PHOSPHOPROTEOMIC PROFILING OF PEDIATRIC T-LINEAGE ACUTE LYMPHOBLASTIC
G Milani, B Accordi, M Giordan, S Bresolin, M Sciro, L Galla, G TeKronnie, G Basso
Background. T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of paediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL paediatric patients remain at risk for early relapse. It is therefore very important to develop more specific and less toxic therapies through the identification of molecules, such as aberrantly activated phosphoproteins that offer the possibility to use specific kinase inhibitors. Aim. The aim of this research was to study the phosphorylation profile of paediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), to find aberrantly activated phosphoproteins. Methods. We analyzed 98 paediatric patients with T-ALL using RPPA technique. This innovative approach allows to investigate post-translation modifications, such as phosphorylation, and thus to characterize the activation state of cellular protein networks. For all patients included in this study informed consent was obtained following the tenets of the Declaration of Helsinki. Twenty-seven samples were from the Padova hospital and were directly processed whereas 71 patients were from other Italian hospitals. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with a different antibody, for a total of 53 antibodies. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features. Results. We first found that the phosphorylation state of samples from Padova is comparable with that of samples from other Italian hospitals. Shipment of samples to Padova at room temperature does not influence in a systematic way protein phosphorylation, thus allowing the study by RPPA also for samples collected in other centers. Comparison between T-ALL patients with and without del(1p32) revealed that mTOR(S2448) is differentially activated between these two groups (t test with Benjamini-Hochberg multiplicity corrections P=0.003). This protein kinase that regulates cell growth and survival is less activated in 1p32 deleted patients. We then compared relapsed vs non relapsed patients. Statistical analysis revealed that PKCα(S657) is hyperactivated in the non relapsed group (t test with Benjamini-Hochberg multiplicity corrections P=0.02). Moreover, we performed a Relapse Free Survival analysis through Kaplan-Meier estimates. We searched a threshold value for PKCα(S657) that resulted in the largest difference in survival between the two groups defined by that threshold: PKCα(S657) threshold 50750.33, log rank test with Holm corrections P=0.02. Patients who show PKCα(S657) lower than 50750.33 have a higher probability to relapse: 15 of 32 (47%) patients with PKCα(S657) lower than 50750.33 relapsed, while among the 58 patients with PKCα(S657) higher than 50750.33 only (17%) relapsed. Of note, mRNA expression analysis for mTOR and PKCα did not reveal any significant difference in gene expression levels among the above studied T-ALL patients. Conclusions. This research identified the different activation state of proteins in subgroups of T-ALL patients with clinical relevance that may be explored for new therapies.