MiR-125a-3p and MiR-320b Differentially Expressed in Patients with Chronic Myeloid Leukemia Treated with Allogeneic Hematopoietic Stem Cell Transplantation and Imatinib Mesylate.
Chronic myeloid leukemia (CML), a hematopoietic neoplasm arising from the fusion of BCR (breakpoint cluster region) gene on chromosome 22 to the ABL (Abelson leukemia virus) gene on chromosome 9 (BCR-ABL1 oncogene), originates from a small population of leukemic stem cells with extensive capacity for self-renewal and an inflammatory microenvironment. Currently, CML treatment is based on tyrosine kinase inhibitors (TKIs). However, allogeneic hematopoietic stem cell transplantation (HSCT-allo) is currently the only effective treatment of CML. The difficulty of finding a compatible donor and high rates of morbidity and mortality limit transplantation treatment. Despite the safety and efficacy of TKIs, patients can develop resistance. Thus, microRNAs (miRNAs) play a prominent role as biomarkers and post-transcriptional regulators of gene expression. The aim of this study was to analyze the miRNA profile in CML patients who achieved cytogenetic remission after treatment with both HSCT-allo and TKI. Expression analyses of the 758 miRNAs were performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Bioinformatics tools were used for data analysis. We detected miRNA profiles using their possible target genes and target pathways. MiR-125a-3p stood out among the downregulated miRNAs, showing an interaction network with 52 target genes. MiR-320b was the only upregulated miRNA, with an interaction network of 26 genes. The results are expected to aid future studies of miRNAs, residual leukemic cells, and prognosis in CML.
Publication Date: 2021-10-14
Journal: International journal of molecular sciences
NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation.
The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 μM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.
Publication Date: 2020-08-12
Journal: PloS one
Mutations in the BCR-ABL1 kinase domain in patients with chronic myeloid leukaemia treated with TKIs or at diagnosis.
The aim of the present study was to analyse the incidence of mutations in the BCR-ABL1 kinase region in patients with newly diagnosed or treated chronic myeloid leukaemia (CML), and the association between mutations clinicopathological characteristics. Samples were collected for mutation analysis from patients who exhibited tyrosine kinase inhibitor resistance following treatment or were in the accelerated or blast phase at diagnosis. The mutations in the breakpoint cluster region (BCR)-ABL proto-oncogene 1 (ABL1) kinase domain were evaluated using conventional sequencing or ultra-deep sequencing (UDS) of peripheral blood samples. Sanger sequencing and UDS of the cDNA region corresponding to the BCR-ABL1 kinase domain was performed. χ
Publication Date: 2020-07-30
Journal: Oncology letters
Optimal Response in a Patient With CML Expressing
The Philadelphia chromosome is considered the hallmark of chronic myeloid leukemia (CML). However, although most patients with CML are diagnosed with the e13a2 or e14a2 breakpoint cluster region (BCR)-Abelson 1 (ABL1) fusion transcripts, about 5% of them carry rare BCR-ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3 and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases; there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype.
We describe a case of atypical BCR-ABL1 e6a2 fusion transcript in a 46-year-old woman with CML.
The use of primers recognizing more distant exons from the common BCR-ABL1 breakpoint region correctly identified the atypical BCR-ABL1 e16a2 fusion transcript. Treatment with second-generation tyrosine kinase inhibitor nilotinib was effective in this patient expressing the atypical e6a2 BCR-ABL1 fusion transcript.
Publication Date: 2020-05-02
Journal: In vivo (Athens, Greece)
Chronic myeloid leukemia: 2020 update on diagnosis, therapy and monitoring.
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with an incidence of 1-2 cases per 100 000 adults. It accounts for approximately 15% of newly diagnosed cases of leukemia in adults.
CML is characterized by a balanced genetic translocation, t(9;22)(q34;q11.2), involving a fusion of the Abelson gene (ABL1) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is known as the Philadelphia chromosome. The molecular consequence of this translocation is the generation of a BCR-ABL1 fusion oncogene, which in turn translates into a BCR-ABL oncoprotein.
Four tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, dasatinib, and bosutinib are approved by the United States Food and Drug Administration for first-line treatment of newly diagnosed CML in chronic phase (CML-CP). Clinical trials with second generation TKIs reported significantly deeper and faster responses, but they had no impact on survival prolongation, likely because of the existence of highly effective salvage therapies for patients who have a cytogenetic relapse with frontline TKI.
For CML post failure on frontline therapy, second-line options include second and third generation TKIs. Although potent and selective, these exhibit unique pharmacological profiles and response patterns relative to different patient and disease characteristics, such as patients' comorbidities, disease stage, and BCR-ABL1 mutational status. Patients who develop the T315I "gatekeeper" mutation display resistance to all currently available TKIs except ponatinib. Allogeneic stem cell transplantation remains an important therapeutic option for patients with CML-CP who have failed at least 2 TKIs, and for all patients in advanced phase disease. Even among older patients who have a cytogenetic relapse post failure on all TKIs, they can maintain long-term survival if they continue on a daily most effective/less toxic TKI, with or without the addition of non-TKI anti-CML agents (hydroxyurea, omacetaxine, azacitidine, decitabine, cytarabine, busulfan, others).
Publication Date: 2020-04-03
Journal: American journal of hematology
Comparison of Real-Time Quantitative PCR and Digital Droplet PCR for BCR-ABL1 Monitoring in Patients with Chronic Myeloid Leukemia.
Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.
Publication Date: 2019-11-02
Journal: The Journal of molecular diagnostics : JMD
FAM168A participates in the development of chronic myeloid leukemia via BCR-ABL1/AKT1/NFκB pathway.
Although the prognosis of chronic myeloid leukemia (CML) has dramatically improved, the pathogenesis of CML remains elusive. Studies have shown that sustained phosphorylation of AKT1 plays a crucial role in the proliferation of CML cells. Evidence indicates that in tongue cancer cells, FAM168A, also known as tongue cancer resistance-associated protein (TCRP1), can directly bind to AKT1 and regulate AKT1/NFκB signaling pathways. This study aimed to investigate the role of FAM168A in regulation of AKT1/NFκB signaling pathway and cell cycle in CML.
FAM168A interference was performed, and the expression and phosphorylation of FAM168A downstream proteins were measured in K562 CML cell line. The possible roles of FAM168A in the proliferation of CML cells were investigated using in vitro cell culture, in vivo animal models and clinical specimens.
We found that the expression of FAM168A significantly increased in the peripheral blood mononuclear cells of CML patients, compared with normal healthy controls. FAM168A interference did not change AKT1 protein expression, but significantly decreased AKT1 phosphorylation, significantly increased IκB-α protein level, and significantly reduced nuclear NFκB protein level. Moreover, there was a significant increase of G2/M phase cells and Cyclin B1 level. Immunoprecipitation results showed that FAM168A interacts with breakpoint cluster region (BCR) -Abelson murine leukemia (ABL1) fusion protein and AKT1, respectively. Animal experiments confirmed that FAM168A interference prolonged the survival and reduced the tumor formation in mice inoculated with K562 cells. The results of clinical specimens showed that FAM168A expression and AKT1 phosphorylation were significantly elevated in CML patients.
This study demonstrates that FAM168A may act as a linker protein that binds to BCR-ABL1 and AKT1, which further mediates the downstream signaling pathways in CML. Our findings demonstrate that FAM168A may be involved in the regulation of AKT1/NFκB signaling pathway and cell cycle in CML.
Publication Date: 2019-07-12
Journal: BMC cancer
Genetic Mutations in a Patient with Chronic Myeloid Leukemia Showing Blast Crisis 10 Years After Presentation.
Since the introduction of tyrosine kinase inhibitors (TKI), the prospects for patients with chronic myeloid leukemia (CML) have improved significantly. Herein we present the case of a patient with CML who experienced blast crisis and development of acute myeloid leukemia (AML) 10 years after presentation. The CML was characterized by the gene fusion of breakpoint cluster region BCR and tyrosine-protein kinase ABL1. During treatment different therapeutic protocols including imatinib, nilotinib, dasatinib and ponatinib were applied due to development of resistance or non-response. Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) were used to describe cytogenetic and molecular aberrations elucidating the development into AML: A loss of chromosome 7, as well as an arising frequency of variants in the gene met proto-oncogene MET (p.T110I) and tyrosine-protein phosphatase non-receptor type 11 PTPN11 (p.Q510L) was observed. This report describes the comprehensive characterization of a clinical case showing multiple therapeutic resistances correlated with genetic aberrations.
Publication Date: 2018-07-05
Journal: Anticancer research
Novel tyrosine-kinase inhibitors for the treatment of chronic myeloid leukemia: safety and efficacy.
Chronic myeloid leukemia (CML) is characterized by a pathognomonic chromosomal translocation, which leads to the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus 1 (ABL1) genes, generating an oncoprotein with deregulated tyrosine kinase activity. Areas covered: In the last two decades, BCR/ABL1 kinase has become the molecular target for tyrosine kinase inhibitors (TKIs), a class of drugs that impressively improved overall survival. Despite these results, a proportion of patients experiences resistance to TKIs and need to change treatment. Furthermore, TKIs are unable to eradicate leukemic stem cells, allowing the persistence of neoplastic clones. Therefore, there is still clinical need for new agents to overcome common resistance mechanisms to available drugs. This review explores the horizon of drugs actually under investigation for CML patients resistant to conventional treatment. Expert commentary: Radotinib is an ATP-competitive TKI that showed significant activity also in front-line setting and could find employment indications in CML. Asciminib, an allosteric ABL1 inhibitor, could demonstrate a higher capacity in overcoming common TKIs resistant mutations, including T315I, but clinical findings are needed. CML stem cell target will probably require new therapeutic strategies: combinations of several compounds acting with different mechanisms of action are actually under investigation.
Publication Date: 2018-03-10
Journal: Expert review of hematology
Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring.
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with an incidence of 1-2 cases per 100 000 adults. It accounts for approximately 15% of newly diagnosed cases of leukemia in adults.
CML is characterized by a balanced genetic translocation, t(9;22)(q34;q11.2), involving a fusion of the Abelson gene (ABL1) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is known as the Philadelphia chromosome. The molecular consequence of this translocation is the generation of a BCR-ABL1 fusion oncogene, which in turn translates into a BCR-ABL1 oncoprotein. Frontline therapy: Four tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, dasatinib, and bosutinib are approved by the United States Food and Drug Administration for first-line treatment of patients with newly diagnosed CML in chronic phase (CML-CP). Clinical trials with second generation TKIs reported significantly deeper and faster responses; this has not translated into improved long-term survival, because of the availability of effective salvage therapies. Salvage therapy: For patients who fail frontline therapy, second-line options include second and third generation TKIs. Second and third generation TKIs, although potent and selective, exhibit unique pharmacological profiles and response patterns relative to different patient and disease characteristics, such as patients' comorbidities, disease stage, and BCR-ABL1 mutational status. Patients who develop the T315I "gatekeeper" mutation display resistance to all currently available TKIs except ponatinib. Allogeneic stem cell transplantation remains an important therapeutic option for patients with CML-CP who have failed at least 2 TKIs, and for all patients in CML advanced phases.
Publication Date: 2018-02-08
Journal: American journal of hematology
Persistent STAT5-mediated ROS production and involvement of aberrant p53 apoptotic signaling in the resistance of chronic myeloid leukemia to imatinib.
The persistent activation of signal transducer and activator of transcription 5 (STAT5) may principally be attributed to breakpoint cluster region (BCR)-Abelson murine leukemia viral oncogene homolog 1 (ABL1), and have multi-faceted effects in the development of chronic myeloid leukemia (CML). The p53 protein network regulates important mechanisms in DNA damage repair, cell cycle regulation/checkpoints, and cell senescence and apoptosis, as demonstrated by its ability to positively regulate the expression of various pro-apoptotic genes, including B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). In the present study, it was observed that the mRNA levels of STAT5A and STAT5B were upregulated in patients with imatinib-resistant CML and in the imatinib-resistant K562/G CML cell line. In addition, increased expression of STAT5 was observed in the BCR-ABL1 mutation group, compared with that in the non-BCR-ABL1 mutation group, regardless of patient imatinib resistance state. Elevated levels of reactive oxygen species (ROS) and DNA double-strand breaks were identified in K562/G cells using flow cytometric and phosphorylated H2AX (γ-H2AX) foci immunofluorescence assays, respectively, compared with the imatinib-sensitive K562 cells. The levels of intracellular ROS and γ-H2AX were decreased by the ROS scavenger (N-acetylcysteine), and ROS levels were also markedly reduced by STAT5 inhibitor (SH-4-54). In addition, imatinib significantly inhibited the proliferation of K562 and K562/G cells, with half maximal inhibitory concentration values of 0.17±0.07 and 14.78±0.43 µM, respectively, and the levels of apoptosis were significantly different between K562 and K562/G cells following treatment with imatinib. The mRNA and protein levels of STAT5 and mouse double minute 2 homolog (MDM2) were upregulated, whereas those of Bax were downregulated in K562/G cells, as determined using western blot analysis. Additionally, although the two cell lines exhibited relatively low protein expression levels of p53, lower levels of p53 and TPp53BP1 transcripts were detected in the K562/G cells. Taken together, these findings suggest that the resistance of CML to the tyrosine kinase inhibitor, imatinib, may be associated with persistent STAT5-mediated ROS production, and the abnormality of the p53 pathway.
Publication Date: 2017-11-09
Journal: International journal of molecular medicine
Current paradigms in the management of Philadelphia chromosome positive acute lymphoblastic leukemia in adults.
Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL) is a biologically, clinically, and genetically distinct subtype of precursor-B ALL. The Ph chromosome, results from a reciprocal translocation of the ABL1 kinase gene on chromosome 9 to the breakpoint cluster region (BCR) gene on chromosome 22. Depending on the translocation breakpoint, typically a p210 BCR-ABL1 or a p190 BCR-ABL onc protein are generated; both are constitutively active tyrosine kinases that play a central role to alter signaling pathways of cell proliferation, survival, and self-renewal, leading to leukemogenesis. In Ph-positive ALL, the p190-BCR-ABL (minor [m]-bcr) subtype is more frequent than the p210-BCR-ABL (major [M]-bcr) subtype, commonly found in chronic myeloid leukemia. The Philadelphia chromosome is the most frequent recurrent cytogenetic abnormality in elderly patients with ALL. Its incidence increases with age, reaching ∼50% in patients with ALL aged 60 years and over. Patients traditionally had a very poor outcome with chemotherapy, particularly if they do not undergo allogeneic hematopoietic cell transplantation (allo-HCT) in first complete remission (CR1). With the availability of multiple tyrosine kinase inhibitors (TKI), the therapeutic armamentarium is expanding quickly. However, there is no consensus on how to best treat Ph-positive ALL. With modern therapy, improved outcomes have led to the emergence of a number of controversies, including the need for intensive chemotherapy, the ideal TKI, and whether all eligible patients should receive an allo-HSCT, and if so, what type. Here, we discuss these controversies in light of the available literature.
Publication Date: 2017-10-04
Journal: American journal of hematology
Influence of BCR-ABL Transcript Type on Outcome in Patients With Chronic-Phase Chronic Myeloid Leukemia Treated With Imatinib.
The prognostic significance of breakpoint cluster region gene-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) transcripts in chronic myeloid leukemia (CML) is still controversial.
All consecutive CML patients in chronic phase treated with imatinib in a single center were analyzed (n = 170). BCR-ABL1 transcript was evaluated using multiplex reverse transcription polymerase chain reaction. Exclusively patients with BCR-ABL transcripts e13a2 and/or e14a2 were included in this analysis.
Patients with e14a2 transcripts presented higher rates of optimal molecular responses at 3 months and higher rates of complete cytogenetic response (CCR) at 6 months. E13a2, e14a2, and e14a2 with e13a2 (e14a2+e13a2) groups presented similar rates of 5-year event-free, progression-free, and overall survival. There was a superior 10-year overall survival in patients with transcripts e13a2 compared with e14a2 (alone or coexpressed with e13a2; 93% vs. 73%; P = .03), although the 5-year overall survival was 96% vs. 88%, respectively (P = not significant). In a multivariate analysis, high/intermediate Sokal score and e14a3/e14a3+e14a2 were independent factors for poor overall survival (hazard risk [HR], 4.63; P = .023 for Sokal score and HR, 10.6; P = .041 for BCR-ABL transcript).
Patients with BCR-ABL transcripts e14a2 transcripts have higher rates of CCR at 6 months and higher rates of optimal molecular response at 3 months compared with e13a2 or with both transcripts, but no difference in 5-year overall, progression-free, and event-free survival. There was a superior 10-year overall survival among patients with transcripts e13a2 compared with e14a2 (alone or coexpressed with e13a2).
Publication Date: 2017-08-22
Journal: Clinical lymphoma, myeloma & leukemia
No influence of BCR-ABL1 transcript types e13a2 and e14a2 on long-term survival: results in 1494 patients with chronic myeloid leukemia treated with imatinib.
The genomic break on the major breakpoint cluster region of chromosome 22 results in two BCR-ABL1 transcripts of different sizes, e14a2 and e13a2. Favorable survival probabilities of patients with chronic myeloid leukemia (CML) in combination with too small patient samples may yet have obstructed the observation of differences in overall survival of patients according to transcript type. To overcome potential power problems, overall survival (OS) probabilities and probabilities of CML-related death were analyzed in 1494 patients randomized to first-line imatinib treatment.
OS probabilities and probabilities of dying of CML were compared using the log-rank or Gray test whichever was appropriate. Both tests were stratified for the EUTOS long-term survival score.
Between the groups with a single transcript, neither OS probabilities (stratified log-rank test: p = 0.106) nor probabilities of CML-related death were significantly different (stratified Gray test: p = 0.256). Regarding OS, the Cox hazard ratio (HR) of transcript type e13a2 (n = 565) to type e14a2 (n = 738) was 1.332 (95% CI 0.940-1.887). Considering probabilities of leukemia-related death, the corresponding subdistribution HR resulted in 1.284 (95% CI 0.758-2.176). Outcome did not change if patients with both transcripts (n = 191) were added to the 738 with type e14a2 only.
The prognostic association of transcript type and long-term survival outcome was weak and without clinical relevance. However, earlier reported differences in the rate and the depth of molecular response could be relevant for the chance of successfully discontinuing TKI treatment. The effect of transcript type on molecular relapse after discontinuation is unknown, yet.
Publication Date: 2017-01-14
Journal: Journal of cancer research and clinical oncology
Current trends in molecular diagnostics of chronic myeloid leukemia.
Nearly 1.5 million people worldwide suffer from chronic myeloid leukemia (CML), characterized by the genetic translocation t(9;22)(q34;q11.2), involving the fusion of the Abelson oncogene (ABL1) with the breakpoint cluster region (BCR) gene. Early onset diagnosis coupled to current therapeutics allow for a treatment success rate of 90, which has focused research on the development of novel diagnostics approaches. In this review, we present a critical perspective on current strategies for CML diagnostics, comparing to gold standard methodologies and with an eye on the future trends on nanotheranostics.
Publication Date: 2016-12-07
Journal: Leukemia & lymphoma
Chronic myeloid leukemia: 2016 update on diagnosis, therapy, and monitoring.
Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm with an incidence of 1-2 cases per 100,000 adults. It accounts for approximately 15% of newly diagnosed cases of leukemia in adults.
CML is characterized by a balanced genetic translocation, t(9;22)(q34;q11.2), involving a fusion of the Abelson gene (ABL1) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is known as the Philadelphia chromosome. The molecular consequence of this translocation is the generation of a BCR-ABL1 fusion oncogene, which in turn translates into a BCR-ABL oncoprotein. Frontline therapy: Three tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, and dasatinib are approved by the United States Food and Drug Administration for first-line treatment of patients with newly diagnosed CML in chronic phase (CML-CP). Clinical trials with 2nd generation TKIs reported significantly deeper and faster responses; their impact on long-term survival remains to be determined. Salvage therapy: For patients who fail frontline therapy, second-line options include second and third generation TKIs. Although second and third generation TKIs are potent and selective TKIs, they exhibit unique pharmacological profiles and response patterns relative to different patient and disease characteristics, such as patients' comorbidities, disease stage, and BCR-ABL1 mutational status. Patients who develop the T315I "gatekeeper" mutation display resistance to all currently available TKIs except ponatinib. Allogeneic stem cell transplantation remains an important therapeutic option for patients with CML-CP who have failed at least two TKIs, and for all patients in advanced phase disease.
Publication Date: 2016-01-23
Journal: American journal of hematology
Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal.
Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.
Publication Date: 2015-12-02
Journal: Proceedings of the National Academy of Sciences of the United States of America
Cytogenetic, fluorescence in situ hybridization, and genomic array characterization of chronic myeloid leukemia with cryptic BCR-ABL1 fusions.
Chronic myeloid leukemia (CML) is characterized by the breakpoint cluster region (BCR)-Abelson murine leukemia (ABL1) fusion gene. In approximately 1% of CML cases, the Philadelphia chromosome associated with the BCR-ABL1 fusion gene is not present, and the BCR-ABL1 fusion gene is generated by cryptic insertion or sequential translocations. In this study, we describe the cytogenetic and molecular features of five CML patients with cryptic BCR-ABL1 fusion genes using karyotype, fluorescence in situ hybridization (FISH), and whole genome single nucleotide polymorphism array techniques. Two cases of CML in the chronic phase (CP) had a normal karyotype, and three cases of CML in the blast phase (BP) had an abnormal karyotype with neither a typical nor variant t(9;22). By BCR-ABL1 metaphase FISH analysis, we found that fusion signals were localized on chromosomes 9 (3 cases), 22 (1 case), and both 9 and 22 (1 case). In two cases of CML-BP, duplication of the BCR-ABL1 fusion gene occurred as a result of mitotic recombination between homologous chromosomes. Copy number losses involving the IKZF1 gene were observed in two patients with CML-BP. This study demonstrates for the first time the acquisition of additional BCR-ABL1 fusion genes through mitotic recombination in CML with cryptic BCR-ABL1.
Publication Date: 2015-07-19
Journal: Cancer genetics
BCR-ABL1 e6a2 transcript in chronic myeloid leukemia: biological features and molecular monitoring by droplet digital PCR.
The BCR-ABL1 fusion on the Philadelphia (Ph) chromosome is a hallmark of chronic myeloid leukemia (CML). More than 95 % of BCR-ABL1 transcripts in CML are either e13a2 or e14a2 (major breakpoint cluster region or M-bcr), whereas rare BCR-ABL1 transcripts are occasionally observed, accounting for less than 1 % of CML cases. Among these, a very rare fusion transcript joining the first 6 exons of BCR to exon 2 of ABL1 (e6a2) has been reported in various hematological malignancies characterized by an aggressive clinical course. We report a new case of blast crisis (BC) CML with an e6a2 fusion transcript characterized by many eosinophil precursors with abnormal granules. Moreover, fluorescence in situ hybridization analysis revealed genomic deletions of 1.3 megabases and 342 kilobases on der(9) of chromosome 9 and 22 sequences, respectively. The fusion transcript was quantified at diagnosis and during follow-up using digital droplet polymerase chain reaction (ddPCR) technology. The patient was treated with Dasatinib (140 mg/day), resulting in a 3-log reduction of the e6a2 transcript molecular burden from the third month after treatment. In this twentieth e6a2 case, characterized by marked eosinophilic dysplasia, deletions on der(9), and responsive to tyrosine kinase inhibitors therapy, we demonstrate that for molecular response monitoring of rare fusion transcripts associated with CML, ddPCR is a very useful technology.
Publication Date: 2015-07-08
Journal: Virchows Archiv : an international journal of pathology