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Sarcopenia is the age-related progressive loss of skeletal muscle mass and strength finally leading to poor physical performance. Impaired myogenesis contributes to the pathogenesis of sarcopenia, while mitochondrial dysfunctions are thought to play a primary role in skeletal muscle loss during aging. Here we studied the link between myogenesis and metabolism. In particular, we analyzed the effect of the metabolic modulator trimetazidine (TMZ) on myogenesis in aging. We show that reprogramming the metabolism by TMZ treatment for 12 consecutive days stimulates myogenic gene expression in skeletal muscle of 22-month-old mice. Our data also reveal that TMZ increases the levels of mitochondrial proteins and stimulates the oxidative metabolism in aged muscles, this finding being in line with our previous observations in cachectic mice. Moreover, we show that, besides TMZ also other types of metabolic modulators (i.e., 5-Aminoimidazole-4-Carboxamide Ribofuranoside-AICAR) can stimulate differentiation of skeletal muscle progenitors in vitro. Overall, our results reveal that reprogramming the metabolism stimulates myogenesis while triggering mitochondrial proteins synthesis in vivo during aging. Together with the previously reported ability of TMZ to increase muscle strength in aged mice, these new data suggest an interesting non-invasive therapeutic strategy which could contribute to improving muscle quality and neuromuscular communication in the elderly, and counteracting sarcopenia.

Brown adipose tissue (BAT) activity plays a key role in regulating systemic energy. The activation of BAT results in increased energy expenditure, making this tissue an attractive pharmacological target for therapies against obesity and type 2 diabetes. Sirtuin 5 (SIRT5) affects BAT function by regulating adipogenic transcription factor expression and mitochondrial respiration. We analyzed the expression of SIRT5 in the different adipose depots of mice. We treated 3T3-L1 preadipocytes and mouse primary preadipocyte cultures with the SIRT5 inhibitor MC3482 and investigated the effects of this compound on adipose differentiation and function. The administration of MC3482 during the early stages of differentiation promoted the expression of brown adipocyte and mitochondrial biogenesis markers. Upon treatment with MC3482, 3T3-L1 adipocytes showed an increased activation of the AMP-activated protein kinase (AMPK), which is known to stimulate brown adipocyte differentiation. This effect was paralleled by an increase in autophagic/mitophagic flux and a reduction in lipid droplet size, mediated by a higher lipolytic rate. Of note, MC3482 increased the expression and the activity of adipose triglyceride lipase, without modulating hormone-sensitive lipase. Our findings reveal that SIRT5 inhibition stimulates brown adipogenesis in vitro, supporting this approach as a strategy to stimulate BAT and counteract obesity.

// Maria Rosaria Ricciardi 1 , Valentina Salvestrini 2 , Roberto Licchetta 1 , Simone Mirabilii 1 , Mattia Forcato 3 , Gabriele Gugliotta 2 , Simona Salati 3 , Fausto Castagnetti 2 , Gianantonio Rosti 2 , Massimo Breccia 4 , Giuliana Alimena 4 , Rossella Manfredini 3 , Silvio Bicciato 3 , Roberto Massimo Lemoli 5 and Agostino Tafuri 1 1 Hematology Unit, Sant'Andrea University Hospital, Department of Clinical and Molecular Medicine, Sapienza University of Rome, Rome, Italy 2 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. and A. Seragnoli,” University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy 3 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy 4 Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy 5 Clinic of Hematology, Department of Internal Medicine (DiMI), University of Genoa, Genoa, Italy Correspondence to: Agostino Tafuri, email: agostino.tafuri@uniroma1.it Keywords: chronic myeloid leukemia; CD34+ cells; proteomic profile; cell signaling; apoptosis Received: July 08, 2017 Accepted: March 06, 2018 Published: April 24, 2018 ABSTRACT Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of quiescent cells which are to some extent resistant to tyrosine kinase inhibitors (TKIs). BCR-ABL oncogene activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, contributing to abnormal proliferation of clonal cells. From this perspective, the aim of this study was to analyze the expression and activation profile of STP involved in the mechanisms of cell proliferation/quiescence and survival of the progenitor CD34+ cells from chronic phase (CP) CML. Our results showed that CP-CML CD34+ progenitors were characterized by significant lower phosphorylation of proteins involved in the regulation of growth and cell survival, such as tyrosine kinases of the Src family and members of STAT family, and by a significant higher phosphorylation of p53 (Ser15), compared to normal CD34+ cells from healthy donors. Consistent with these results, cell cycle analysis demonstrated that CP-CML CD34+ cells were characterized by higher percentage of cells in G0-phase compared to normal CD34+ cells. Analysis of expression profile on proteins involved in the apoptotic machinery revealed that, in addition, CD34+ cells from CP-CML were characterized by a significant lower expression of catalase and higher expression of HSP27 and FADD. In sum, we report that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence through the inhibition of proliferation and the promotion of survival. This differential signaling activation network may be addressed by novel targeted therapies aimed at eradicating CML stem cells.

Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.

Vitiligo is characterized by death or functional defects of epidermal melanocytes through still controversial pathogenic process. Previously, we showed that mitochondria-driven pre-senescent phenotype diminishes the capability of vitiligo melanocytes to cope with stressful stimuli. In the current study, we investigated markers of mitochondrial energy metabolism including the PGC1a axis, and then we determined the index of mitochondrial impairment using a cytomic approach. We found in cultured epidermal vitiligo melanocytes, compared to healthy ones, low ATP, increased proton leakage, and altered expression of several glycolytic enzymes (hexokinase II, pyruvic dehydrogenase kinase 1 and pyruvic kinase M2), We suggest that the low ATP production may be sufficient in steady-state conditions but it is unable to cover further needs. We also found in vitiligo melanocyrtes hyper-activation of the PGC1α axis, finalized to counteract the energy defect. Cytomic analysis, supported by MitoTracker Red pattern and ex-vivo immunohistochemistry, suggested an increased mitochondrial mass, possibly useful to ensure the essential ATP level. Finally, pharmacological cardiolipin stabilization reverted the energetic impairment, confirming the initial mitochondrial role. In conclusion, we report new insight in the pathogenetic mechanism of viitligo and indicate that the mitochondrial failure rescue by cardiolipin manipulation may be a new intriguing target in treatment development.

Over the last few decades, there has been significant progress in the understanding of the pathogenetic mechanisms of the Acute Myeloid Leukemia (AML). However, despite important advances in elucidating molecular mechanisms, the treatment of AML has not improved significantly, remaining anchored at the standard chemotherapy regimen “3 + 7”, with the prognosis of patients remaining severe, especially for the elderly and for those not eligible for transplant procedures. The biological and clinical heterogeneity of AML represents the major obstacle that hinders the improvement of prognosis and the identification of new effective therapeutic approaches. To date, abundant information has been collected on the genetic and molecular alterations of AML carrying prognostic significance. However, not enough is known on how AML progenitors regulate proliferation and survival by redundant and cross-talking signal transduction pathways (STP). Furthermore, it remains unclear how such complicated network affects prognosis and therapeutic treatment options, although many of these molecular determinants are potentially attractive for their druggable characteristics. In this review, some of the key STP frequently deregulated in AML, such as PI3k/Akt/mTOR pathway, GSK3 and components of Bcl-2 family of proteins, are summarized, highlighting in addition their interplay. Based on this information, we reviewed new targeted therapeutic approaches, focusing on the aberrant networks that sustain the AML blast proliferation, survival and drug resistance, aiming to improve disease treatment. Finally, we reported the approaches aimed at disrupting key signaling cross-talk overcoming resistances based on the combination of different targeting therapeutic strategies.

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Mushrooms represent a formidable source of bioactive compounds. Some of these may be considered as biological response modifiers ; these include compounds with a specific biological function: antibiotics (e.g. plectasin), immune system stimulator (e, g, lentinan), antitumor agents (e.g. krestin, PSK) and hypolipidemic agents (e.g. lovastatin) inter alia. In this study, we focused on the Chinese medicinal mushroom "yun zhi", Trametes versicolor, traditionally used for (cit.) "replenish essence and qi (vital energy)". Previous studies indicated the potential activity of extracts from culture filtrate of asexual mycelia of T. versicolor in controlling the growth and secondary metabolism (e.g. mycotoxins) of plant pathogenic fungi. The quest of active principles produced by T. versicolor, allowed us characterising an exo- polysaccharide released in its culture filtrate and naming it Tramesan. Herein we evaluate the biological activity of Tramesan in different organisms: plants, mammals and plant pathogenic fungi. We suggest that the bioactivity of Tramesan relies mostly on its ability to act as pro antioxidant molecule regardless the biological system on which it was applied.

Scientific Reports 5: Article number: 18137; published online: 17 December 2015; updated: 05 February 2016 This Article contains errors in the Author Contributions Statement. “M.A. designed and performed the research, analyzed data and wrote the manuscript; M.R.R. designed the research, analyzed data and edited the manuscript; S.

Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML.

The mammalian target of rapamycin (mTOR) is a central processor of intra- and extracellular signals, regulating many fundamental cellular processes such as metabolism, growth, proliferation, and survival. Strong evidences have indicated that mTOR dysregulation is deeply implicated in leukemogenesis. This has led to growing interest in the development of modulators of its activity for leukemia treatment. This review intends to provide an outline of the principal biological and molecular functions of mTOR. We summarize the current understanding of how mTOR interacts with microRNAs, with components of cell metabolism, and with controllers of apoptotic machinery. Lastly, from a clinical/translational perspective, we recapitulate the therapeutic results in leukemia, obtained by using mTOR inhibitors as single agents and in combination with other compounds.

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL.

We have demonstrated that selective MEK inhibitors (MEK-I) significantly inhibit in vitro the growth of AML cell lines and primary AML blasts (Ricciardi et al., JMM 2012). However, these effects were mostly related to the inhibition of cell cycle progression, while apoptosis induction requires higher concentrations of the inhibitor and longer times of exposure. Among the many downstream effectors of MAPK pathway, metabolism is one of the key aspects that can be targeted at several levels for therapeutic perspectives (Ricciardi et al., Blood 2015). In this study, we have explored on AML cells the functional effects of combining the MEK1/2 inhibitor, PD0325901 (PD), with a metabolic modulator, dichloroacetate (DCA), which acts as pyruvate dehydrogenase kinase (PDHK) inhibitor. AML cell lines (OCI-AML3, U937, MOLM13, HL60) and AML primary samples were exposed to PD (2.5-1000nM) and DCA (0.5-2mM), alone or in combination. PD dose-dependently inhibited cell growth of OCI-AML3, HL60 and MOLM13, showing a constitutive activation of RAS/RAF/MEK/ERK pathway. This activation was not seen in U937, thus exhibiting resistance to PD. DCA shows a pro-apoptotic activity on AML cells, only at the highest concentration. The combination between PD and DCA synergistically enhanced the antiproliferative effect, with a combination index (CI) ranging between 0.37 and 0.41 in OCI-AML3, as measured by isobologram analysis from MTT data. Conversely, in U937 the combination resulted antagonistic. Co-administration of non- or sub-toxic concentrations of DCA (2mM) and of PD (10nM) resulted in a strong increase of cell death in OCI-AML3 (Annexin V). The PD/DCA combination significantly increased Annexin V+ cells, at 72 hours, (65.7±25.0%), compared to single compounds: 16.4±8.7% with 10nM PD (p=0.03) and 28.0±12.4% with 2mM DCA (p=0.04). Similar results were obtained in HL60 and MOLM13 (data not shown). In the U937 cell line, resistant to PD, the levels of apoptosis observed with DCA alone were not further enhanced by the combination: 6.19±1.7% (1000nM PD), 23.63±6.7% (2mM DCA), 31.28±9.9% (PD/DCA). At a protein level, densitometric analysis of Western blot carried out on OCI-AML3 demonstrated that PD/DCA was able to further increase PD-induced ERK inhibition (from 40% to 54%), while DCA alone, as expected, had no effect. Real time metabolic data, obtained by a Seahorse XF24 on OCI-AML3, showed that the combination of PD and DCA affected mitochondrial metabolism: basal respiration and ATP production were impaired by 37.34±10.4% (p=0.012) and by 56.31±10.0% (p=0.003), respectively, compared to control. On the other hand, both maximal respiration and spare respiratory capacity were increased by 28.80±9.5% (p=0.025) and 68.78±14.2% (p These preliminary results suggest that the combination of a signal transduction and a metabolic inhibitor spares mitochondrial machinery while impairing at the same time its activity, thus resulting in an enhanced antiproliferative and pro-apoptotic effect on AML cells. Disclosures No relevant conflicts of interest to declare.

Abstract 3733 The tyrosine kinase inhibitors (TKIs) represent the successful molecular therapy for patients with chronic myeloid leukemia (CML), targeting the Bcr-Abl oncogenic product. However, this disease may remain not curable for the presence of residual refractory cells, persisting during the history of the disease treatment. Several mechanisms have been associated with resistance to TKIs, including the presence of rare quiescent leukemic stem cells, less susceptible to TKIs. Moreover, Bcr-Abl activates additional downstream pathways involved in the apoptotic and proliferation control of CML cells, such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5 pathways, potentially contributing to CML TKIs drug resistance. Therefore, in this study we aimed to investigate, at the protein level, proliferative and apoptotic signal transduction pathways (STP) in CML CD34+ cells, as compared to normal CD34+ cells, in order to identify additional aberrant signals, potentially therapeutic targetable. CD34+ cells were purified from peripheral blood (PB) of five newly diagnosed, chronic phase (CP) CML patients, three normal cord blood (CB) and one leukapheretic product of a normal volunteer (PBSC). The phosphorylation status of 46 proteins from various STP and the expression of 32 proteins of the apoptotic machinery were assessed by using a customized direct phase proteome profiler antibody array. The resulting dots were visualised using ECL and quantified by densitometric analysis. CML samples were collected from patient in CP, with a WBC count ranging between 41900–135400 per microliter. The Sokal risk category was low (1/5) and intermediate (4/5). The comparison between normal CD34+ cells obtained from CB and PBSC showed that the first cells were characterized by a lower expression of STAT, Tyrosine-protein kinase and MAPK protein families. The phospho-proteomic profile of CD34+ cells from CML samples showed remarkably similarity when compared to normal CB CD34+ cells, while only two proteins resulted differently expressed in CP CD34+ cell vs. PBSC: CREB-S133, involved in the pro-survival/anti-apoptotic gene control (p=0.025) and p70S6K-T389, along the PI3k/Atk pathway and involved in the cell proliferation (p=0.049). The analysis of the 32 apoptotic proteins revealed that 10 of them were statistically significant lower in CML CD34+ cells compared to PBSC. Most of them were related to the Bcl-2 family and caspase inhibitors family, such as cIAP-1 (p=0.025), cIAP-2 (p=0.0003) and livin (p=0.016). The expression of the cyclin-dependent kinase inhibitors (CKI) was found significantly lower in CML CD34+ (p=0.05 and p=0.02 for p21/CIP1 and p27/Kip1, respectively). In conclusion, we have reported in this study that proteins and/or phosphoproteins controlling apoptosis and proliferation STP, such as Bcl-2, IAP, MAPK, PI3K/Akt, STATs and CKI families, are differentially expressed in CD34+ from CML CP, compared to PBSC. The understanding of these differences in the proteomic profile may confirm that additional multiple aberrant STP are involved in the CML and therefore must be taken into account for targeted therapies, especially of resistant cases. Disclosures: Petrucci: Jansse-Cilag, Celgene: Honoraria. Castagnetti:Novartis Pharma: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau. Rosti:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of kinetic quiescent cells, frequently resistant to tyrosine kinase inhibitors (TKIs). The Bcr-Abl oncogene and the resulting fusion protein, in fact, activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, potentially contributing to TKIs drug resistance. Since increasing evidence reports the cooperation of numerous STP in the control of cell proliferation and survival in CML, the aim of this project was to analyze, at the protein level, the expression and activation profile of various STP involved in the mechanisms of cell proliferation and survival of CML CD34+ cells, as compared to different sources of normal CD34+ cells. CD34+ cells were purified by immunomagnetic separation from peripheral blood (PB) of 7 newly diagnosed chronic phase (CP) CML patients and compared to the normal counterpart obtained from normal bone marrow of three healthy donors (NBM) and/or from umbilical cord blood (CB) of three donors. The phosphorylation status of 46 different proteins belonging to numerous STP and the expression of 32 proteins of the apoptotic machinery were assessed by using a customized direct phase proteome profiler antibody array. The resulting dots were visualised using ECL and quantified by densitometric analysis. CP-CML samples were obtained from patients with WBC counts ranging between 41,900 to 421,400; Sokal score resulted intermediate in six patients and low in one. The comparison between the phospho-proteomic profile of CP-CML CD34+ cells and NBM CD34+ cells showed that the former are characterized by: 1) lower phosphorylation of STAT2 (p=0.023), Chk-2 (p=0.036), a serine/threonine-protein kinase required for checkpoint-mediated cell cycle arrest, and tyrosine kinases of the Src family - Lck, Fyn, Src, particularly Yes (p=0.04) - involved in the regulation of growth and cell survival; 2) higher phosphorylation of p53, both at Ser15 (p=0.047) and at Ser46 (p=0.039), p70S6 kinase (p=0.035), RSK (p=0.046), a mediator of mitogens- and stress-induced activation of several transcription factors, and Pyk-2 (p=0.036), a tyrosine kinase involved in cell adhesion and migration. The analysis of the 32 apoptotic proteins revealed that CD34+ cells from CP-CML, compared to CD34+ cells from NBM, are characterized by: 1) lower expression of the catalase (p=0.044), an enzyme that protects cells from the toxic effects of hydrogen peroxide and promotes growth of normal and neoplastic cells including myeloid leukemia cells; 2) higher expression of some members of the heat shock proteins family - HSP60 and HSP70 (p=0.033). We then compared CD34+ cells obtained from CP-CML with the other normal CD34+ cell source represented by the CB. The proteomic profile indicated a remarkable similarity between the CD34+ from CP-CML and those from CB. Accordingly, the two normal CD34+ cells sources showed some differences: in particular, as for CP-CML CD34+ cells, those from CB had significantly lower phosphorylation of STAT2 (p=0.026), of Chk-2 (p=0.014) and higher phosphorylation of p53 at Ser15 (p=0.05), compared to NBM CD34+ cells. In summary, we reported that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence status through the inhibition of proliferation. A striking similarity was found between CD34+ cells obtained from CP-CML and those from CB. The two normal sources of CD34 displayed differences in the activation status of selected proteins. The presence of these additional and complex changes in the signaling network of CP-CML must be taken into account for the investigation on novel targeted therapies. Disclosures: Castagnetti: Novartis Farma : Consultancy, Honoraria; Bristol Myers Squibb : Consultancy, Honoraria. Rosti:Bristol Myers Squibb : Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Acute myeloid leukemia (AML) has been characterized by a growing number of recurrent genetic alterations, frequently causing the constitutive activation of signal transduction pathways, which result in enhanced blast proliferation and prolonged survival. Despite the increased understanding of AML biology, prognosis of these patients remains, generally, severe. Therefore, novel therapies targeted on aberrant signaling are now under evaluation. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a pivotal role in the control of cell growth, proliferation and apoptosis of AML, and is often found constitutively activated. Currently, several inhibitors of this pathway, especially those inhibiting the activity of mTOR, have been investigated showing a limited efficacy due to the reactivation of upstream nodes. Since a novel PI3K inhibitor, BKM120, has been evaluated with encouraging results on solid tumors and lymphoid malignancies, we aimed in this study to investigate the pre-clinical activity of BKM120 on cell lines and primary AML samples. BKM120, kindly provided by Novartis, was tested on myeloid leukemia cell models - U937, MOLM13, OCIAML3 and KG1 - and on 4 primary AML samples at concentrations ranging from 0.5 to 5µM. Cell cycle changes and apoptosis induction were analyzed using the Acridine-Orange technique (AO) and the Annexin V (AnnV) binding assay. Intracellular signaling modulations induced by BKM120 were evaluated at different times (3h and 24h) by Western blot analysis. BKM120 exhibited dose- and time-dependent anti-proliferative and pro-apoptotic effects on the all analyzed leukemia cell models, although with different sensitivity (IC50s of 1.6, 2.4, 2.7 and 3.2μM for the U937, MOLM13, OCIAML3 and KG1 cell lines, respectively). On the most sensitive cell line U937, BKM120 in vitro exposure resulted, at 24h, in a significant cell growth reduction of 48.6% (p=0.006) and 73.9% (p=0.006), at 1 and 2µM, respectively. After 72h of treatment, AnnV positive cells significantly increased from 7.4% ± 0.03 (vehicle) to 43.5% ± 0.08 (p Disclosures: No relevant conflicts of interest to declare.

Glycolysis is the central axis of cellular metabolism. The cancer cell bioenergetic status heavily relies on high glycolytic rates, even in aerobic conditions, thus sustaining the expensive processes of cell growth and proliferation. Growing evidences show that signaling aberrations - especially those involving PI3K/Akt/mTOR, HIF1a, Ras/Raf/MEK/ERK - are strictly connected to the establishment of a pro-glycolytic metabolism, through a multi-level crosstalk between proteins and metabolites that contribute to the acquisition of an energetic background granting a proliferative advantage. Here we investigated the glycolytic rate of resting and activated normal peripheral blood lymphocytes (NPBLs) and of acute myeloid leukemia (AML) cell lines. In an attempt to modulate the cellular metabolism for therapeutic intervention, we tested the following compounds that directly interfere with major metabolic or signaling pathways: dichloroacetate (DCA), a glycolysis inhibitor; aminooxyacetate (AOA), a glutaminolysis inhibitor; ST1326 (kindly given by Sigma-Tau), a fatty acid oxidation (FAO) inhibitor; and the MEK inhibitor PD0325901 (Selleck Chemicals). The cytotoxic drug effects were evaluated on two human leukemia cell lines, U937 and OCI-AML3, characterized by PI3K/Akt/mTOR and Ras/Raf/MEK/ERK hyperactivation, respectively. Cell counts, apoptosis (AnnexinV), glucose and lactate levels (GEM4000, Instrumentation Laboratory, UK) were measured. The glucose consumption rate (GCR) and lactate production rate (LPR) were calculated according to Li et al. (Biotechol. Appl. Biochem., 2005, 42, 73-80). Resting NPBLs were characterized by a very low glycolytic rate, according to their quiescent state, while cultured phytohemagglutinin-activated NPBLs displayed a remarkable increase in glycolytic rate: the GCR calculated over 72 hours showed a 25 fold-increase, while LPR had a of 10 fold-increase. Acute myeloid cell lines showed a high glucose catabolism: at 24h the U937 cell line, compared to activated NPBLs, had a 6.7 fold higher GCR, while the OCI-AML3 cell line showed a 4-fold increase. DCA exposure showed at 24h no detectable effect on GCR, LPR and apoptosis at concentrations ranging from 0.01 to 0.5mM on the U937 cell line. Apoptosis effects were detected only at higher concentrations: AnnexinV positive cells increased from 4.3 ± 1.5 (control) to 62.8 ± 16.4 (5mM) and 88.1 ± 16.8 (10mM). Exposure to AOA (24h at 1000µM) slightly increased GCR (1.23-fold) and LPR (1.22-fold) on U937 cells, followed by apoptotic effects at 72h: from 6.24 ± 4.2 (control) to 10.4 ± 0.8 at 100µM to 83.5 ± 0.7 at 1000µM. The FAO inhibitor ST1326 (10µM at 24h) induced a 3-fold increase of GCR in the U937 cell line. Apoptotic effects were seen in the U937 cells at 72h, from 5.0 ± 2.3 (control) to 35.9 ± 5.7 at 5µM to 64.1 ± 20.5 at 10µM. Conversely, GCR, LPR and apoptosis did not change on the OCI-AML3 line following ST1326 exposure. The MEK inhibitor PD0325901 caused a reduction of GCR and LPR on OCI-AML3 cells (6-fold GCR decrease, 2-fold LPR decrease at 100nM); apoptosis at 72h ranged from 6.3 ± 1.1 (control) to 15.5 ± 3.9 at 10nM to 45.3 ± 0.1 at 100nM. The U937 cells proved resistant to this compound, showing no metabolic perturbation and absence of apoptotic effects. In summary, this study indicates that exploiting the metabolism as a target for therapeutic intervention appears to be a promising new strategy. In fact, the inhibition of glycolysis by blocking either the activity of the enzymes that directly participate to the metabolic pathway or key components of cell signaling has proven to be effective in inducing apoptosis in AML cells. Interestingly, the opposing response to the various compounds observed in the two AML models may likely reflect their divergent signaling network, prompting further studies to evaluate the correlation between aberrant signal transduction pathways and peculiar metabolic profiles. Disclosures: Nicolai: Sigma Tau Pharmaceuticals: Employment.

Abstract 1371 Aberrant signal transduction pathways influence metabolic changes in neoplastic cells. It is well established, in fact, that cancer cells are characterized by a pro-glycolytic metabolic phenotype and recent evidences confirmed that also leukemia cells may switch to a non-oxidative metabolism. Among the different substrates used to sustain the anabolic processes and to keep the tricarboxylic acid cycle (TCA) active, the fatty acid oxidation (FAO) may represent an alternative carbon source. The carnitine palmitoyl transferase 1a (CPT1a) catalyzes the first step of FAO, by loading long chain fatty acyl groups onto carnitine, transporting them through the mitochondrial membrane. Moreover, CPT1a has been previously demonstrated to interact with members of the apoptotic machinery, such as Bcl-2 and t-Bid, and its inhibition can cause an accumulation of the toxic metabolite palmitate, resulting in mitochondrial damage and cell death. In diabetic patients the use of a recently discovered antihyperglycemic agent characterized by the selective and reversible inhibition of CPT1a has been reported. Therefore, we aimed in this study to investigate the in vitro anti-leukemic effect of the CPT1a inhibition. Particularly, we evaluated the activity of two CPT1a-inhibitors, the novel ST1326 (kindly provided by Sigma-Tau) and the previously known Etomoxir on the proliferation and apoptosis of leukemia cell lines and primary cells obtained from patients affected by acute leukemias. The drug concentration inducing a 50% cell killing (IC50) was calculated from the dose-response curve. The cytotoxic drug effects on leukemia cell lines (HL-60, HL-60/MX2, U937, K562, CEM S, CEM R, MOLT-4) and primary cells of acute myeloid (AML) and lymphoid (ALL) leukemia were evaluated using the MTT test. Flow cytometry techniques Acridine-Orange staining, AnnexinV binding assay and Chloromethyl-X-Rosamine (CMXRos)/MitoTracker Green (MTGreen) staining were used to examine cell cycle changes, apoptosis and the loss of mitochondrial membrane potential (ΔΨm). We evaluated the activity of ST1326 (1–50 μM) on leukemia models, demonstrating a dose- and time-dependent cell growth arrest, caused by mitochondrial damage and apoptosis induction. In fact, 6 hours of ST1326 exposure were able to induce a dramatic loss of ΔΨm, as observed in MOLT-4 cells (cells affected increased from 15% in the control to 59.7% at 50 μM). Following 72 hours of ST1326 exposure, the same cells showed then an increase in the subG1 peak from a baseline value of 10% to 8.1%, 7.3%, 10.5%, 37% and 95.8% at 1, 5, 10, 20 and 50 μM, respectively (IC50= 39.2 μM). Etomoxir failed to show any activity (data not shown). The myeloid cell lines HL60, HL60/MX2 and U937 were more sensitive to ST1326, showing an IC50 of 11.8, 8.2 and 8.8 μM, respectively. A milder activity was found in the other lymphoid-derived model, the CEM S cell lines (IC50= 71.1 μM). Instead, both the lymphoid CEM R and the myeloid K562 cell lines proved resistant (IC50= n.d.). We then exposed in vitro (72 hours) primary cells from 12 AML and found in all a significant pro-apoptotic activity (AnnexinV positive cells): from 23.27% ± 13.63 (control) to 36.59% ± 19.97 (p=0.23), 40.51% ± 18.66 (p=0.0074), 43.43% ± 19.81 (p=0.0071) and 75.30% ± 11.52 (p=0.00018) following 72 hours of exposure to 5, 10, 20 and 50 μM ST1326, respectively. ALL primary samples (6/8 samples), instead, showed a significant (p Disclosures: Petrucci: Jansse-Cilag, Celgene: Honoraria. Nicolai:Sigma Tau Pharmaceutical Industries SpA: Employment. Tafuri:Sigma Tau Pharmaceuticals: Research Funding.