase-activating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity.

Conditional Activation of Bcr-Abl Kinase Delineates Time-Dependent Transforming Events in Hematopoietic Cells.

Abstract 2162Poster Board II-139Cytoplasmic Bcr-Abl kinase evokes a diverse array of transforming signals and is responsible for development of Philadelphia chromosome-positive (Ph+) leukemias. In this fusion protein, disruption or deletion of a N-terminal coiled-coil (CC) domain of Bcr results in substantially decreased or absent tyrosine kinase activity and defective cellular transformation, indicating the crucial role of Abl oligomerization in constitutive activation of its intrinsic kinase activity. Fusion of the estrogen receptor (ER) ligand binding domain (LBD) to the C-terminus of Abl generates a ligand-activatable and transformation-competent tyrosine kinase dimer. However, additional sequences in Bcr may also be required for cellular transformation since Bcr-Abl mutants containing just the extreme CC domain cannot transform fibroblasts.To revisit the mechanism of Bcr-Abl-induced leukemogenesis and especially to delineate early events upon Bcr-Abl activation, we applied this fusion technology to construct DCCp190ER, a p190Bcr-Abl mutant including ER-LBD at the C-terminus but not CC domain at the N-terminus. Cytokine-dependent murine Ba/F3 and human TF-1 cells were retrovirally transduced with DCCp190ER as well as wild-type p190 (wt), DCCp190 and vector control, respectively. Such a series of transformants were subjected to not only biological assays but also biochemical analysis such as anti- phosphotyrosine immunoblot and NF-kB assay.DCCp190ER, but not DCCp190, can support cytokine-independent growth of both Ba/F3 and TF-1 cells in a dose-dependent manner on 4-HT, a synthetic ER-ligand, although excess of 4-HT is rather toxic to these cell lines. Optimal concentration of 4-HT has been determined as 0.5mM. The growth stimulatory effect of 4-HT on DCCp190ER -transduced cells was efficiently canceled by imatinib. Expectedly, upon 4-HT binding, a number of phosphotyrosil proteins could be detected using 4G10 MAb. Protein tyrosine phosphorylation was induced within 10 min after ligand stimulation and a part of proteins revealed a biphasic phosphorylation pattern during the time course. The overall protein profiles of DCCp190ER-induced tyrosine phosphorylation including CrkL and Stat5 resembled p190wt- induced those, but a significant time-interval beyond a day was required for the former to be comparable to the latter in signal intensity (Figure 1). Curiously this holds true for autophosphorylated DCCp190ER. [Display omitted] It is evidenced that Bcr-Abl constitutively activates NF-kB signaling pathway via unknown mechanism. TF-1/DCCp190ER cells were lentivirally transduced with NF-kB/luciferase (kB/Luc) reporter construct and were subjected to monitoring Luc activity upon ligand stimulation. Unstimulated TF-1/DCCp190ER-kB/Luc cells revealed a weak but significant Luc activity over the background. GM-CSF, but not 4-HT, moderately enhanced their Luc activities after 24 hrs of stimulation. On the contrary, 4-fold up-regulation of Luc activity was observed after 6 days culture with 4-HT (Figure 2), suggesting the delayed cellular transformation by DCCp190ER, which is compatible with delayed enhancement of tyrosine phosphorylation. Finally, we investigated gene expression profiling of TF-1 cells induced by DCCp190ER upon 4-HT binding. As a result, expression of a number of genes including TRAIL, GATA2 and endoglin were altered in response to Bcr-Abl activation by treatment with 4-HT for 4 days. The present results suggested that Bcr-Abl kinase may evoke early transforming signals in a unique time-dependent manner, leading to established transformed phenotype. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The function of the pleckstrin homology domain in BCR–ABL-mediated leukemogenesis

The BCR–ABL tyrosine kinase is central to the pathogenesis of chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). Depending on the breakpoint in BCR, several BCR–ABL variants can be generated (Figure 1a), such as p190BCR–ABL and p210BCR–ABL, which are detected in the majority of cases of Ph-positive ALL and CML, respectively.1 The p190BCR–ABL isoform was previously reported to show elevated tyrosine kinase activity,2, 3 although the structural basis for increased kinase activity has not been established and kinase activity alone fails to explain the predilection of p190BCR–ABL for transformation of lymphoid cells. At the sequence level, the genetic information retained in p210BCR–ABL but not in p190BCR–ABL encodes the diffuse poorly differentiated B-cell lymphoma (DBL)-like and pleckstrin homology (PH) domains of BCR. Although earlier studies have suggested a positive role for the DBL-like domain in p210BCR−ABL-induced transformation,4 the function of the PH domain in leukemogenesis has not been studied in isolation. In this study, we used a rare, naturally occurring variant of BCR–ABL (p200BCR–ABL; Figure 1a) that retains the DBL-like, but not the PH domain to analyze the role of the PH domain in BCR–ABL-induced leukemia.

Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr–Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr–Abl proteins, and an intact OD in Bcr–Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr–Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr–Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr–Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni +–NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr–Abl, and potently inhibited the phospho-tyrosine pathways of Bcr–Abl oncoprotein at a low concentration of 4 μM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.

Xeroderma Pigmentosum Group B Binding Contributes to p210 BCR/ABL Mediated Transformation in CML.

Patients with chronic myelogenous leukemia (CML) typically carry a balanced reciprocal translocation between chromosomes 9 and 22, resulting in the production of an in-frame fusion protein known as p210 BCR/ABL. Expression of p210 BCR/ABL in myeloid cells is associated with a variety of transformed cellular phenotypes including changes in nucleotide excision repair (NER). Consistent with this, previous studies have demonstrated that p210 BCR/ABL interacts directly with xeroderma pigmentosum group B (XPB), a protein necessary for both transcription and NER. In the current study we have mapped the docking site for XPB within the RhoGEF domain of BCR, and have constructed a p210 BCR/ABL mutant that is deficient in XPB binding. The mutant has normal kinase activity, and retains Grb2 binding ability, but can no longer phosphorylate XPB. When expressed in 32Dc13 cells, the mutant still confers IL-3 independent growth, but abrogates the resistance to ultraviolet C (UVC)-induced apoptosis seen in wild-type p210 BCR/ABL expressing cells. In a bone marrow transplantation model for CML, mice that express the mutant have significantly extended lifespans. Whereas p210 BCR/ABL transplanted mice became moribund within 28–35 days of transplantation, mutant mice survived for an average of 78 days. Necropsies performed at death revealed splenomegaly and clear evidence of myeloproliferation in the mutant mice, however tissue infiltration and lung hemorrhage was less extensive than in the p210 BCR/ABL expressing mice. These results suggest that the interaction between p210 BCR/ABL and XPB regulates disease progression, and may represent a novel target for therapeutic intervention.

Characterization of BCR-ABL Deletion Mutants from Patients with Chronic Myeloid Leukemia.

Background: The Bcr-Abl tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, in some patients cells are selected with point mutations in the Bcr-Abl kinase domain that decrease drug sensitivity. A deletion mutant lacking amino acids 248–274 was recently reported in 2 patients with the L248V mutation (Gruber et al, Leukemia, 2006). In addition, missplicing of BCR to ABL exon 4 or 5 has been reported to be common in CML patients (Volpe et al, Cancer Research, 2007). However, the incidence and functional relevance of deletion mutants involving ABL exons including the kinase domain are unknown. Wild type Bcr-Abl coexpression with kinase dead mutants that retain the coiled-coil domain of Bcr may result in hetero-dimer and -tetramer formation. In turn, this could produce a dominant-negative effect through decreased Bcr-Abl transphosphorylation.Methods: Bone marrow and peripheral blood samples were obtained following informed consent from randomly selected CML patients followed in the OHSU outpatient clinic. Nested RT-PCR for BCR-ABL was performed using 2 sets of primers from the breakpoint to the kinase domain. Sequencing covered the Cap, SH3, SH2, linker and kinase domains. Deletions involving the P-loop of the kinase domain were further characterized. To test activity, the mutations introduced into Abl were expressed in HEK293 cells and immunoprecipitated for kinase assays. To test for dominant-negative effects, we generated Ba/F3 lines coexpressing low (L), medium (M) and high (H) levels of deletion mutant Bcr-Abl together with native Bcr-Abl. These cells were tested for IL-3 independence, autophosphorylation and imatinib sensitivity.Results: We found 14 of 101 (14%) patients to have a deletion mutation. Five patients were found to have a deletion of amino acids 184–274 (Δ184–274), corresponding to loss of ABL exon 4. Other mutants were Δ27–183 (out of frame, stop at 296) (N=3), Δ248–274 with the L248V mutant (N=2) and Δ342–383 (N=2). Also, one patient each had Δ362–444 (loss of ABL exon 7, out of frame, stop at 458) and Δ151–183. Immunoprecipitated Abl proteins with the P-loop deletion mutations were kinase inactive (Figure 1A). P-loop deletion mutants were transformation defective, while coexpression of deletion mutant and native Bcr-Abl conferred growth factor independence and Bcr-Abl signaling was unimpaired. Unexpectedly, imatinib sensitivity was consistently increased in cells coexpressing native and deletion mutant Bcr-Abl (Figure 1B).Conclusions:1.Deletion mutants are relatively common in patients on imatinib therapy (14% in our series) and most frequently involve missplicing.2.P-loop deletion mutants are kinase inactive.3.Coexpression of deletion mutants with wild type Bcr-Abl confers increased sensitivity to imatinib and thus may improve response to the drug. [Display omitted]

A BCR–JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of a t(9;22)(q34;q11.2), which leads to the well-known BCR-ABL1 fusion protein. We describe a patient who was diagnosed clinically with a typical CML but on cytogenetic analysis was found to have a t(9;22)(p24;q11.2). Chromosomal fluorescence in situ hybridization showed that the BCR gene locus spanned the breakpoint at band 22q11.2 but that the ABL1 gene was not rearranged. By means of a candidate gene approach, the JAK2 gene, at 9p24, was identified as the fusion partner of BCR in this case. The BCR-JAK2 fusion protein contains the coiled-coil dimerization domain of BCR and the protein tyrosine kinase domain (JH1) of JAK2. The patient's disease did not respond to Imatinib, and this unresponsiveness was most likely a result of the BCR-JAK2 fusion protein.

BCR-ABL Lacking the Pleckstrin Homology (PH) Domain of BCR Induces a More Aggressive Leukemia Than P210BCR-ABL in a Murine Model of CML.

Background: The two common types of BCR-ABL fusion protein, p190 and p210, are associated with Ph-positive ALL and CML, respectively. Compared to p190, p210 retains the Dbl-like, CDC24 homology and PH domains. Dbl-like and CDC24 homology domains are thought to serve as a guanidine exchange factor (GEF) for small G proteins, including Rac, CDC42 and RhoA. It has been suggested that the loss of GEF activity may be responsible for the more aggressive phenotype of p190-positive leukemia (McWhirter and Wang, Oncogene 1997; 15(14):1625–34). PH domains bind phosphoinositides and have been implicated in targeting proteins to cellular membranes as well as modulating the activity of the adjacent Dbl-like GEFs. It is not known, however, if the PH domain of BCR plays a role in determining disease features. We therefore decided to study the biology of p200BCR-ABL, a naturally occurring variant identified in several CML patients that lacks the PH domain but retains the Dbl-like and CDC24 homology domains.Materials and methods: p190, p210 and p200BCR-ABL cDNAs were cloned into pSRa and MIGR1 for expression in cell lines and primary murine bone marrow (BM) cells, respectively. The activation of signaling pathways downstream of BCR-ABL was studied in Ba/F3 and 32D cells expressing the various constructs. For colony-forming assays, murine BM cells were infected with p210, p200 or p190 retroviral vectors and seeded in methylcellulose in the presence or absence of cytokines (SCF, IL3 & IL6) and colonies were counted on day 12. For in vivo studies, BM cells isolated from 5-fluorouracil treated 5-6 week-old female Balb/c mice were infected with the various retroviral constructs in the presence of cytokines (SCF, IL3 & IL6), and 4.0 x 105 cells were injected retro-orbitally into lethally irradiated recipient mice. Disease latency was determined by monitoring white blood counts at 48h intervals. Moribund animals were sacrificed and studied by FACS and histopathology.Results: As expected, p200 transformed Ba/F3 and 32D cells to IL-3 independence. Immunoblotting of whole cell lysates with a phosphotyrosine antibody revealed no significant differences in global protein phosphorylation between the three variants, but phosphorylation of BCR-ABL was consistently less sensitive to imatinib inhibition in cells expressing p190 compared to p200 and p210. Analysis of downstream signaling showed approximately 15 fold higher levels of phosphorylated STAT6 in Ba/F3 cells expressing p200 and p190 vs. p210, while STAT5 and AKT phophorylation were similar in all variants. In the absence of cytokines, BM cells from Balb/c mice transduced with p200 and p190 generated significantly more CFU-GM than cells transduced with p210 (p<0.001, Student's t-test). The significance of these findings was tested in vivo in a murine CML model. Mice transplanted with p210-transduced cells survived 16–20 days, significantly longer than mice transplanted with p200 or p190 expressing cells (13–15 days) (p = 0.01, log-rank test).Conclusion: p200 is closely related to p190 but very distinct from p210 with respect to downstream signaling and in vivo transforming potential. These data suggest a critical role for the PH domain of BCR in attenuating the phenotype of the leukemia. We speculate that deletion of the PH domain may disrupt the ability of the Dbl-like and CDC24 homology domains to function as a GEF towards small G proteins.

CML with E8A2 BCR-ABL Fusion: The Fourth Breakpoint Cluster Region?.

In Ph+ leukemia, the breakpoints within BCR cluster in 3 distinct breakpoint cluster regions (bcr). Rearrangements in the minor (m−), major (M−) and micro (m−) breakpoint cluster region (bcr) give rise to e1a2, e13/14a2 and e19a2 BCR-ABL fusion proteins that are associated with Ph+ ALL, CML and chronic neutrophilic leukemia, respectively. Atypical BCR-ABL fusions have also been reported, mostly in isolated cases. Here, we describe eight patients with an unusual e8a2 BCR-ABL transcript and provide an overview of their common characteristics. The index case (#1) was a 56 year old man diagnosed with CML in 1993. Therapy with busulfan and hydrea induced a complete hematologic remission (CHR) that was maintained until March 2001, when accelerated disease developed. Treatment with imatinib and subsequently AML-type chemotherapy were only transiently effective and the patient died from myeloid blast crisis in March 2003. Cytogenetics demonstrated a standard t(9;22)(q34;q11). Unexpectedly, FISH was consistent with an m-bcr rearrangement and multiplex PCR showed an unusually large band. Sequencing identified a fusion between BCR exon e8 and ABL exon a2, with a 55 bp insert corresponding to an inverted segment of ABL intron Ib (corresponding to nt 29861–29915, Genbank U07561). Immunoblotting of bone marrow mononuclear cells with anti-ABL antibody identified a 200 kDa protein (p200BCR-ABL).Seven additional patients with an e8a2 BCR-ABL fusion have been identified (table 1). These patients tend to have relatively high platelet counts (median 569 x 109/L, range 216 – 1123) and relatively low white counts (median 42 x 109/L, range 4.7 – 146) but no other distinguishing characteristics at diagnosis. With a median follow-up of 36 months (range, 4 –97), two patients had progressed to blast crisis and died, one died from an unrelated cause, and five were alive, one after allogeneic BMT. Remarkably, none of the patients treated with interferon-a achieved even a minor cytogenetic remission, similar to patients with p190-positive CML. Two patients besides case #1 have received imatinib, one has achieved a CHR at four months, and information on the other is not available. Compared to P190 and P210, p200 lacks the pleckstrin homology (PH) domain but retains the Dbl-like and CDC24 homology domains of BCR. Deletion of the PH domain may disrupt the function of the Dbl-like domain to serve as a GTP exchange factor for Rac, Cdc42 and Rho. This may lead to disruption of the actin cytoskeleton and would be expected to cause a disease that is biologically closer to p190 than to p210. [Display omitted]

P38 MAPK-mediated activation of NF-kappaB by the RhoGEF domain of Bcr.

The oncogenic fusion protein p210 Bcr-Abl is causally associated with virtually all cases of chronic myelogenous leukemia. The wild-type Bcr product has several recognizable structural and functional motifs including a domain that contains guanine nucleotide exchange activity for Rho family GTPases (DH/PH domain). Although this domain is retained within p210 Bcr-Abl, it has no known signaling activities in vivo. Here we report that a fragment of Bcr that encodes the isolated DH/PH domain is a potent activator of the NF-kappaB transcription factor. Within the context of full length Bcr, this activity is regulated by proximal flanking sequences that suppress the DH/PH domain encoded guanine nucleotide exchange activity. NF-kappaB activation by Bcr is not mediated by nuclear translocation, but rather by p38 mitogen-activated protein kinase (MAPK)-dependent modification of the RelA/p65 transactivation domain. Although we were able to demonstrate that Bcr can function as an exchange factor for Cdc42 in vivo, NF-kappaB activation appears to occur via a Cdc42-independent mechanism. These studies constitute direct evidence that the Bcr RhoGEF domain can function in vivo, and identify a new signaling activity that may contribute to the transforming potential of p210 Bcr-Abl.

CHRONIC NEUTROPHILIC LEUKAEMIA: A DISTINCT CLINICAL ENTITY?

CHRONIC NEUTROPHILIC LEUKAEMIA: A DISTINCT CLINICAL ENTITY?

The Dbl Homology Domain of BCR Is Not a Simple Spacer in P210BCR-ABL of the Philadelphia Chromosome

The Dbl homology (DH) domain of BCR in P210BCR-ABL (P210/WT) has been thought to have a negative effect on the activation of BCR-ABL because P185BCR-ABL, in which this region is physically deleted, has stronger biochemical and biological activities. To study the role of the DH domain of BCR in the background of P210/WT, the region was replaced with homologous sequences derived from Dbl (P210/Dbl) or CDC24 (P210/CDC24) or with irrelevant sequences from LacZ (P210/LacZ) or luciferase (P210/Luci). Surprisingly, the abilities to transform Rat1 cells or mouse bone marrow cells and induce growth factor independence in interleukin 3-dependent mouse Ba/F3 cells were retained only in P210/Dbl. However, even P210/Dbl could not achieve the wild type level of surviving potential against genotoxins in Rat1 cells and in Ba/F3 cells. Activation of Akt correlated with the biological changes in Rat1 cells but did not correlate with the biological changes in Ba/F3 cells. The DH domain was not tyrosine-phosphorylated in vitro, nor could we find any differences in peptide mapping between in vitro phosphorylated P210/WT and P210/Dbl. Although functions of the DH domain remain to be discovered, we propose that the DH domain makes positive contributions to P210BCR-ABL.

Bcl-2 expression restores the leukemogenic potential of a BCR/ABL mutant defective in transformation

AbstractGrowth factor–dependent hematopoietic cell lines expressing the BCR/ABL oncoprotein of the Ph chromosome show growth factor–independent proliferation and resistance to apoptosis. Apoptosis resistance of BCR/ABL-expressing cells may depend on enhanced expression of anti-apoptotic proteins as well as reduced expression and/or inactivation of pro-apoptotic proteins. Compared to myeloid precursor 32Dcl3 cells expressing wild type BCR/ABL, cells expressing a BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185ΔBCR) are susceptible to apoptosis induced by interleukin-3 (IL-3) deprivation. These cells exhibited the hypophosphorylated apoptotic BAD and markedly reduced levels of Bcl-2. Upon ectopic expression of Bcl-2, these cells showed no changes in BAD phosphorylation, but they became apoptosis-resistant and proliferated in the absence of IL-3, albeit more slowly than cells expressing wild type BCR/ABL. Moreover, the p185ΔBCR/Bcl-2 double transfectants were leukemogenic when injected into immunodeficient mice, but Bcl-2 expression did not restore the leukemia-inducing effects of p185ΔBCR to the levels of wild type BCR/ABL. Leukemic cells recovered from the spleen of mice injected with p185ΔBCR/Bcl-2 cells did not show rearrangements in the Bcl-2 genomic locus, but they exhibited enhanced proliferation in culture and induced a rapidly fatal disease process when inoculated in secondary recipient mice. Together, these data support the importance of anti-apoptotic pathways for BCR/ABL-dependent leukemogenesis and suggest that Bcl-2 expression promotes secondary changes leading to a more aggressive tumor phenotype.

Bcl-2 expression restores the leukemogenic potential of a BCR/ABL mutant defective in transformation

Growth factor-dependent hematopoietic cell lines expressing the BCR/ABL oncoprotein of the Ph chromosome show growth factor-independent proliferation and resistance to apoptosis. Apoptosis resistance of BCR/ABL-expressing cells may depend on enhanced expression of anti-apoptotic proteins as well as reduced expression and/or inactivation of pro-apoptotic proteins. Compared to myeloid precursor 32Dcl3 cells expressing wild type BCR/ABL, cells expressing a BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185 delta BCR) are susceptible to apoptosis induced by interleukin-3 (IL-3) deprivation. These cells exhibited the hypophosphorylated apoptotic BAD and markedly reduced levels of Bcl-2. Upon ectopic expression of Bcl-2, these cells showed no changes in BAD phosphorylation, but they became apoptosis-resistant and proliferated in the absence of IL-3, albeit more slowly than cells expressing wild type BCR/ABL. Moreover, the p185 delta BCR/Bcl-2 double transfectants were leukemogenic when injected into immunodeficient mice, but Bcl-2 expression did not restore the leukemia-inducing effects of p185 delta BCR to the levels of wild type BCR/ABL. Leukemic cells recovered from the spleen of mice injected with p185 delta BCR/Bcl-2 cells did not show rearrangements in the Bcl-2 genomic locus, but they exhibited enhanced proliferation in culture and induced a rapidly fatal disease process when inoculated in secondary recipient mice. Together, these data support the importance of anti-apoptotic pathways for BCR/ABL-dependent leukemogenesis and suggest that Bcl-2 expression promotes secondary changes leading to a more aggressive tumor phenotype. (Blood. 2000;96:3915-3921)

The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein.

The previously uncharacterized CDC24 homology domain of BCR, which is missing in the P185 BCR-ABL oncogene of Philadelphia chromosome (Ph1)-positive acute lymphocytic leukemia but is retained in P210 BCR-ABL of chronic myelogeneous leukemia, was found to bind to the xeroderma pigmentosum group B protein (XPB). The binding appeared to be required for XPB to be tyrosine-phosphorylated by BCR-ABL. The interaction not only reduced both the ATPase and the helicase activities of XPB purified in the baculovirus system but also impaired XPB-mediated cross-complementation of the repair deficiency in rodent UV-sensitive mutants of group 3. The persistent dysfunction of XPB may in part underlie genomic instability in blastic crisis.

Haemopoietic transformation by the TEL/ABL oncogene.

Rare, novel forms of activated ABL kinase, the result of a fusion between TEL (or ETV6, a member of the ETS transcription factor family), and the non-receptor tyrosine kinase ABL, have been identified. We have analysed the TEL/ABL fusion protein (type A) cloned from an acute lymphoblastic leukaemia patient. In contrast to a second TEL/ABL fusion (type B) identified in two cases of myeloid leukaemia, the portion of TEL contained in the type A TEL/ABL fusion was smaller and did not contain a potential Grb2 binding site. The type A TEL/ABL cDNA we used in this study encoded a 155 kD protein with elevated tyrosine kinase activity and was responsible for the phosphorylation of a number of proteins in vivo. Its expression in factor-dependent murine haemopoietic precursor cells efficiently converted these cells to factor independence for both survival and growth. These cells continued to express high levels of myc mRNA after growth factor depletion. We also demonstrated that type A TEL/ABL self-associated in stably expressing haemopoietic cells. Although the TEL portion of the TEL/ABL fusion protein has no sequence similarity to that of BCR in the BCR/ABL protein, all forms of these fusion proteins contain a structure implicated in oligomerization. Our results support the conclusion that the protein interaction domain of BCR and TEL, but not the Grb2 binding site, are the important functional components in the activation of ABL kinase in haemopoietic discase.