The Philadelphia Chromosome Is Not a Stable Structure and Is Vulnerable to Further Rearrangements during Disease Progression in CML

Abstract Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder characterized by the expression of the BCR/ABL1 fusion gene, which commonly results from formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or related variant rearrangement. BCR/ABL1 is a constitutively activated tyrosine kinase and its amplification has been described in association with resistance to imatinib in CML patients. BAC array CGH analysis on CML patients and CML cell lines (Brazma et al., 2007) revealed unexpected genomic imbalances in form of duplications and high copy number gains affecting the region immediately downstream of the ABL1 gene at the Philadelphia (Ph) chromosome in patients at the blast crisis stage. We aimed to confirm and map these amplifications by fluorescence in situ hybridization (FISH) on 19 CML patients in accelerated phase/blast crisis and 10 CML cell lines (KU812, K562, MEG-01, MC3, BV173, EM-2, LAMA-84, KCL-22, JK-1 and CML-T1) with more than 1 copy of the BCR/ABL1 fusion gene. We used a range of BAC probes and 9q and 22q sub-telomeric probes in order to do the FISH mapping. While the majority of the analysed patients and cell lines (12/19 patients and 6/10 CML cell lines) had 2 identical Ph chromosomes, 2 main groups of abnormalities were identified. Firstly, gains of the Ph chromosome taking the form of ider(22)t(9;22) chromosome were detected in 1 or more copies in a subset of bone marrow cells of 5/19 patients and, secondly, high copy number gains were seen in 2/19 patients and 2/10 cell lines (K562 and KU812). The amplified region was variable in size spanning from 400 Kb up to 1.5 Mb downstream of the ABL1 gene. In 1 patient, 7 different cell sub-clones harbouring increasing levels of amplification were found. The gains resulted in formation of different chromosome structures-from an ider(22)t(9;22) to markers with tandem amplifications, which included the BCR/ABL1 fusion with variable in length sequences downstream of the ABL1. Duplication of some 571 Kb downstream of ABL1 was also detected in 1 of the 2 apparently normal Ph chromosomes in the MC3 cell line, while a larger duplication (5.16 Mb) was found in another cell line (MEG-01). These findings confirm the presence of duplications and high level amplifications at the der(22) t(9;22) in CML patients and that the sequences involved are variable in length, indicating that the Ph chromosome is an unstable structure and vulnerable to further rearrangements during disease progression.

Download Full-text

Molecular Characterization Of The Anti-Leukemic Effects Of Single Agent Eltrombopag

Blood ◽

10.1182/blood.v122.21.4923.4923 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4923-4923

Author(s):

Uwe Platzbecker ◽

Katja Sockel ◽

Claudia Schönefeldt ◽

Daniel Nowak ◽

Susann Helas ◽

...

Keyword(s):

Complete Remission ◽

Disease Progression ◽

Cell Line ◽

Cell Lines ◽

Copy Number ◽

Single Agent ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Dose Dependent

Abstract Introduction Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist which has been shown in-vitro to inhibit leukemia cell growth. The underlying mechanism is still under investigation. Methods We report a patient with NPM1 mutated/FLT3 negative refractory AML who achieved a complete remission during treatment with single agent EP within the PMA112509 trial. In this patient we conducted sequential molecular analyses out of the bone marrow to study the underlying molecular mechanisms. Therefore, samples prior to EP, at remission and relapse were subjected to genome-wide copy number analysis using Affymetrix SNP 6.0 array in search for acquired copy number alterations (CNA). To screen for alterations in commonly mutated genes in AML, samples were further subjected to a next generation deep sequencing assay (NGS) of mutational hotspots in the genes ASXL1, CBL, DNMT3A, ETV6, EZH2, IDH1/2, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Sequencing was performed on the 454 GS Junior platform (Roche applied science). Moreover we investigated the expression of TPO-R (CD110) by different assays in cell lines and primary AML samples. To study the TPO-R dependency of potential antineoplastic EP effects we studied the effects of lentiviral TPO-R knockdown and single agent EP on the vitality and cell cycling of TPO-expressing and non-expression leukemia cell lines. Results By using NGS we followed the NPM1+ mutation (NPM1 c.864incTCTG) load in this patient and found a concomitant decline (prior EP: 12.6%, at CR: 1.1%) but not disappearance of NPM1+ cells and a reemergence (15.2%) together with a clonal evolution and development of a NRAS c.37G>C mutation during disease progression (Figure 1) while a SNP-array demonstrated no additional CNA at disease progression. Real time PCR analysis demonstrated TPO-R expression at all time points analyzed which declined during complete remission(TPO-R/GAPDH: prior EP: 56.7%, at CR: 32.3%). These results prompted us to study TPO-R expression of blasts by flow cytometry in de novo AML samples (n=72) at diagnosis. In fact, TPO-R was expressed only in 33 of 72 AML patients but across all FAB and cytogenetic subgroups. The median MFI (mean fluorescence intensity) of CD110 was 2-fold higher on blasts than on CD110 positive lymphocytes. Interestingly, there were some differences with regards to the mutational status, since the NPM mutation was documented more frequently in CD110 negative than in CD110 positive AML cases (26% vs. 10%). These data were confirmed by Taqman-PCR in an independent cohort (n=57) with a nearly three fold lower expression of TPO-R on NPM1+/FLT3- compared to NPM1-/FLT3- (p=0.0163) cases. Next, we sought to clarify if TPO-R expressing AML cell lines are dependent on TPO-R expression. Knockdown of TPO-R by lentivirally transferred shRNA resulted in down-regulation and rapid cell death in the TPO-R expressing megakaryoblastic cell line (CMK). However, treatment with EP in-vitro at doses ranging from 1 to 10 µg/ml lead to a dose-dependent decrease in the cell division rate and vitality not only in CMK but also in cell-lines with weak or absent surface TPO-R expression (e.g. KG1a, a human acute myeloid leukemia cell line or OCI-AML3, a NPM1+ myeloid cell line). In parallel, a significant counterregulatory upregulation of TPO-R mRNA was observed which was dose-dependent (KG1a, p=0.0014). Conclusion These data demonstrate that TPO-R is heterogeneously expressed across all AML subtypes but absent in the majority of NPM1+/FLT3- cases. The clinical response seen in our patient with a refractory NPM1+ AML further provides evidence to the fact that single agent EP can exert potent anti-leukemic effects in-vivo. These effects seem to be mediated rather independently of TPO-R expression. Disclosures: Platzbecker: GlaxoSmithKline: Honoraria, Research Funding.

Download Full-text

Genomic Copy Number Variants in CML Patients With the Philadelphia Chromosome (Ph+): An Update

Frontiers in Genetics ◽

10.3389/fgene.2021.697009 ◽

2021 ◽

Vol 12 ◽

Author(s):

Heyang Zhang ◽

Meng Liu ◽

Xiaoxue Wang ◽

Yuan Ren ◽

Young Mi Kim ◽

...

Keyword(s):

Copy Number ◽

Array Cgh ◽

Fusion Gene ◽

Philadelphia Chromosome ◽

Copy Number Variants ◽

Chromosomal Anomaly ◽

Comparative Genomic ◽

Ph Chromosome ◽

Patient Prognosis

BackgroundSubmicroscopic segmental imbalances detected by array-comparative genomic hybridization (array-CGH) were discovered to be common in chronic myeloid leukemia (CML) patients with t(9;22) as the sole chromosomal anomaly. To confirm the findings of the previous study and expand the investigation, additional CML patients with t(9;22) as the sole chromosomal anomaly were recruited and copy number variants (CNVs) were searched for.MethodsKaryotyping tests were performed on 106 CML patients during January 2010–September 2019 in our Genetics Laboratory. Eighty-four (79.2%) patients had the Philadelphia (Ph) chromosome as the sole chromosomal anomaly. Only 49(58.3%) of these 84 patients had sufficient marrow or leukemia blood materials to additionally be included in the array-CGH analysis. Fluorescence in situ hybridization (FISH) was carried out to confirm the genes covered by the deleted or duplicated regions of the CNVs.Results11(22.4%) out of the 49 patients were found to have one to three somatic segmental somatic segmental (CNVs), including fourteen deletions and three duplications. The common region associated with deletions was on 9q33.3-34.12. Identified in five (45.5%) of the 11 positive patients with segmental CNVs, the deletions ranged from 106 kb to 4.1 Mb in size. Two (18.2%) cases had a deletion in the ABL1-BCR fusion gene on der (9), while three (27.3%) cases had a deletion in the ASS1 gene. The remaining CNVs were randomly distributed on different autosomes.ConclusionSubtle genomic CNVs are relatively common in CML patients without cytogenetically visible additional chromosomal aberrations (ACAs). Long-term studies investigating the potential impact on patient prognosis and treatment outcome is underway.

Download Full-text

Imbalances of the 9q34 Region Downstream of ABL Gene in CML Are Associated with Blast Crisis.

Blood ◽

10.1182/blood.v108.11.2118.2118 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2118-2118

Author(s):

Elisabeth P. Nacheva ◽

Diana Brazma ◽

Ruth Benson ◽

Julie Howard ◽

Anna Virgil ◽

...

Keyword(s):

Fusion Gene ◽

Cell Clone ◽

Blast Crisis ◽

In Situ Hybridisation ◽

Philadelphia Chromosome ◽

Chronic Phase ◽

Prognostic Scores ◽

Haematopoietic Stem Cell ◽

Clinical Criteria ◽

Genomic Imbalances

Abstract Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR-ABL fusion gene, a constitutively activated tyrosine kinase. The fusion gene commonly results from formation of the Philadelphia chromosome after a t(9;22)(q34;q11) or related variant rearrangement. The disease follows a biphasic course with an initial benign chronic phase (CP) of 3–4 years treatable with agents such as Imatinib, followed by a rapidly fatal transformation resembling an acute leukaemia (blast crisis, BC). The CML blast crisis has been linked to a number of secondary chromosome changes leading to genome imbalances, such as additional Ph, +8, +9, iso17q. Prognostic scores based on clinical criteria (Sokal and Hasford scores) have been replaced with the discovery of other cytogenetic abnormalities occurring simultaneously at the time of translocation, such as deletions flanking the ABL/BCR breakpoint at the der(9)t(9;22) chromosome that lead to a significantly worse prognosis. We undertook array CGH analysis using an oligo based platform (Agilent) on 46 CML patients (25 CP, 19 BC & 8 in both phases) and 12 CML cell lines revealed a range of recurrent genomic imbalances. Two sets of recurrent cryptic imbalances were identified. These represented both gains and high-level amplifications affecting the 9q34.12 region immediately downstream of ABL gene, as well as losses or gains in the distal part of the 9q34-qter segment. These imbalances were found only in patients at the BC stage. Furthermore, in cases with follow-up samples, the 9q34 imbalances were present in the advanced stage of disease thus confirming their secondary nature. Among the genes found to be amplified in CML/BC is NUP-214, a nucleoporin gene, recently implicated in T-cell acute lymphoblastic leukaemia, thus suggesting an important role for cell clone survival. Confirmation of these amplifications was sought using fluorescent in situ hybridisation (FISH). A dual fusion probe targeting the ABL-NUP214 region was applied to a selection of twenty three patients with confirmed CML and features such as deletion of der(9) chromosome or resistance to treatment with Imatinib, shown to be more prone to variation within the CML phenotypeAmplification of the NUP214 region was found in five patients. These findings represent the first reports of NUP214 amplifications in CML, paving the way for future work.

Download Full-text

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line

Blood ◽

10.1182/blood.v82.2.600.600 ◽

1993 ◽

Vol 82 (2) ◽

pp. 600-605 ◽

Cited By ~ 104

Author(s):

DS Snyder ◽

Y Wu ◽

JL Wang ◽

JJ Rossi ◽

P Swiderski ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Biology ◽

Fusion Gene ◽

Therapeutic Potential ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Myelogenous Leukemia ◽

A Cell

Abstract The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

Download Full-text

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line

Blood ◽

10.1182/blood.v82.2.600.bloodjournal822600 ◽

1993 ◽

Vol 82 (2) ◽

pp. 600-605 ◽

Cited By ~ 1

Author(s):

DS Snyder ◽

Y Wu ◽

JL Wang ◽

JJ Rossi ◽

P Swiderski ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Biology ◽

Fusion Gene ◽

Therapeutic Potential ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Myelogenous Leukemia ◽

A Cell

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

Download Full-text

Liposomal delivery of methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia [see comments]

Blood ◽

10.1182/blood.v84.2.601.601 ◽

1994 ◽

Vol 84 (2) ◽

pp. 601-607 ◽

Cited By ~ 35

Author(s):

AM Tari ◽

SD Tucker ◽

A Deisseroth ◽

G Lopez-Berestein

Keyword(s):

Growth Inhibition ◽

Cell Lines ◽

Chronic Myelogenous Leukemia ◽

Fusion Gene ◽

Hematologic Malignancy ◽

Control Cell ◽

Selective Inhibition ◽

Inhibitory Effect ◽

Myelogenous Leukemia ◽

Ph Chromosome

Abstract Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by the presence of the Philadelphia (Ph) chromosome. Bcr- abl, the fusion gene associated with the Ph chromosome, expresses a p210bcr-abl protein that promotes a selective expansion of mature myeloid progenitor cells. Methylphosphonate (MP) oligodeoxynucleotides complementary to specific regions of the bcr-abl mRNA were incorporated in liposomes. We studied the effects of liposomal MP (L-MP) on the growth inhibition of CML-like cell lines. L-MP targeted to the breakpoint junctions of the bcr-abl mRNA inhibited the growth of CML cells. Fifty percent inhibition was achieved at approximately 1 mumol/L of L-MP oligonucleotide concentrations. The inhibitory effect was selective because growth inhibition was observed only with CML but not with control cell lines. Moreover, CML cell growth inhibition was dependent on the sequence of the MP oligodeoxynucleotides incorporated in the liposomes. The growth inhibition of CML cells by L-MP resulted from selective inhibition of the expression of the p210bcr-abl protein.

Download Full-text

Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs

Scientific Reports ◽

10.1038/s41598-019-52143-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sho Nakai ◽

Shutaro Yamada ◽

Hidetatsu Outani ◽

Takaaki Nakai ◽

Naohiro Yasuda ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Fusion Gene ◽

Primary Tumour ◽

Chromosomal Rearrangements ◽

Basic Research ◽

Metastatic Potential ◽

Original Tumour

Abstract Approximately 60–70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX4. CIC-DUX4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

Download Full-text

Constitutive expression of SOCS3 confers resistance to IFN-α in chronic myelogenous leukemia cells

Blood ◽

10.1182/blood-2002-01-0073 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2926-2931 ◽

Cited By ~ 67

Author(s):

Ikuya Sakai ◽

Kazuto Takeuchi ◽

Hayato Yamauchi ◽

Hirosi Narumi ◽

Shigeru Fujita

Keyword(s):

Cell Line ◽

Cell Lines ◽

Chronic Myelogenous Leukemia ◽

Blast Crisis ◽

Constitutive Expression ◽

Myelogenous Leukemia ◽

Cytokine Signaling ◽

Interferon Α ◽

Socs3 Expression ◽

Blast Cells

Because suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine-induced signaling, it has been hypothesized that aberrant SOCS expression confers resistance against cytokine therapy. This study reports on the constitutive expression of SOCS3 in most chronic myelogenous leukemia (CML) cell lines, which are resistant to treatment with interferon α (IFN-α). In contrast, the KT-1/A3 cell line, in which constitutive expression of SOCS3 is barely detectable, is sensitive to IFN-α treatment. Forced expression of SOCS3 in the KT-1/A3 cell line confers resistance to IFN-α treatment. Furthermore, most of the blast cells from patients in CML blast crisis, which are usually resistant to IFN-α therapy, showed constitutive expression of SOCS3. These findings indicate that constitutive SOCS3 expression affects the IFN-α sensitivity of CML cell lines and blast cells from patients with CML blast crisis.

Download Full-text

Addition of Drug-Response Specific Micro-RNAs to the International Prognostic Index Improves Prognostic Stratification of GCB-DLBCL Patients Treated with R-CHOP

Blood ◽

10.1182/blood-2019-122351 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1623-1623 ◽

Cited By ~ 1

Author(s):

Karen Dybkær ◽

Hanne Due ◽

Rasmus Froberg Brøndum ◽

Ken H. Young ◽

Martin Bøgsted

Keyword(s):

Disease Progression ◽

Cell Line ◽

Cell Lines ◽

Mirna Expression ◽

Drug Response ◽

Drug Exposure ◽

International Prognostic Index ◽

Prognostic Index ◽

Expression Data ◽

Prognostic Stratification

Background: Patients with Diffuse large B-cell lymphoma (DLBCL) in approximately 40% of cases suffer from primary refractory disease and treatment induced immuno-chemotherapy resistance demonstrating that standard provided treatment regimens are not sufficient to cure all patients. Early detection of resistance is of great importance and defining microRNA (miRNA) involvement in resistance could be useful to guide treatment selection and help monitor treatment administration while sparing patients for inefficient, but still toxic therapy. Concept and Aims: With information on drug-response specific miRNAs, we hypothesized that multi-miRNA panels can improve robustness of individual clinical markers and serve as a prognostic classifier predicting disease progression in DLBCL patients. Methods: Fifteen DLBCL cell lines were tested for sensitivity towards rituximab (R), cyclophosphamide (C), doxorubicin (H), and vincristine (O). Cell line specific seeding concentrations was used to ensure exponential growth and each cell line was subjected to 16 concentrations in serial 2-fold dilutions and number of metabolic active cells was evaluated after 48 hours of drug exposure using MTS assay. For each drug, we ranked the cell lines according to their sensitivity and categorized them as sensitive, intermediate responsive, or resistant. Differential miRNA expression analysis between sensitive and resistant cell lines identified 43 miRNAs to be associated with response to compounds of the R-CHOP regimen, by selecting probes with a log fold change larger than 2. Baseline miRNA expression data were obtained for each cell line in untreated condition, and differential miRNA expression analysis identified 43 miRNAs associated to response to R-CHOP. Using the Affymetrix HG-U133+2 platform, expression levels of the miRNA precursors were assessed in 701 diagnostic DLBCL biopsies, and miRNA-panel classifiers were build using multiple Cox regression or random survival forest. Results: Generated prognostic miRNA-panel classifiers were tested for predictive accuracies and were subsequently evaluated by Brier scores and time varying area under the ROC curves (tAUC). Progression-free survival (PFS) was chosen as the outcome, since it is a treatment evaluation parameter as closely as possible to the time of drug exposure and the tested miRNAs were all associated directly to drug specific response. Furthermore, overall survival (OS) was used for verification of findings. Comparison of analyses conducted for the respective cohorts (All DLBCL, ABC, and GCB patients) showed the lowest prediction errors for all models within the GCB subclass with a multivariate Cox miRNA-panel model including miR-146a, miR-155, miR-21, miR-34a, and miR-23a~miR-27a~miR-24-2 cluster performed the best and successfully stratified GCB-DLBCL patients into high- and low-risk of disease progression. In addition, combination of the miRNA-panel and international prognostic index (IPI) substantially increased prognostic performance in GCB classified patients, indicating a prognostic signal from the response-specific miRNAs independent of IPI. In conclusion: We found as proof of concept that adding gene expression data detecting drug-response specific miRNAs to the clinically established IPI improved the prognostic stratification of GCB-DLBCL patients treated with R-CHOP. Disclosures No relevant conflicts of interest to declare.

Download Full-text

IKZF1 (Ikaros) Deletions Are a Hallmark of BCR-ABL1 Positive Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v110.11.721.721 ◽

2007 ◽

Vol 110 (11) ◽

pp. 721-721

Author(s):

Charles G. Mullighan ◽

Christopher B. Miller ◽

Letha A. Phillips ◽

James Dalton ◽

Jing Ma ◽

...

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Cell Lines ◽

Progenitor Cell ◽

Blast Crisis ◽

Dominant Negative ◽

Pcr Analysis ◽

Rt Pcr ◽

Snp Arrays ◽

Progenitor Cell Line

Abstract Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature2007;446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

Download Full-text