Effect of IKZF1 deletions on signal transduction pathways in Philadelphia chromosome negative pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL)

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Acute lymphoblastic leukemia; IKZF1; Kinome profiling; Signaling.

Fig. 1

Patients’ characteristics. Clinical and genetic characteristics as well as copy number alterations identified using multiplex ligation-dependent probe amplification (MLPA) analysis are shown for 45 patients who were included in the kinome analysis.

Fig. 2

Kinome profile in Philadelphia chromosome negative pediatric BCP-ALL patients. a Unsupervised hierarchical clustering of 45 pediatric BCP-ALL cases; 13 IKZF1 deleted, 1 IKZF1 gain, and 31 IKZF1 wild type Philadelphia chromosome negative patients based on 1,008 unique target peptides. No distinct clustering could be observed based on IKZF1 status, neither on genetic background or other copy number alterations. b Unsupervised hierarchical clustering of 44 pediatric BCP-ALL cases; 13 IKZF1 deleted and 31 IKZF1 wild type patients based on 38 differentially phosphorylated peptides identified by t test. Each row represents a peptide, each column represents a single ALL sample. The magnitude of deviation from the median is represented by the color saturation with each variable normalized to mean 0 and variance 1. Red and green spots display the phosphorylation intensity above and below the median, respectively.

Fig. 3

Signal transduction in IKZF1 deleted versus wild type cases in Philadelphia chromosome negative pediatric BCP-ALL patients. Shown are peptide phosphorylation intensities of proteins involved in important signal transduction pathways for BCP-ALL cell proliferation and survival. Results are expressed in gray scale representing the mean peptide phosphorylation intensity of all category peptides.

Fig. 4

Protein phosphorylation profiles in Philadelphia chromosome negative pediatric BCP-ALL patients. Western blot analysis of 14 pediatric BCP-ALL samples; seven Philadelphia negative IKZF1 wild type, six Philadelphia negative IKZF1 deleted patients, and one Philadelphia positive IKZF1 deleted patient showing phosphorylation expression levels of a selection of proteins. The results show a great diversity in phosphorylation levels between the different ALL samples independently of IKZF1 status and other copy number alterations.