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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, we first explored through Reverse Phase Protein Arrays (RPPA) the phosphoproteomic profile of B-cell precursor (BCP)-ALL pediatric patients. This technique represents a powerful tool to profile protein pathways activation in order to identify novel diagnostic and prognostic biomarkers and new therapeutics targets. RPPA approach leads to measure the activation levels/phosphorylation of large numbers of signalling proteins and to identify critical interactions within the protein network. This is a potential starting point for drug development and/or design of individual therapy regimens. The principal aim of my PhD research was the discovery of new candidate biomarkers and therapeutic targets involved in the leukemic process starting from the study of the phosphoproteomic profiles of 118 BCP-ALL pediatric patients through RPPA. We screened 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 patients revealed BCL-2 hyperphosphorylation caused by AMPK activation, indicating that AMPK could be responsible of apoptosis inhibition in these patients and thus it 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 relapse, who usually show an unfavourable prognosis. We decided to first 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. Indeed its inhibition, using a commercial inhibitor (Compound C), induces cell death through the intrinsic apoptosis pathway in both cell lines and primary samples. Moreover, Compound C is able to synergize with drugs commonly used in pediatric ALL therapy. The second aim was thus to study in deep the AMPK principal activator that was also observed to be hyperphosphorylated in the previous study, LKB1. LKB1 was found to be overexpressed in MLL-rearranged leukemia at protein level, but not at mRNA level. Its specific silencing brought cell cycle block at S phase and apoptosis. Very interestingly we also found that LKB1 is hyperphosphorylated in cells during mitosis and is localized at centrosome. This result opens the possibility that LKB1 could play a key role in the control of cell cycle through regulation of elongation and stability of microtubules probably following the AMPK-MRLC pathway. Moreover LKB1 probably controls also DNA damage in MLL-rearranged leukemia, indeed its inhibition induces mitotic catastrophe. LKB1 is a master kinase and has broad known functions, even in cellular metabolism and circadian rhythm. The third aim of my PhD was thus focused on the role played by LKB1 in circadian rhythm. We report that LKB1 is a key factor in circadian control, interacting with clock-proteins. LKB1 is able to regulate clock-proteins phosphorylation, function and localization in the cell in a circadian manner. Therefore, a comprehensive understanding of the molecular links that connect LKB1 and the circadian clock could provide therapeutic benefit against cancer. For example, specific LKB1 inhibitors or activators can be more effective in the treatment of cancer if administered at a particular time of the day (chronotherapy). In my PhD thesis I showed that identification and functional studies of new aberrant molecular players involved in cancer will be useful in order to highlight the connection between these novel markers and the goal to discover more effective patient-tailored treatments.

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., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

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.

Alzheimer&prime, s disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the &gamma, secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (A&beta, ) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, PSEN1 FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to PSEN2 FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca2+ signaling and mitochondrial function. By exploiting neurons derived from a PSEN2 knock-out (PS2&ndash, /&ndash, ) mouse model, we found that, upon IP3-generating stimulation, cytosolic Ca2+ handling is not altered, compared to wild-type cells, while mitochondrial Ca2+ uptake is strongly compromised. Accordingly, PS2&ndash, neurons show a marked reduction in endoplasmic reticulum&ndash, mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2&ndash, and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.

Summary Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described., Graphical Abstract, Highlights • Donor substitution in a mitochondrial Ca2+ sensor improves photo-physical properties • Mitochondria-targeting sequence amelioration enhances the sensor localization • Donor substitution allows FLIM-FRET analysis, with a compensation for pH bias • The performance of the sensor is improved in situ, ex vivo, and in vivo, Biological Sciences Tools; Cell Biology; Optical Imaging