Lack of Protein Kinase C Alpha Is Associated with Poor Prognosis in Pediatric T-Lineage Acute Lymphoblastic Leukemia

T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL pediatric patients remain at high risk for early relapse. It is therefore very important to identify new prognostic biomarkers and to develop new therapeutic approaches for these patients. More specific and less toxic therapies might be developed through the identification of molecular targets, such as aberrantly activated phosphoproteins, that offer the possibility to use specific kinase inhibitors.

In this research we first explored the phosphoproteomic profile of pediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), and we further investigated the origin and the functional significance of Protein Kinase C alpha (PKCα) downregulation in patients that are liable to relapse.

We analyzed 98 pediatric T-ALL patients at diagnosis using RPPA. This innovative approach allows to profile phosphorylation modifications and to characterize the activation state of cellular protein networks. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with one of 53 different antibodies included in the study. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features through statistical analyses. Gene expression analysis was performed by means of HG-U133 Plus 2.0 array. Permission for this study was obtained following the tenets of the Declaration of Helsinki. A set of commercially available human T-ALL cell lines was studied by Western Blot and cells were treated with Ro-32-0432, a commercial PKC inhibitor.

The phosphoproteomic profile of T-ALL patients was compared between patients subgroups at diagnosis. Comparison between relapsed vs non relapsed patients samples revealed the downregulation of PKCα(S657) in relapsed patients (t test with BH multiplicity corrections p=0.02) and Relapse Free Survival analysis through Kaplan-Meier estimates showed that patients with low PKCα(S657) have a high probability to relapse. Notably, 54.5% of patients with low PKCα(S657) relapsed whereas no relapses were observed among patients with high PKCα activity. Multivariate analysis showed no correlation between PKCα(S657) levels and other parameters currently used for clinical characterization of T-ALL patients.

Differences in PKCα(S657) between relapsed and non relapsed patients were confirmed by Western Blot in two independent sets of T-ALL patients. Analysis of PKCα total protein form revealed concordance between total protein expression and PKCα(S657) levels. Furthermore, gene expression analysis revealed a significant difference in PKCα gene expression levels, comparable with levels of protein expression among T-ALL patients studied by RPPA. Gene expression data were validated by Sybr Green Real-Time Quantitative PCR. Moreover, gene pathway analysis in a larger cohort of T-ALL pediatric patients, divided by high and low PKCα mRNA levels, revealed the phosphatidylinositol signalling pathway as the most important deregulated pathway. Of note, this pathway is involved in the production of DAG and Ca²⁺, the PKCα cofactors that play an essential role in mediating its translocation from the cytosol to the plasmatic membrane and subsequent activation.

PKCα and PKCα(S657) were then analysed by Western Blot in five T leukemic cell lines (DND41, P12 ICHIKAWA, MOLT3, TALL1 and CEM). These cell lines were treated with a PKCα inhibitor and PKCα inhibition effects were studied by the means of MTT assay and Trypan Blue cell count. An increase in cell proliferation was observed in each treated cell line 9 hours after PKCα inhibition.

PKCα is differentially expressed in T-ALL pediatric patients. Low levels of PKCα(S657) are associated with a higher probability to relapse and downregulation or absence of PCKα seems to be related to a more aggressive T-ALL phenotype. Ongoing studies are needed to better define its role as a new prognostic marker.

No relevant conflicts of interest to declare.