Functional Protein Network Activation Mapping Reveals New Potential Molecular Drug Targets for Poor Prognosis Pediatric BCP-ALL

Abstract

BackgroundIn spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.Methodology/Principal FindingsWe employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and 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, thus modulating LCK activity 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 early relapsed patients, who usually show an unfavourable prognosis.Conclusions/SignificanceWe conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

Low PKCα expression within the MRD-HR stratum defines a new subgroup of childhood T-ALL with very poor outcome

“…Cell lysis, protein extraction and quantification, samples dilution and RPPA procedure were performed as described by Accordi et al [26]. Protein lysates were loaded into a 384-well plate and serially diluted with dilution buffer into four-point dilution curves (from undiluted to 1:8).…”

“…53 antibodies specific for key signalling molecules involved in different cell pathways were tested (Table S2). Each primary antibody used in RPPA was previously subjected to an extensive validation for single band specificity by Western Blot [26]. The TIF images of antibody or Fast Green FCF stained slides were analyzed using MicroVigeneTM software (VigeneTech Inc, Boston, MA) and every protein expression or activation signal was quantified for each sample.…”

Sox4 cooperates with CREB in myeloid transformation

“…21 In brief, patients and cell lines samples were lysed in an appropriate lysis buffer and printed in 4-point dilution curves in duplicate on nitrocellulose-coated slides (FAST slides; Whatman Schleicher & Schuell) with the 2470 Arrayer (Aushon BioSystems). Slides were stained for total protein content (Fast Green FCF; Sigma-Aldrich) and with 3 different previously validated antibodies using the Catalyzed Signal Amplification System kit (Dako North America).…”

Microwave and Magnetic (M2) Proteomics of Childhood B-ALL

“…Previous studies have included measurements using genomic, transcriptomic [27][28][29][30] and proteomic [9][10][11][12][13][14] approaches. This study utilizes a new approach, M 2 proteomics, to examine differences in protein expression between cytogenetic subtypes of childhood B-ALL.…”

“…In contrast, there are only a few reports of proteomic analysis of childhood ALL [6,7,[9][10][11][12][13][14]. Significantly, few previous studies have employed isobaric labeling techniques such as 'tandem mass tagging' (TMT) [15,16] or 'super' stable isotopic labeling of amino acids in culture (SILAC) [17,18] for quantitative MS/MS-based proteomics.…”