Abstract
Background. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and a major cause of childhood cancer-related mortality. Although the cure rate now approaches 90%, certain pediatric ALL subgroups present subsequent relapse. For this reason, analyses of cell signaling pathways will help to identify new markers and/or targets for tailored therapy. PI3K/AKT/mTOR activation is frequently found in both B-ALL and T-ALL. Protein kinase Ck2 (CSNK2) activity in pediatric ALL was increased and its inhibition restored PTEN phosphatase activity with subsequent inactivation of AKT. Moreover, Ck2 may serve the activity of oncogenes such as BCR-ABL and c-MYC, control the activation of other critical signaling cascades (JAK-STAT), and sustain multiple cellular stress-elicited pathway such as the proteotoxic stress, unfolded protein and DNA-damage responses. Ck2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment (Piazza F et al, Oncogene2016).
Material and Methods. We analyzed cDNA collected from 46 patients with B-ALL [19 High Risk (HR) for Minimal Residual Disease (MRD) and 27 NON-HR] and 25 with T-ALL (8 HR and 17 NON-HR), respectively, diagnosed in our Center from 2000 to 2012. The latter subgroup was screened fro PTEN-Exon7 mutations and TXL3 rearrangements. We evaluated the gene expression of Ck2 and c-Myc genes using RQ-PCR with Sybr-Green and a relative quantification method (ΔΔCt method), comparing gene's expression from patients with samples from 6 healty donors (HDs). In order to demonstrate the correlation between genetic alteration and signaling transduction, specifically in HR patients , we analyzed some phosphoproteins by Phospho-flow approach. We profiled 5 proteins (STAT3, STAT5, CREB, PTEN and pS6) in 4 T-ALL cases (3 with PTEN-Exon 7 mutation).
Results. We observed a significant difference of Ck2 expression in T-ALL NON-HR patients vs HDs (Mean Ck2 Fold-Changes 3.494 vs 1.17, p=0.0315) and in T-ALL HR patients (6.384 vs 1.17, p=0.0219) vs HDs (Fig 1A and B). Comparing NON-HR vs HR cases, we found a statistically significant difference (p<0.0001) (Fig 1C). c-Myc mean expression was very similar between the two T-ALL subgroups. Moreover, among T-ALL cases, we identified 5 patients with PTEN-Exon7 mutations and 6 with TLX3 rearrangements. We observed that cases with PTEN-Exon7 mutation showed lower c-Myc expression than cases with PTEN-Exon7 wild-type (mean c-Myc 8.550 vs 1.920) whereas patients with TLX3 rearrangements showed higher c-Myc expression than TLX3 negative (mean c-Myc 18.260 vs 5.502) (p<0.005) (Fig 2A and B). We did not observe any correlation between these rearrangements and Ck2 expression. We also performed Ck2 and c-Myc expression analysis in B-ALL (NON-HR and HR) subgroups. We surprisingly observed a Ck2 overexpression in both NON-HR and HR B-ALLs compared to HDs. On the other side, we did not observed significant difference about c-Myc expression in cases with B-ALL vs HDs; whilst we observed an overexpression of c-Myc in HR vs NON-HR patients with B-ALL (mean 7.075 vs 2.095, respectively)(p<0.0004). Phospho-Flow analysis, in 3 cases with PTEN-Exon7 mutation (1 Ck2+/normal Myc, 1 normal Ck2/normal Myc, 1 Ck2-/normal Myc) showed PTEN null, very lower pS6 basal level and higher CREB basal level than in case with PTEN-Exon7 wild-type. Moreover, we observed that the latter patient, presented with a TLX3 rearrangements (Ck2+/Myc+) with higher c-Myc expression, showed higher STAT3 basal state level confirming that STAT3 induces the expression of c-Myc.
Conclusions. Based on our preliminary findings, Ck2 could be considered as a marker and /or a potential candidate for targeted therapy, specifically in HR-ALL, as confirmed by the use of CK2 inhibitor (CX-4945) in ongoing clinical trials. c-Myc overexpression confirmed its association with HR features. The potential role as markers of both genes needs to be demonstrated in a larger population study. Combined application of genomic and phosphoproteomic strategies will lead us to better profile diagnostic samples of HR-ALL, addressing future tailored treatments.
Disclosures
No relevant conflicts of interest to declare.
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