Lack of Protein Kinase C alpha is associated with poor prognosis in pediatric T-ALL: a phosphoproteomic discovery

Acute Lymphoblastic Leukemia (ALL), the most common pediatric cancer, is an aggressive malignancy of lymphopoietic cells characterized by a clonal proliferation of blast cells originated from lymphoid precursors arrested at early stages of differentiation. Nowadays the introduction of risk-directed treatment and intensified cure protocols has improved the outcome of ALL pediatric patients. Although current therapies achieve five-year event-free survival rate of about 80% in children, the rest of patients experience treatment resistance and risk of early relapse. In order to develop new additional therapeutic supports and to assemble specific and personalized therapies, improving drug response and reducing drug toxicity, it is extremely important to identify novel therapeutic targets and new prognostic biomarkers on leukemic cells. To pursue this aim, Reverse Phase Protein Arrays (RPPA) approach represents a powerful tool to profile protein pathways in order to identify novel diagnostic and prognostic biomarkers. RPPA can measure the activation levels/phosphorylation of large numbers of signalling proteins from small clinical samples in a very reproducible, precise, sensitive and high-throughput manner. The identification of critical nodes or interactions within the protein network is a potential starting point for drug development and/or design of individual therapy regimens. In RPPA, protein lysates are immobilized on a nitrocellulose-coated glass slide. Each group of spots represents an individual test sample, such that an array is comprised of multiple, different samples. Each RPPA slide is probed with a single detection molecule (one slide = one antibody) and a single analyte is concurrently measured across multiple samples. This format allows multiple samples to be analyzed under the same experimental conditions for any given analyte. This approach facilitates comparisons between samples, since experimental variability is eliminated. During my PhD research I applied the Reverse Phase Protein Arrays (RPPA) technique to the study of patients affected by Acute Leukemia. The principal aim of this research was the discovery of new candidate biomarkers and therapeutic targets involved in the leukemic process through the study of the phosphoproteomic profiles of ALL pediatric patients (T-ALL and B-ALL) by means of the RPPA technique. In the study of pediatric T-ALL at diagnosis reported in the “Main Topic”, we identified PKC alpha as a new prognostic marker of relapse. This promising protein kinase has been identified through a retrospective RPPA screening of fifty-three different proteins/phophoproteins in 98 T-ALL specimens. PKC alpha resulted to be downregulated in the group of patients with a higher incidence of relapse. We showed that low PKC alpha activation is a consequence of a low PKC alpha "gene expression" Moreover, we induced PKC alpha inhibition in T-ALL cell lines using a PKC alpha commercial inhibitor and an increase in proliferation rate was detected. RPPA thus proved to be a useful approach to discover new molecules aberrantly expressed in leukemia pathways. The identification of players involved in malignant mechanisms allows focusing on novel prognostic factors: PKC alpha resulted to be a promising marker of risk of relapse in pediatric T-ALL. A study of PKC alpha expression in a larger T-ALL patients cohort should confirm this relevant data and to propose PKC alpha as a new prognostic marker for T-ALL patients stratification. In the pediatric B-cell precursor (BCP)-ALL study we used RPPA to map in 118 pediatric BCP-ALL patients for the expression/activation of 92 different proteins/phophoproteins part of key signalling “hubs”. Correlation of signalling activation with clinical response and known genetic information enabled us to identify new protein pathway biomarkers that, when validated in larger clinical sets, could be used for patients stratification and targeted therapy trials. In detail, we observed an increased activation/expression of several pathways involved in cell proliferation in patients with a clinical poor phenotype. First, MLL-rearranged leukemia revealed BCL-2 hyperphosphorylation caused by AMPK activation, indicating that AMPK could be responsible of apoptosis inhibition in MLL-rearranged patients and thus could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to Prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors that could be considered for further studies as a new additional therapy for Prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in patients with early relapsed, who usually show an unfavourable prognosis. We decided to explore in deep the functional significance of AMPK activation in MLL-rearranged patients. We showed that the AMPK pathway contributes directly to the survival of MLL-rearranged BCP-ALL cells and AMPK can be considered a new druggable target in MLL-rearranged leukemias. RPPA reliability in detection of novel targets and markers has been demonstrated also in collaborative studies with national and international research groups, regarding also adult diseases such as Acute Myeloid Leukemia. In my PhD research I showed that the application of Reverse Phase Protein Arrays is a reliable, sensitive and high-throughput method to identify new aberrant molecular players involved in haematological malignancies. These findings will be useful in order to discover novel prognostic markers and therapeutic targets to propose for the development of more effective patient-tailored treatments