Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the in vitro differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α5β1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27Kip1. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27Kip1, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.

Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity

β1-integrin engagement on normal (NL) CD34+ cells increases levels of the cyclin-dependent kinase inhibitor (cdki), p27Kip, decreases cdk2 activity, and inhibits G1/S-phase progression. In contrast, β1-integrin engagement on chronic myelogenous leukemia (CML) CD34+ cells does not inhibit G1/S progression. We now show that, in CML, baseline p27Kip levels are significantly higher than in NL CD34+ cells, but adhesion to fibronectin (FN) does not increase p27Kip levels. p27Kip mRNA levels are similar in CML and NL CD34+ cells and remain unchanged after adhesion, suggesting posttranscriptional regulation. Despite the elevated p27Kip levels, cdk2 kinase activity is similar in CML and NL CD34+ cells. In NL CD34+ cells, >90% of p27Kip is located in the nucleus, where it binds to cdk2 after integrin engagement. In CML CD34+ cells, however, >80% of p27Kip is located in the cytoplasm even in FN-adherent cells, and significantly less p27Kip is bound to cdk2. Thus, presence of BCR/ABL induces elevated levels of p27Kip and relocation of p27Kip to the cytoplasm, which contributes to the loss of integrin-mediated proliferation inhibition, characteristic of CML.

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth.

Tumorigenic activity of the BCR-ABL oncogenes is mediated by BCL2.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.

Alterações genéticas com interesse prognóstico nas doenças mieloproliferativas crónicas bcr-abl negativas

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The analysis of Bcr-Abl—Nox signalling in chronic myeloid leukaemia

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterised by increased proliferation of haematopoietic stem cells. CML results following generation of the chimeric protein Bcr-Abl, a constitutively active tyrosine kinase which induces oncogenesis in part by promoting increased cell survival and proliferation. Since the development of Bcr-Abl-specific tyrosine kinase inhibitors (TKIs) there has been a substantial improvement in the clinical treatment of CML. Unfortunately, residual disease and the development of TKI resistance has become an ever growing concern, resulting in the need for a greater understanding of the disease in order to develop new treatment strategies. Interestingly, constitutive expression of the Bcr-Abl in CML is known to produce elevated levels of Reactive Oxygen Species (ROS) which are known to influence a variety of cellular processes. Previous studies have demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, enhancing survival signalling. The objective of this study was to elucidate how Nox protein activity was influenced downstream of Bcr-Abl while examining how Nox-derived ROS influenced CML disease phenotype to identify the potential in targeting these proteins to improve CML treatment. These studies demonstrated that inhibition of Bcr-Abl signalling, led to a significant reduction in ROS levels which was concurrent with the GSK-3dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. siRNA knockdown of p22phox identified it to have a significant role in cellular proliferation and cell viability, demonstrating the importance of Nox protein activity in CML disease phenotype. Furthermore, removal of p22phox was demonstrated to make cells significantly more susceptible to Bcr-Abl-specific TKI treatment, while pharmacological silencing of Nox activity in combination with TKIs was demonstrated to produce substantial, synergistic increases in cell death through augmentation of apoptosis, demonstrating the therapeutic potential of targeting Nox proteins in combination with Bcr-Abl inhibition.

Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

Interstitial insertion of varying amounts of ABL-containing genetic material into chromosome 22 in Ph-negative CML.

We studied the cells from three selected patients with Ph-chromosome-negative chronic myeloid leukemia (CML) by Southern blotting, polymerase chain reaction, and in situ hybridization of informative probes to metaphase chromosomes. All three patients had rearrangement of M-BCR sequences in the BCR gene and expression of one or other of the mRNA species characteristic of Ph-positive CML. Leukemic metaphases studied after trypsin-Giemsa banding were indistinguishable from normal. The ABL probe localized both to chromosome 9 and 22 in each case. A probe containing 3' M-BCR sequences localized only to chromosome 22, and not to chromosome 9 as would be expected in Ph-positive CML. Two new probes that recognize different polymorphic regions distal to the ABL gene on chromosome 9 in normal subjects localized exclusively to chromosome 9 in two patients and to both chromosomes 9 and 22 in one patient. These results show that Ph-negative CML with BCR rearrangement is associated with insertion of a variable quantity of chromosome 9 derived material into chromosome 22q11; there is no evidence for reciprocal translocation of material from chromosome 22 to chromosome 9.

Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

Abl expression, tumour grade, and apoptosis in chondrosarcoma

AIMS: To determine whether Abl immunoreactivity correlates with grade and cell kinetics (apoptosis and mitosis) in chondrosarcoma.

METHODS: Sections from 16 chondrosarcomas were stained immunohistochemically using a polyclonal antibody to the c-Abl/Bcr-Abl oncoprotein. Apoptotic indices and mitotic indices were assessed in all tumours. Sections from 24 paraffin wax blocks of human fetal rib (gestational ages, 15-42 weeks) were also stained to determine whether the Abl protein is synthesised consistently throughout endochondral ossification.

RESULTS: Abl staining in immature fetal rib chondrocytes at all stages of development was predominantly nuclear, and 70% of cells showed moderate to strong staining. Abl immunoreactivity was minimal or absent in hypertrophic chondrocytes about to undergo apoptosis at the growth plate. There was strong Abl staining in grade 1 and grade 2 chondrosarcomas but staining was greatly reduced or absent in grade 3 chondrosarcomas. There was a very significant linear correlation between apoptotic index (mean, 0.68%; range, 0-3.2%) and mitotic index (mean, 0.23%; range, 0-0.9%), and both indices were significantly lower in grade 1 than in grade 2 and grade 3 chondrosarcomas.

CONCLUSIONS: These data suggest that abl gene expression is associated with differentiation and apoptosis inhibition in fetal and neoplastic chondrocytes. However, these putative effects cannot be ascribed solely to the Abl protein, because several additional factors contribute to the regulation of both differentiation and apoptosis.

Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT)

The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

Studies on the role of BCR in Philadelphia chromosome-positive leukemias

It is well established that the chimeric Bcr-Abl oncoprotein resulting from fusing 3$\sp\prime$ ABL sequences on chromosome 9 to 5$\sp\prime$ BCR sequences on chromosome 22 is the primary cause of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Although it is clear that the cis-Bcr sequence present within Bcr-Abl is able to activate the tyrosine kinase activity and F-actin binding capacity of Bcr-Abl which is critical for the transforming ability of BCR-ABL, the biological role of normal BCR gene product (P160 BCR) remains largely unknown. The previous finding by our lab that P160 BCR forms stable complexes with Bcr-Abl oncoprotein in Ph$\sp1$-positive leukemic cells implicated P160 BCR in the pathogenesis of Ph$\sp1$-positive leukemias. Here, we demonstrated that P160 BCR physically interacts with P210 BCR-ABL and become tyrosine phosphorylated when co-expressed with P210 BCR-ABL in COS1 cells while no tyrosine phosphorylation of P160 BCR can be detected when it is expressed alone. The results suggest that P160 BCR is a target for the Bcr-Abl tyrosine kinase. Although we were unable to detect stable physical interaction between P160 BCR and P145 c-ABL (Ib) in COS1 cells overexpressing both proteins, P160 BCR was phosphorylated on tyrosine residues when co-expressed with activated tyrosine kinase of P145 c-ABL (Ib). In addition, studies of tyrosine phosphorylation of BCR deletion mutants and 2-dimensional tryptic mapping of in vitro phosphorylated wild type and mutant (tyrosine to phenylalanine) Bcr-Abl indicated that tyrosine 177, 283 and 360 of Bcr represent some of the phosphorylation sites. Even though the significance of tyrosine phosphorylation of residues 283 and 360 of Bcr has not been determined, tyrosine phosphorylation of residue 177 within Bcr-Abl has been reported to be critical for its interaction with Grb2 molecule and subsequent activation of Ras signaling pathway. Here, we further demonstrated that tyrosine 177 phosphorylated P160 BCR is also able to bind to Grb2 molecule suggesting the role of P160 BCR in the Ras signaling pathway.^ Surprisingly, using 3$\sp\prime$ BCR antisense oligonucleotide to reduce the expression of P160 BCR without interfering with the expression of BCR-ABL resulted in increased growth or survival of B15 cells and M3.16 cells expressing either P185 BCR-ABL or P210 BCR-ABL respectively. The results provided strong arguments that P160 BCR may function as a negative regulator for cell growth.^ Considering all these results, we hypothesize that P160 BCR negatively regulate cell growth and tyrosine phosphorylation of P160 BCR turns off its growth suppressor function and turns on its growth stimulatory function. We further speculate that Bcr-Abl oncoprotein in leukemia cells stably interacts with and constitutively phosphorylates portions of P160 BCR converting it into a growth stimulatory state. In normal cells, the growth suppressor effects of P160 BCR could only be transiently and conditionally switched to growth stimulatory action by a strictly regulated cellular tyrosine kinase such as c-ABL. The model will be further discussed in the text. ^