Hepatocyte growth factor receptor c-MET is associated with FAS and when activated enhances drug-induced apoptosis in pediatric B acute lymphoblastic leukemia with TEL-AML1 translocation.

Expression of c-MET, the HGF (hepatocyte growth factor) tyrosine kinase receptor, was investigated in pediatric B-acute lymphoblastic leukemia (ALL) patients. c-MET was found to be expressed in normal B cells and in B-ALL patients with the t(12;21) TEL-AML1 translocation, but it is not expressed in the most part of B-ALL without the t(12;21). We also found that c-MET, related to proliferation and protection from apoptosis, is associated with the pro-apoptotic protein FAS in TEL-AML1 B-ALL cells and in normal B lymphocytes. The possible role of this protein complex in drug-induced apoptosis was thus investigated in REH TEL-AML1 B-ALL cell line. REH cells prestimulated with HGF and treated with doxorubicin had shown a higher apoptotic rate than non-HGF-prestimulated ones (p = 0.03). REH cells stimulated with IL-3 and treated with doxorubicin did not undergo apoptosis more than nonstimulated cells, demonstrating that increased proliferation in itself is not directly related to the higher apoptotic sensitivity observed with HGF stimulation. These results indicate that c-MET activation enhances specifically FAS-mediated apoptosis in TEL-AML1 ALL cells and, considering that the c-MET/FAS complex is present only in normal B lymphocytes and in TEL-AML1 leukemias, this implies that it may have an important contribution in cellular homeostasis and in high sensitivity of TEL-AML1 ALL to chemotherapeutic regimens

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase 1 (ChoK 1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoK 1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.This work was supported by funds from Istituto di Ricerca Pediatrica (IRP)-Città della Speranza and Cassa di Risparmio di Padova e Rovigo—CARIPARO Foundation (project IRP13/05) and by the University of Granada, (Cei-Biotic project CEI2013-MP-1), and Associazione Italiana per la Ricerca sul Cancro (AIRC) MFAG 18459 grant (R.R.). E.M. was supported by AIRC (21101) and V.S. by FIRC (16616) fellowships

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

JRC.I.5-Systems Toxicolog

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations

Protein microarray analysis of aberrant signaling pathways in Acute Myeloid Leukemia to predict the patients responsiveness to PI3K/Akt/mTOR inhibitors

Mapping of deregulated kinases and protein signalling networks within tumors can provide a means to stratify patients with shared biological characteristics to the most optimal treatment, and identify drug targets. In particular, the PI3K/AKT/mTOR signaling pathways are frequently activated in blast cells from patients with acute myelogenous leukemia (AML), a neoplastic disorder characterized by the accumulation of genetically altered myelogenous cells displaying deregulated intracellular signalling pathways and aggressive clinical behavior with poor prognosis. Using Reverse Phase Protein Microarrays (RPMA), we have analyzed the phosphorylated epitopes of signal pathway proteins of 81 peripheral blood and bone marrow specimens with newly diagnosed AML. Patients are diagnosed according to blast content, FAB classification and cytogenetic analysis. Samples are enriched for leukemic cells by performing Ficoll separation to yield a mononuclear fraction with >60% blast cells. The objective of the study was to predict the sensitivity of each patient to PI3K/Akt/mTor inhibitors, to avoid unnecessary and toxic ineffective treatment of non-responsive patients. To this goal, fresh blast cells were grown for 16 h untreated or treated with phase I or phase II mTor or Akt inhibitors either alone or in combination. Remarkably, by unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. This confirms that this pathway might indeed represent a pharmacological target in many patients. Moreover, treatment with the above inhibitors had no effect on the phosphorylation of other selected targets, demonstrating the specificity of the above results (more than one different inhibitor was used to avoid off-target effects). More importantly, by the use of the above drugs, we have been able to discriminate within the “high pAkt” population a PI3K/Akt/mTOR inhibitor-responsive group of patients and a PI3K/Akt/mTOR inhibitor non-responsive group. In addition, our data indicate that the Akt pathway is hyper-activated in M4, M5 patients, compared to M0, M2 patients, and that a strong activation of most upstream and downstream Akt effectors correlates with an over-expression of the c-kit receptor (CD117). We believe these data are important because they, have the potential to define a profile for the personalized administration of targeted drugs

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

Background: In 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 Findings: We 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/Significance: We 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

Hovering between death and life: Natural apoptosis and phagocytes in the blastogenetic cycle of the colonial ascidian Botryllus schlosseri

Colonies of the compound ascidian Botryllus schlosseri undergo recurrent generation changes during which massive, natural apoptosis occurs in zooid tissues: for this reason the species is emerging as an interesting model of invertebrate chordate, phylogenetically related to vertebrates, for studies of apoptosis during development. In the present work, we carried out a series of morphological, cytofluorimetrical and biochemical analyses, useful for a better characterization of Botryllus apoptosis. Results are consistent with the following viewpoints: (i) both intrinsic and extrinsic pathways, probably connected by the BH3-only protein Bid, are involved in cell death induction; (ii) phagocytes, once loaded with senescent cells, frequently undergo apoptosis, probably as a consequence of oxidative stress caused by prolonged respiratory burst, and (iii) senescent phagocytes are easily recognized and ingested by other phagocytes, responsible for their clearance. In addition, results suggest the conservation of apoptosis induction mechanisms throughout chordate evolution

Differential expression of hERG1A and hERG1B genes in pediatric acute lymphoblastic leukemia identifies different prognostic subgroups.

none7nomixedPillozzi S.; Accordi B.; Rebora P.; Serafin V.; Valsecchi M.G.; Basso G.; Arcangeli A.Pillozzi, S.; Accordi, B.; Rebora, P.; Serafin, V.; Valsecchi, M. G.; Basso, G.; Arcangeli, A