Effect of IKZF1 deletions on signal transduction pathways in Philadelphia chromosome negative pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL)
© van der Sligte et al. 2015
Received: 24 July 2015
Accepted: 27 July 2015
Published: 12 August 2015
IKZF1 deletions are an unfavorable prognostic factor in children with Philadelphia chromosome positive (Ph+) as well as negative (Ph−) acute lymphoblastic leukemia (ALL). Although IKZF1 deletions occur in 10–15% of Ph− ALL cases, effects of IKZF1 deletions on signaling pathways in this group have not been extensively studied. Therefore, in this study we aimed to study the effect of IKZF1 deletions on active signal transduction pathways.
Multiplex ligation-dependent probe amplification (MLPA) was used to determine IKZF1 deletions and other copy number alterations in 109 pediatric B-Cell Precursor ALL (BCP-ALL) patients. Kinase activity profiling of 45 primary Ph− BCP-ALL patients (31 IKZF1 wild type patients and 14 patients harboring an IKZF1 alteration) and western blot analysis of 14 pediatric BCP-ALL samples was performed to determine active signal transduction pathways.
Unsupervised hierarchical cluster analysis of kinome profiles of 45 pediatric Ph− ALL cases showed no clustering based on IKZF1 status. Comparing the phosphorylation intensities of peptides associated with signaling pathways known to be involved in BCP-ALL maintenance, we did not observe differences between the two groups. Western blot analysis of 14 pediatric BCP-ALL samples showed large variations in phosphorylation levels between the different ALL samples, independent of IKZF1 status.
Based on these results we conclude that, although IKZF1 deletions appear to be an important clinical prognostic factor, we were unable to identify a unique IKZF1 dependent protein expression signature in pediatric Ph− ALL and consequently no specific targets for future therapy of Ph− IKZF1 deleted BCP-ALL could be identified.
KeywordsAcute lymphoblastic leukemia IKZF1 Signaling Kinome profiling
Overall survival rates for children with Acute Lymphoblastic Leukemia (ALL), the most common type of cancer in children, are approaching 90% . Historically, risk stratification of newly diagnosed children was based on age and white blood cell count (WBC), but nowadays includes extensive cytogenetic and molecular analyses. In the past 5 years, genome wide approaches, studying DNA copy number alterations in ALL, have identified novel molecular markers that can be used for further risk stratification, including IKZF1 deletions as a predictor of poor outcome. IKZF1 deletions can be identified in approximately 70% of the children with Philadelphia chromosome positive (Ph+) ALL and in 10–15% of the children with Philadelphia chromosome negative (Ph−) ALL and are associated with an increased relapse risk and decreased overall survival in both groups [2–4]. More recent studies indicate that the genomic context in which IKZF1 deletions occur is more important for prognosis as for example CRLF2 and JAK2 mutations are more common in IKZF1 deleted BCP-ALL [5–7]. In pediatric B-cell progenitor ALL (BCP-ALL), 80% of the IKZF1 deletions are found in a Philadelphia chromosome negative background.
IKZF1, which encodes the transcription factor Ikaros, is essential for normal lymphoid development, whereas for erythroid and myeloid lineage differentiation IKZF1 is less critical . Mice deficient for IKZF1 show a complete arrest in B-lymphocyte development while mice heterozygous for a dominant-negative mutation of IKZF1 develop T cell leukemia and lymphoma with a 100% penetrance [9, 10]. During normal development, Ikaros restricts the G1-S transition of the cell cycle when it binds to the DNA, by regulating transcription of cell cycle regulator genes e.g. a positive effect on cell cycle inhibitors CDKN1A (p21Cip1) and CDKN1B (p27Kip1) . Phosphorylation of Ikaros by casein kinase II (CK2) temporarily reduces Ikaros binding to DNA and thereby facilitates progression through the S phase of the cell cycle . Furthermore, Ikaros can be phosphorylated by spleen tyrosine kinase (SYK) and bruton’s tyrosine kinase (BTK) [12, 13]. These phosphorylation events are essential for nuclear localization, regulation of DNA binding activity, and an optimal transcriptional function of Ikaros [12, 13].
IKZF1 deletions observed in BCP-ALL are typically mono-allelic, either resulting in a loss of function or the expression of a dominant-negative isoform . The dominant-negative isoforms lack the DNA binding N-terminal zinc fingers, preventing DNA binding after dimerization with Ikaros . As a result, the control of Ikaros on the G1-S transition is abolished leading to hyperproliferation and the development of leukemia .
Although the cure rates for children with BCP-ALL have improved substantially, the outcome after ALL relapse remains poor. Since IKZF1 deletions increase the risk of relapse, new therapeutic options aiming to improve cure rates for this specific subtype of ALL are needed. We have previously shown that insight into active signal transduction pathways allows identification of interesting targets for future therapy [16–19]. At the level of signal transduction, Iacobucci et al. showed on western blot analysis a higher STAT5 phosphorylation in IKZF1 deleted compared to IKZF1 wild type adult BCP-ALL patients with unknown cytogenetic background . However, this observation might also be associated with BCR-ABL1 activity as in adult BCP-ALL patients IKZF1 deletions are more common in Ph+ ALL . Additionally, Ikaros-reconstitution in two IKZF1 deleted Philadelphia positive ALL patients resulted in an upregulation of the B-cell receptor (BCR) signaling pathway and a concomitant cell cycle arrest; showing that in Ph+ ALL pre-B cell receptor signaling suppresses proliferation through an Ikaros-mediated cell cycle arrest . Although IKZF1 deletions in children are most commonly found in a Philadelphia negative background, the effect of IKZF1 deletions on signaling pathways in Philadelphia negative ALL have not been extensively studied. Therefore, in this study we aimed to study the effect of IKZF1 deletions on active signal transduction pathways in Philadelphia negative pediatric BCP-ALL using kinome profiling.
Primary blood and bone marrow samples from newly diagnosed ALL patients were collected after getting written informed consent in accordance with the regulations and protocols of the medical ethics committee of the University Medical Center Groningen. Overall, we collected material of 109 Philadelphia negative BCP-ALL patients. Mononuclear cells were isolated by Lympho-prep (Nycomed, Zürich, Switzerland) density gradients and cryopreserved in liquid nitrogen until use. The cryopreserved leukemia cells were thawed rapidly at 37°C and diluted in a 6 ml volume of newborn calf serum, as described earlier .
Genomic DNA was extracted from mononuclear cells using the QIAamp DNA easy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All isolated DNA was quantified by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).
Multiplex ligation-dependent probe amplification (MLPA)
Targeted copy number screening of eight selected loci was performed in the cohort by multiplex ligation-dependent probe amplification (MLPA) using the P335-B2 SALSA MLPA kit (MRC-Holland, Amsterdam, The Netherlands). The assay includes probes for each of the eight exons of the IKZF1 gene and is able to detect deletions of the whole gene as well as all types of focal intragenic deletions. Selected exons of the genes BTG1, CDKN2A/B, EBF1, ETV6, PAX5, RB1 and the PAR1 region (approx. 230 kbp downstream of SHOX, CRLF2, CSF2RA and IL3RA) are also covered. Probe mix and hybridization buffer (MRC-Holland) were added in equal amounts to 50 ng of genomic DNA followed by heat denaturation and overnight hybridization of the probes at 60°C. Subsequently, ligation was performed and the ligation products were amplified by PCR using a 6-FAM fluorophore-labeled primer set (MRC-Holland). The amplification products were quantified and identified by capillary electrophoresis on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA).
Data analysis: Data were analyzed using Gene Mapper v.4.0 software (Applied Biosystems). Normalization of the data was carried out by dividing the peak area of each probe by the average peak area of the control probes. This normalized peak pattern was divided by the average peak pattern of all the samples in the same experiment. The resulting values were 1 for every wild-type peak, 0.5 for heterozygous deletions and 1.5 for heterozygous duplications.
Kinase activity profiles of 45 primary Ph− BCP-ALL patients were determined using the PepChip™ Kinomics microarray system (Pepscan, Lelystad, the Netherlands) and performed as described previously [18, 19]. The microarray contain 1,024 peptides in triplicate (1,008 unique target peptides and 16 peptides used for production) derived from known phosphorylation sites from human protein sequences that can be phosphorylated by the kinases in the sample lysate. Per sample, 0.5 × 106 cells were lysed in 100 μl of M-PER Mammalian Protein Extraction Buffer containing 1 μl Phosphatase Inhibitor and 1 μl Protease Inhibitor (Pierce, Rockford, IL, United States). Peptide array incubation mix was produced by adding 10 μl of filter-cleared activation mix onto 90 μl cell lysate. Peptide array incubation mix was loaded on the chip and incubated for 2 h at 37°C in a closed humid box at saturated humidity. Subsequently, the peptide array was washed and blow dried with compressed air or N2 and the chips were exposed to a phospho-storage screen for 24–96 h. The amount of bound 33P-labelled ATP to the peptides specifies the amount of peptide phosphorylation and was analyzed with array software (ScanAlyze, Eisen Lab, University of California at Berkely, Berkely, CA, United States).
Data analysis: Data were analyzed as described previously [18, 19]. In short, background was subtracted and the spot intensities were quantile normalized. A Pearson’s correlation coefficient was determined over the triplicates (Excel 2003, Microsoft Office, Redmond, WA, United States). Slides with a correlation <0.6 over the triplicates were excluded from further analysis. The correlation over the triplicates was <0.6 in none of the samples. Cluster, statistical and heatmap analysis were performed using Qlucore Omics Explorer 3.0 (Qlucor AB, Lund, Sweden). The file containing the processed raw data can be found in the additional information (Additional file 1: Table S1).
Western blot analysis
Primary ALL cells were solved in laemmli sample buffer (Bio-Rad laboratories, Veenendaal, the Netherlands). Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and transported to nitrocellulose membranes. Membranes were blocked in 7.5% skimmed milk and incubated overnight with primary antibodies for phospho-Src_Y416, phospho-Syk_Y323, phospho-ERK1/2_T202/Y204, phospho-CREB_S133, phospho-p38_T180/Y182, phospho-Akt_S473, phospho-mTOR_S2448, phospho-GSK3α/β_S21/S9, phospho-MDM2_S166, phospho-Chk2_T68, phospho-RB1_S807/811, phospho-STAT3_Y705, phospho-STAT5_Y694 (Cell Signaling, Danvers, MA, USA), or phospho-p27_T187 (Abgent, San Diego, CA, United States) and for 1 h with HRP conjugated secondary antibodies (Dako, Glosturp, Denmark). Protein bands were visualized using the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, United States) and ImageLab software (version 5.0, Bio-Rad Laboratories). Loading control was visualized using β-actin (Santa Cruz Biotechnology, Dallas, TX, United States).
Generation of kinase activity profiles in IKZF1 deleted versus IKZF1 wild type Philadelphia negative pediatric BCP-ALL
Signal transduction pathway activation in response to IKZF1 status
Phosphorylation levels of key signaling proteins in IKZF1 deleted and wild type BCP-ALL
IKZF1 deletions are found in approximately 70% of the children with Philadelphia chromosome positive (Ph+) ALL and in 10–15% of the children with Philadelphia chromosome negative (Ph−) ALL. In both groups, IKZF1 deletions are associated with an increased risk on relapse and decreased overall survival [2–4]. In Ph− ALL, the effect of IKZF1 deletions on outcome is most pronounced in children with an intermediate treatment response based on the minimal residual disease at days 42 and 84 . Although multiple studies have established IKZF1 as a prognostic factor in pediatric BCP-ALL, the effect of IKZF1 deletions on signaling pathways in Philadelphia negative B-cell precursor ALL is poorly understood. In previous studies, we have shown that kinome profiling can be used successfully to describe active signal transduction pathways in pediatric malignancies [16–19]. In this study we used kinome profiling to elucidate the effect of IKZF1 deletions on active signal transduction pathways.
Unexpectedly, unsupervised hierarchical cluster analysis revealed no clustering between IKZF1 deleted and wild type patients. Furthermore, peptide phosphorylation intensities between IKZF1 deleted and wild type Ph− BCP-ALL patients were very comparable as shown by the phosphorylation intensities of the thirty-eight differentially phosphorylated peptides and by the list of top 100 most highly phosphorylated peptides. While focusing on important signaling pathways involved in cell proliferation and survival of BCP-ALL cells we showed activity of all these pathways, however, no differences between the IKZF1 deleted versus the IKZF1 wild type group could be observed. Moreover, western blots of several key proteins involved in ALL signaling showed a variety of phosphorylation events, clearly unrelated to IKZF1 status.
Although no clear differences in peptide phosphorylation intensities could be observed, kinome profiles showed a remarkable high phosphorylation of peptides derived from Cytohesin-1_S394 and Cytohesin-2_S392. Cytohesins have been described as ErbB receptor activators . It has been described that Cytohesin overexpression enhances epidermal growth factor receptor (EGFR) signaling in human cancers including lung cancer and colorectal cancer [25, 26]. Although kinase domain mutations of ErbB receptors are uncommon in acute leukemias, in vitro inhibition of ErbB2 reduces cell proliferation especially when combined with BCR-ABL tyrosine kinase inhibitors in Ph+ ALL, suggesting a role for ErbB signaling pathway activation in leukemia [27, 28]. Therefore, it will be interesting to further explore the role of Cytohesin in BCP-ALL.
Although we did not identify a unique kinome signature as a result of IKZF1 status, effects of IKZF1 deletions on gene expression were described previously. Iacobucci et al. showed that IKZF1 deletions display a unique gene expression signature in a cohort of adult B-ALL patients, including patients with a Philadelphia translocation and B-ALL patients negative for known molecular rearrangements . The gene expression signature was characterized by the downregulation of genes regulating B-cell lineage development and DNA repair upon DNA damage response genes and upregulation of cell cycle/apoptosis genes, JAK/STAT signaling and stem cell self-renewal . More recently, besides an upregulation of genes associated with B-cell proliferation, an upregulation of genes involved in cell adhesion and communication was also observed in pediatric Ph− ALL .
In addition to a unique IKZF1 dependent gene expression profile, a subtype of precursor BCP-ALL with a similar gene expression profile compared to Philadelphia-positive ALL was identified several years ago (Ph-like ALL) [2, 30]. Importantly, 68% of the patients with Ph-like ALL showed deletions in IKZF1 . Within the Ph-like ALL subtype, 5-year event-free survival rates in patients with IKZF1 alterations were inferior compared to Ph-like IKZF1 wild type ALL patients . Recently, Roberts et al. defined the genomic landscape of Ph-like ALL in a large cohort of children and adolescents to elucidate kinase-activating genetic alterations which might include potential leads for targeted therapy . Genomic alterations activating kinase signaling were identified in 91% of the Ph-like ALL patients (N = 156) including ABL-class fusions, rearrangements of JAK2 or CRLF2, genetic alterations including IL7R and FLT3, and Ras pathway mutations . Patients harboring IKZF1 alterations (N = 96, 61.5%) were distributed over all groups of different kinase alterations, indicating a high degree of heterogeneity within the group of IKZF1 deleted patients . Our results and the results of Roberts et al. suggest that patients harboring IKZF1 alterations represent a heterogeneous subgroup when evaluated at the level of active signal transduction pathways. The identification of potential targets for tyrosine kinase inhibitors therefore appears to be dictated by upstream genomic alterations that activate kinase signaling or cytokine receptor pathways rather than IKZF1 status per se.
The aim of this study was to elucidate the effect of IKZF1 deletions on active signal transduction pathways using kinome profiling and western blot analysis in children with Ph− BCP-ALL. Although IKZF1 deletions are an important clinical prognostic factor we were unable to identify a unique IKZF1 associated protein expression signature in pediatric Ph− BCP-ALL and consequently no specific targets for future therapy of Ph− IKZF1 deleted BCP-ALL were identified.
acute lymphoblastic leukemia
B-cell progenitor acute lymphoblastic leukemia
Bruton’s tyrosine kinase
casein kinase II
multiplex ligation-dependent probe amplification
- Ph− :
Philadelphia chromosome negative
- Ph+ :
Philadelphia chromosome positive
spleen tyrosine kinase
NEvdS performed research, collected data, analyzed data and wrote the paper. FJGS performed research and collected data. AtE supervised pepchip data analysis. VG performed quantile normalization and supervised pepchip data analysis. FNvL supervised and edited the paper. ESJMdB designed research, analyzed data, supervised and edited the paper. All authors read and approved the final manuscript.
We thank the Junior Scientific Masterclass, University of Groningen, Groningen, The Netherlands for financial support.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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