Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.
Ph-positive leukemias are caused by the aberrant fusion of the BCR and ABL genes. Nilotinib is a selective Bcr/Abl tyrosine kinase inhibitor related to imatinib, which is widely used to treat chronic myelogenous leukemia. Because Ph-positive acute lymphoblastic leukemia only responds transiently to imatinib therapy, we have used mouse models to test the efficacy of nilotinib against lymphoblastic leukemia caused by the P190 form of Bcr/Abl.
After transplant of 10,000 highly malignant leukemic cells into compatible recipients, untreated mice succumbed to leukemia within 21 days, whereas mice treated with 75 mg/kg nilotinib survived significantly longer. We examined cells from mice that developed leukemia while under treatment for Bcr/Abl kinase domain point mutations but these were not detected. In addition, culture of such cells ex vivo showed that they were as sensitive as the parental cell line to nilotinib but that the presence of stromal support allowed resistant cells to grow out. Nilotinib also exhibited impressive anti-leukemia activity in P190 Bcr/Abl transgenic mice that had developed overt leukemia/lymphoma masses and that otherwise would have been expected to die within 7 days. Visible lymphoma masses disappeared within six days of treatment and leukemic cell numbers in peripheral blood were significantly reduced. Treated mice survived more than 30 days.
These results show that nilotinib has very impressive anti-leukemia activity but that lymphoblastic leukemia cells can become unresponsive to it both in vitro and in vivo through mechanisms that appear to be Bcr/Abl independent.
Although the pathogenesis of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is mainly related to the expression of the BCR–ABL1 fusion transcript, additional cooperating genetic lesions are supposed to be involved in its development and progression. Therefore, in an attempt to investigate the complex landscape of mutations, changes in expression profiles and alternative splicing (AS) events that can be observed in such disease, the leukemia transcriptome of a BCR–ABL1-positive ALL patient at diagnosis and at relapse was sequenced using a whole-transcriptome shotgun sequencing (RNA-Seq) approach. A total of 13.9 and 15.8 million sequence reads was generated from de novo and relapsed samples, respectively, and aligned to the human genome reference sequence. This led to the identification of five validated missense mutations in genes involved in metabolic processes (DPEP1, TMEM46), transport (MVP), cell cycle regulation (ABL1) and catalytic activity (CTSZ), two of which resulted in acquired relapse variants. In all, 6390 and 4671 putative AS events were also detected, as well as expression levels for 18 315 and 18 795 genes, 28% of which were differentially expressed in the two disease phases. These data demonstrate that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations potentially involved in ALL.
sequencing; ALL; BCR-ABL1
The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR.
We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient.
Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells.
Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre–B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre–B ALL and normal bone marrow pre–B cells by serial analysis of gene expression, many genes involved in pre–B cell receptor signaling are silenced in the leukemia cells. Although normal pre–B cells are selected for the expression of a functional pre–B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre–B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre–B cell receptor signaling and the expression of a truncated isoform of the pre–B cell receptor–associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre–) B cell receptor.
immunoglobulin; SLP65; differentiation; STI571; V(D)J recombination
In the Philadelphia positive bcr negative acute leukemias (Ph1+bcr- AL), the chromosomal breakpoints on chromosome 22 have been shown clustered within 10.8kb (bcr2) and 5kb (bcr3) fragments of the first intron of the BCR gene. We previously reported that the breakpoints were localized in Alu repeats on chromosomes 9 and 22 in a Ph1+bcr- acute lymphoblastic leukemia with a rearrangement involving bcr2. Molecular data of two other Ph1 translocations, one a Ph1+bcr- acute myeloblastic leukemia in the bcr2 region, and the other an acute lymphoblastic leukemia in the bcr3 region are presented. In the former, the breakpoints on chromosomes 9 and 22 are localized in Alu repeats, in regions with two inverted Alu sequences, as in our previously reported case. In the second leukemia, the breakpoints are not located in Alu sequences, but such repeats are found in their vicinity. The implications of these findings are discussed.
The translocation of the c-abl oncogene from chromosome 9 to the bcr gene on chromosome 22 in cases of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) generates an aberrant bcr-abl fusion transcript which may be intimately related to the pathogenesis of CML. Because factors controlling normal bcr expression might also be involved in the expression of this aberrant bcr-abl transcript, we studied the patterns of expression of the normal bcr gene in different cell types. We found that the normal bcr gene was expressed in many different types of human cells. Moreover, the bcr gene was evolutionarily conserved, and homologous bcr genomic sequences and RNA transcripts were readily detected in chick tissue. The highest level of bcr expression in chick tissue was in brain tissue, the lowest level was in liver tissue, and a truncated bcr mRNA was noted in chick testes. Normal bcr transcripts, in addition to the aberrant bcr-abl hybrid transcripts, have been found in all Philadelphia chromosome-positive CML cells studied to date. Within a given CML sample, the relative amounts of normal bcr RNA and aberrant bcr-abl RNA were similar. In addition, the normal bcr and the aberrant bcr-abl hybrid transcripts demonstrated similarly prolonged half-lives compared with that of the normal abl-related transcripts in CML cells. These findings suggest that in CML cells, similar cellular mechanisms control the steady-state levels of both the normal bcr and the bcr-abl fusion RNAs.
The BCR–ABL fusion kinase is the driving mutation of chronic myelogenous leukemias and is also expressed in a subset of acute lymphoblastic leukemias. Recent advances in elucidating the structure, regulation, and signaling of BCR–ABL have led to the identification of allosteric sites that are distant from the ATP-binding pocket and are critical for BCR–ABL–dependent oncogenic transformation. Here, we review the available data regarding the molecular mechanism of action and the specificity of ATP-competitive tyrosine kinase inhibitors targeting BCR–ABL. In addition, we discuss how targeting of allosteric sites could provide new opportunities to inhibit resistant BCR–ABL mutants, either alone or in combination with conventional ATP-competitive inhibitors.
This study aimed to verify the association between human leukocyte antigens and the bcr-abl fusion protein resulting from t(9;22)(q34;q11) in chronic leukemia myeloid and acute lymphoblastic leukemia patients.
Forty-seven bcr-abl positive individuals were evaluated. Typing was performed bymicrolymphocytotoxicity and molecular biological methods (human leukocyte antigens Class I and Class II). A control group was obtained from the data of potential bone marrow donors registered in the Brazilian Bone Marrow Donor Registry (REDOME).
Positive associations with HLA-A25 and HLA-B18 were found for the b2a2 transcript, as well as a tendency towards a positive association with HLA-B40 and a negative association with HLA-A68. The b3a2 transcript showed positive associations with HLA-B40 and HLA-DRB1*3.
The negative association between human leukocyte antigens and the BCR-ABL transcript suggests that binding and presentation of peptides derived from the chimeric protein are effective to increase a cytotoxic T lymphocyte response appropriate for the destruction of leukemic cells.
HLA antigens; Fusion proteins, BCR-ABL; Genetic predisposition to disease; Philadelphia chromosome; Leukemia, myelogenous, chronic, BCR-ABL positive; Precursor cell lymphoblastic leukemia-lymphoma
Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.
The Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. ALL cells are derived from B cell precursors in most cases and typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on activation-induced cytidine deaminase (AID). Studying AID expression in 108 cases of ALL, we detected AID mRNA in 24 of 28 Ph+ ALLs as compared with 6 of 80 Ph− ALLs. Forced expression of BCR-ABL1 in Ph− ALL cells and inhibition of the BCR-ABL1 kinase showed that aberrant expression of AID depends on BCR-ABL1 kinase activity. Consistent with aberrant AID expression in Ph+ ALL, IGH V region genes and BCL6 were mutated in many Ph+ but unmutated in most Ph− cases. In addition, AID introduced DNA single-strand breaks within the tumor suppressor gene CDKN2B in Ph+ ALL cells, which was sensitive to BCR-ABL1 kinase inhibition and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1–induced mutator in Ph+ ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.
Hematopoietic myeloproliferative neoplasms (MPNS) with rearrangements of the receptor tyrosine kinase FGFR1 gene, located on chromosome 8p11, are uncommon and associated with diverse presentations such as atypical chronic myeloid leukemia, acute myeloid leukemia, or an acute T- or B-lymphoblastic leukemia, reflecting the hematopoietic stem cell origin of the disease. A review of MPN patients with the t(8;22) translocation that results in a chimeric BCR-FGFR1 fusion gene reveals that this disease either presents or rapidly transforms into an acute leukemia that is generally unresponsive to currently available chemotherapeutic regimens including tyrosine kinase inhibitors (TKIS). The first case of a rare BCR-FGFR1 MPN presenting in a B-acute lymphoblastic phase who underwent allogeneic hematopoietic stem cell transplantation (HSCT) with a subsequent sustained complete molecular remission is described. Allogeneic HSCT is currently the only available therapy capable of achieving long-term remission in BCR-FGFR1 MPN patients.
Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to completely eradicate Bcr-Abl+ leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl+ leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib in CML cells. This screen identified numerous components of a Wnt/Ca2+/NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl+ acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl+ leukemias.
The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. In this study we report the frequencies of BCR-ABL1 fusion transcript variants studied in 43 CML patients from Sudan. The study includes 46 Sudanese patients, three of which negative for the BCR-ABL1 fusion transcript. More than half of 43 positive patients showed b2a2 fusion transcript (53.5%), while (41.9%) showed b3a2 transcript and the remaining (4.6%) coexpression of b3a2/ b2a2 and b3a2/b2a2/e19a2. We detected neither coexpression of p210/p190 nor e1a2 alone. Male patients showed a tendency to express b2a2, while female tende to express b3a2 (p = 0.017). Moreover, a single nucleotide polymorphism was detected in BCR exon 13 in one out of four patients and this patient showed only b2a2 expression. In conclusion, we observed a significant correlation between sex and type of BCR-ABL1 transcript, an observation that deserves further investigation.
BCR-ABL; chronic myeloid leukemia; Ph chromosome; Sudanese; RT-PCR
The aims of this study were to estimate the incidences of BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions in childhood acute lymphoblastic leukemia (ALL), to identify new abnormalities, and to demonstrate the usefulness of interphase fluorescence in situ hybridization (FISH). We performed G-banding analysis and FISH using probes for BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions on 65 childhood ALL patients diagnosed and uniformly treated at a single hospital. Gene rearrangements were identified in 73.8% of the patients using the combination of G-banding and FISH, while the chromosomal abnormalities were identified in 49.2% using G-banding alone. Gene rearrangements were disclosed by FISH in 24 (72.7%) of 33 patients with normal karyotype or no mitotic cell in G-banding. Among the gene rearrangements detected by FISH, the most common gene rearrangement was p16 deletion (20.3%) and the incidences of others were 14.1% for TEL/AML1, 11.3% for MLL, and 1.8% for BCR/ABL translocations. Infrequent or new aberrations such as AML1 amplification, MLL deletion, ABL deletion, and TEL/AML1 fusion with AML1 deletion were also observed. We established the rough incidences of gene rearrangements in childhood ALL, found new abnormalities and demonstrated the diagnostic capability of interphase FISH to identify cryptic chromosome aberrations.
In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Acute; Childhood; Gene Rearrangements
Based on the site of breakpoint in t(9;22) (q34;q11), bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules.
The study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test.
Significant negative associations (p < 0.05) were observed with HLA-A*02 (b2a2, e1a2), -A*68 (b2a2, b3a2, e1a2), -B*14 (b2a2, b3a2, e1a2), -B*15 (b2a2, b3a2), -B*40 (b2a2), -DQB1*0303 (b2a2, b3a2), -DQB1*0603 (b2a2), -DRB1*0401 (e1a2), -DRB1*0701 (b3a2), and -DRB1*1101 (b2a2).
The negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22) (q34;q11)-positive leukemia.
PECAM-1 (CD31) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing surface glycoprotein expressed on various hematopoietic cells as well as on endothelial cells. PECAM-1 has been shown to play roles in regulation of adhesion, migration and apoptosis. The BCR/ABL fusion tyrosine kinase is expressed in chronic myeloid leukemia and Philadelphia-positive (Ph+) acute lymphoblastic leukemia cells, and its inhibition by the clinically used tyrosine kinase inhibitors imatinib or dasatinib induces apoptosis of these cells. In the present study, we demonstrate that PECAM-1 is tyrosine phospho rylated in its ITIM motifs in various BCR/ABL-expressing cells including primary leukemia cells. Studies using imatinib and dasatinib as well as transient expression experiments in 293T cells revealed that PECAM-1 was phosphorylated directly by BCR/ABL, which was enhanced by the imatinib-resistant E255K and T315I mutations, or partly by the Src family tyrosine kinases, including Lyn, which were activated dependently or independently on BCR/ABL. We also demonstrate by using a substrate trapping mutant of SHP2 that tyrosine phosphorylated PECAM-1 binds SHP2 and is a major substrate for this tyrosine phosphatase in BCR/ABL-expressing cells. Overexpression of PECAM-1 in BCR/ABL-expressing cells, including K562 human leukemia cells, enhanced cell adhesion and partially inhibited imatinib-induced apoptosis involving mitochondria depolarization and caspase-3 cleavage, at least partly, in an ITIM-independent manner. These data suggest that PECAM-1 may play a role in regulation of apoptosis as well as adhesion of BCR/ABL-expressing cells to modulate their imatinib sensitivity and would be a possible candidate for therapeutic target in Ph+ leukemias.
PECAM-1; BCR/ABL; imatinib; apoptosis; leukemia
In recent years, the panel of known molecular mutations in acute lymphoblastic leukemia (ALL) has been continuously increased. In Philadelphia-positive ALL, deletions of the IKZF1 gene were identified as prognostically adverse factors. These improved insights in the molecular background and the clinical heterogeneity of distinct cytogenetic subgroups may allow most differentiated therapeutic decisions, for example, with respect to the indication to allogeneic HSCT within genetically defined ALL subtypes. Quantitative real-time PCR allows highly sensitive monitoring of the minimal residual disease (MRD) load, either based on reciprocal gene fusions or immune gene rearrangements. Molecular diagnostics provided the basis for targeted therapy concepts, for example, combining the tyrosine kinase inhibitor imatinib with chemotherapy in patients with Philadelphia-positive ALL. Screening for BCR-ABL1 mutations in Philadelphia-positive ALL allows to identify patients who may benefit from second-generation tyrosine kinase inhibitors or from novel compounds targeting the T315I mutation. Considering the central role of the molecular techniques for the management of patients with ALL, efforts should be made to facilitate and harmonize immunophenotyping, cytogenetics, and molecular mutation screening. Furthermore, the potential of high-throughput sequencing should be evaluated for diagnosis and follow-up of patients with B-lineage ALL.
Chromosomal translocations resulting in chimeric fusion genes are prototypic for pediatric leukemia patients. The most known fusions are ETV6-RUNX1 or BCR-ABL1 in B-cell progenitor (BCP)-ALL, and rearrangements of MLL in pediatric ALL and AML. Genome-wide sequencing projects have revealed additional, recurrent gene mutations in B cell malignancies. One of these mutations comprises the IKZF1 gene, encoding the IKAROS transcription factor which is one of the essential transcription factors driving lymphoid development. IKZF1 deletions were first identified by SNP arrays in ALL patients, and later identified with a high prevalence in BCR-ABL1+ patients. IKZF1 deletions turned out to be an independent prognostic marker associated with a poor outcome. Here, we characterized IKZF1 deletions in pediatric BCP-ALL patients by combining MLPA mapping experiments with long distance inverse PCR. The aim of our study was also to compare existing methods with our approach. Our attempt confirmed many of the existing data but revealed a more complex pattern of recombination sites, including a total of 4 recombination hotspots. This extended knowledge was translated into a novel, multiplex PCR assay that allows to perform IKZF1 deletion analyses by using a 2-tube PCR approach.
Childhood leukemia; cancer genetics; gene deletion; IKAROS; IKZF1; leukemia markers
We report here on a rare case of BCR-ABL1-negative atypical chronic myeloid leukemia with a t(9;22)(p24;q11.2)translocation and a BCR-JAK2 fusion gene, with resistance to the tyrosine kinase inhibitors imatinib and dasatinib.At two years of follow-up, the patient showed no hematologic response and was submitted to an allogeneic bonemarrow transplantation. Fifty-three days after the procedure, he died due to acute graft-versus-host disease. This BCR-JAK2 fusion gene has so far been found in only five patients in the whole world, with three clinical presentations: myeloproliferative neoplasm, acute lymphoblastic leukemia and acute myeloid leukemia.
Leukemia, myeloid, chronic, atypical, BCR-ABL negative; Myelodysplastic-myeloproliferative diseases; Proto-oncogene proteins c-bcr
Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling.
Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1.
Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.
The nonreceptor tyrosine kinases Abl and Arg are among the most well-characterized tyrosine kinases in the human genome. The activation of Abl by N-terminal fusions with Bcr (Bcr-Abl) or Gag (v-Abl) is responsible for chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia and mouse leukemia virus, respectively. In addition, aberrant Abl and Arg activation downstream of several oncogenic growth factor receptors contributes to the development and progression of a variety of human cancers, often associated with poor clinical outcome, drug resistance, and tumor invasion and metastasis. Abl activation can occur by a variety of mechanisms that include domain interactions involving structural remodeling of autoinhibited conformations as well as direct phosphorylation by upstream kinases and phosphatases. Constitutive activation of Abl plays a significant role in regulating the actin cytoskeleton by modulating cell adhesion, motility, and invadopodia. This review addresses the role of Abl and Arg in tumor progression with particular emphasis on the roles of Crk and Abi1 adapter proteins as distinct molecular switches for Abl transactivation. These insights, combined with new insights into the structure of these kinases, provide the rationale to envision that Crk and Abi1 fine-tune Abl regulation to control signaling to the cytoskeleton.
Crk; Abi1; Abl kinase; cytoskeleton; WAVE complex
Chronic myeloid leukemia (CML) is initially driven by the bcr–abl fusion oncoprotein. The identification of bcr–abl led to the discovery and rapid translation into the clinic of bcr–abl kinase inhibitors. Although, bcr–abl inhibitors are efficacious, experimental evidence indicates that targeting bcr–abl is not sufficient for elimination of minimal residual disease found within the bone marrow (BM). Experimental evidence indicates that the failure to eliminate the leukemic stem cell contributes to persistent minimal residual disease. Thus curative strategies will likely need to focus on strategies where bcr–abl inhibitors are given in combination with agents that specifically target the leukemic stem cell or the leukemic stem cell niche. One potential target to be exploited is the Janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) pathway. Recently using STAT3 conditional knock-out mice it was shown that STAT3 is critical for initiating the disease. Interestingly, in the absence of treatment, STAT3 was not shown to be required for maintenance of the disease, suggesting that STAT3 is required only in the tumor initiating stem cell population (Hoelbl et al., 2010). In the context of the BM microenvironment, STAT3 is activated in a bcr–abl independent manner by the cytokine milieu. Activation of JAK/STAT3 was shown to contribute to cell survival even in the event of complete inhibition of bcr–abl activity within the BM compartment. Taken together, these studies suggest that JAK/STAT3 is an attractive therapeutic target for developing strategies for targeting the JAK–STAT3 pathway in combination with bcr–abl kinase inhibitors and may represent a viable strategy for eliminating or reducing minimal residual disease located in the BM in CML.
STAT3; bone marrow microenvironment; drug resistance; transformation; chronic myeloid leukemia
Chromosomal translocations generating the BCR-ABL oncogene cause chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia. The BCR-ABLT315I mutation confers drug resistance to FDA-approved targeted therapeutics imatinib mesylate, dasatinib, and nilotinib. We tested the ability of a recombinant yeast-based vaccine expressing the T315I-mutated BCR-ABL antigen to stimulate an anti-BCR-ABLT315I immune response. The yeast-based immunotherapy significantly reduced or eliminated BCR-ABLT315I leukemia cells from the peripheral blood of immunized animals and extended leukemia-free survival in a murine model of BCR-ABL+ leukemia compared to animals receiving sham injection or yeast expressing ovalbumin. With immunization, leukemic cells harboring BCR-ABLT315I were selectively eliminated after challenge with a mixed population of BCR-ABL and BCR-ABLT315I leukemias. In summary, yeast-based immunotherapy represents a novel approach against the emergence of cancer drug resistance by the pre-emptive targeted ablation of tumor escape mutants.
T315I; BCR-ABL; Immunotherapy
The use of a DNA vaccine encoding the BCR/ABL fusion gene is thought to be a promising approach for patients with chronic myeloid leukemia (CML) to eradicate minimal residual disease after treatment with chemotherapy or targeted therapy. In this study, our strategy employs genetic technology to create a DNA vaccine encoding the BCR/ABL fusion and human interleukin-2 (hIL-2) genes. The successfully constructed plasmids BCR/ABL-pIRES-hIL-2, BCR/ABL-pIRES, and pIRES-hIL-2 were delivered intramuscularly to BALB/c mice at 14-day intervals for three cycles. The transcription and expression of the BCR/ABL and hIL-2 genes were found in the injected muscle tissues. The interferon-γ (IFN-γ) serum levels were increased, and the splenic CD4+/CD8+ T cell ratio was significantly decreased in the BCR/ABL-pIRES-hIL-2-injected mice. Furthermore, specific antibodies against K562 cells could be detected by indirect immunofluorescence. These results indicate that a DNA vaccine containing BCR/ABL and hIL-2 together may elicit increased in vivo humoral and cellular immune responses in BALB/c mice.