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// 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.

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.

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.

Adult acute lymphoblastic leukemia (ALL) is characterized by a high relapse rate, with the majority of patients developing chemo-resistance and ultimately dying of the disease with a 5-year survival rate of 40% (Faderl et al., 2010). Significant advances, however, have been made in cases carrying the acquired genetic alteration BCR-ABL (ALL-Ph+) targeted by tyrosine-kinase inhibitors (Ottmann & Pfeifer, 2009). Therefore, several studies have recently been carried out to look for additional, therapeutically exploitable, genetic lesions. Aberrant activation of signal transduction pathways (STP) implicated in proliferation and survival mechanisms are generally involved in leukemogenesis and drug resistance (Zhao et al., 2010). Genes in the PI3K/PTEN/AKT/mTOR, RAS/RAF/MEK/ERK, and Jak/STAT pathways are frequently mutated and their expression is often altered in hematopoietic malignancies, including ALL (McCubrey et al., 2011; Steelman et al. 2008). In addition, deregulation of survival mechanisms may confer chemo-resistance to leukemic cells, particularly involving alterations of the Bcl-2 signaling cascade, which may represent one of the most important, potentially druggable, pathways for therapeutic intervention in ALL. Starting from our studies on chemo-resistance in ALL, particularly on multidrug resistance (MDR1) expression and prognostic significance, in this chapter we will illustrate the major pathways aberrantly activated in ALL - PI3K/PTEN/AKT/mTOR, RAF/RAS/MEK/ERK, and the Bcl-2 family of proteins - with the ultimate goal of summarizing novel targets for molecular therapies, especially in resistant/refractory cases. Controversies on and potential benefits of novel approaches based on STP inhibitors will be discussed. Epigenetic changes will be examined to address their importance in association with post-translational modifications. Future perspectives will encompass review of Reverse Phase Protein Arrays (RPPA) as a promising new tool for translational studies in clinical sample series, helping define the functional proteomic profile at steady-state; this approach may lead to the identification of aberrant molecular target(s), possibly guiding the choice of specific molecular therapies, on one hand, and leading to the elucidation of post-treatment changes, on the other, with the overall objective of improving the strategies currently employed to counteract leukemia chemo- and targeted drug-resistance.

Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects in vitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1's binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions.

Abstract 3228 Although they frequently achieve complete remission (CR), adult patients with acute lymphoblastic leukemia (ALL) subsequently experience leukemia relapse, which represents an unresolved therapeutic problem. Based on the observation that ALL cells are frequently characterized by the deregulation of the apoptotic machinery, we and others have evaluated pre-clinically the activity of ABT-737 (kindly provided by Abbott Laboratories), a BH3-mimetic Bcl-2/Bcl-XL inhibitor, displaying a potent growth-inhibitory activity in ALL cell lines and primary cells. However ABT-737 binds to the anti-apoptotic protein Mcl-1 with low affinity and Mcl-1 expression may mediate resistance to ABT-737. Since ALL is also characterized by the aberrant activation of the mTOR and related signalling pathways, in the present study we further evaluated the combined Bcl-2/Bcl-XL (by ABT-737) and mTOR (by CCI-779) inhibition focusing, in particular, on the activity of combined molecularly targeted therapies on resistant cells. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed, at higher concentrations (250-500nM), by apoptosis induction. In contrast, the CEM-S, CEM-R, JURKAT, DAUDI and RAJI cells proved resistant (IC-50 >5 μM). When we explored the effects of CCI-779 on the aforementioned cell lines, only minor cytostatic effects were observed (IC-50 0.5–28.2μM). MOLT-4 cells, for example, showed a flat dose-response curve (35-55% growth inhibition) at concentrations ranging between 1 and 5000 nM (IC50=9,87μM) and apoptosis induction was not seen until 5000 nM. We next investigated the effects of the combined use of ABT-737 and CCI-779 (each at 1000nM) in the ABT-resistant JURKAT cells. A significant (p= 0.04) induction of apoptosis was observed with the combination, as compared with single agents, after 24 h (47.7% ±5.9 of cells with sub-G1 DNA content with ABT-737 + CCI-779, compared to 17.4% ±1.5 and 4.2% ±1.5 with ABT-737 and CCI-779 as single agents, respectively). Similarly, when we exposed CEM-R cells to the drug combination (ABT-737 1000nM and CCI-779 5000nM) for 24 h, a strikingly stronger apoptosis induction (sub-G1 peak= 75.3% ±16.8) was observed, compared to single agents (15.8% ±7.2 and 4.2% ±1.9 with ABT-737 and CCI-779 alone, respectively) (p=0.0003). These effects were confirmed by measuring Annexin V binding. WB analysis showed decreased Mcl-1 levels, following exposure to CCI-779 and further downregulation in response to combined ABT-737+CCI-779 in the CEM-R cell line. These effects, however, were not seen in the parental CEM-S cell line. Primary cells, obtained from 10 ALL patients, showed an increase of the sub-G1 peak in 7/10 and in 4/10 samples, after exposure to ABT-737 (50nM) and CCI-779 (5000 nM), while synergistic effects on apoptosis induction were observed in 4/10 samples after exposure to the combination. In summary, we observed that the combined use of Bcl-2/Bcl-XL and mTOR inhibitors may exert synergistic cytotoxic effects in some resistant ALL models and this effect is associated with CCI-779-induced Mcl-1 down-regulation. Synergistic effects between these inhibitors were also found in a proportion of primary ALL samples, thus supporting further studies of combined Bcl-2/Bcl-XL and mTOR inhibitors, to overcome ALL resistance. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Tafuri:Sigma-Tau: Research Funding.

Abstract 4946 Histone deacetylase inhibitors (HDAC-I) are a class of agents with the capacity to induce acetylation of histone and non-histone proteins. These molecules have been intensively investigated in a variety of malignancies because of their ability to inhibit proliferation, induce differentiation and apoptosis in tumor cells. However, clinical response to clinically available HDAC-I have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds and at defining the molecular mechanisms of response to this class of agents. Acetyl-L-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane, thereby contributing to the regulation of energy production and metabolism. ALCAR activity as a modulator of cellular stress response has prompted its use to protect against chemotherapy-induced neurotoxicity. However, ALCAR effects on neoplastic cells are still not defined, especially in combination with chemotherapy. Here we investigated the effects of MS-275, a HDAC-I, on cell proliferation and apoptosis in cell line models of acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), in comparison with vorinostat also known as SAHA (suberoyl anilid hydroxamic acid), the most widely used HDAC-I in clinical setting. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and selected sub-toxic concentration of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing 50 % cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine- Orange (AO) technique. Results indicated that the tested compound MS-275 significantly inhibited cell growth, as assessed by MTT assay, when used at of 5000nM. Comparative analysis of the efficacy of the two different HDAC-I compounds indicated that MS-275 was the more effective agent and the only one with clear dose-dependent activity, while SAHA displayed a flat dose-response curve, which dropped only at the highest concentration. In particular, the myeloid cell line Molm-13 was strikingly sensitive to MS-275 (IC50: < 15 nM), U937 and HL60 myeloid cell lines, the lymphoid cell line Jurkat and the MM cell line ARH-77 showed intermediate sensitivity (IC50: < 1000 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). SAHA showed no activity in U937 cells when used at concentrations ranging from 100 to 1000 nM, with a dramatic reduction of absorbance at 5000 nM (>80% reduction compared to the control). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR reveled a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction, as determined by the percentage of cells with a sub-G1 DNA content. MS-275 dose-dependently induced apoptosis in HL-60 cells (4.2%, 17.1%, 60.8%, and 87.5% in the presence of 100, 500, 1000, 5000 nM of MS-275, respectively). Conversely, SAHA induced minimal apoptosis (< 10%) at concentration ranging from 100 to 1000 nM, although > 75% of cells became apoptotic after treatment with the compound at 5000 nM. In summary, our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in cell lines derived from myeloid leukemia and MM. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in selected hematological malignancies. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Pisano: Sigma-Tau: Employment. Tafuri: Sigma-Tau: Research Funding.

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.

Abstract 3581 ALL cells are frequently characterized by deregulation of several intracellular signaling pathways involved in the control of proliferation and apoptosis. Among these, the constitutive activation of the mTOR signaling cascades has been frequently reported in the majority of ALL patients and molecular therapies targeting mTOR have been therefore proposed with variable preclinical efficacy, generally inhibiting proliferation in a large number of cell lines and only in some instances leading to apoptosis. To improve pro-apoptotic effects of the mTOR inhibition, especially in ALL cells, a combined Bcl-2 family inhibition approach can be explored, based on the pivotal role of Bcl-2 in lymphoproliferative disease. In addition, Mcl-1 protein levels may be controlled by the AKT/mTOR pathway, since the prosurvival AKT, negatively regulate Gsk-3. In fact, following AKT inactivation, Gsk-3 promotes apoptosis by Mcl-1 phosphorylation and its proteasome degradation. Moreover, Mcl-1 overexpression is a know resistance mechanism to the BH-3 mimetic, the ABT-737. Interestingly prolonged exposure to mTOR inhibitor rapamycin is reported to inactivate the AKT signaling pathway. Aim of this study was to evaluate the combined effects of mTOR (CCI-779) and Bcl-2/Bcl-XL (ABT-737) inhibition in ALL cell lines and primary samples (21). In MOLT-4 cell line exposure to CCI-779, up to 72 hours, induced a flat dose-response curve (35–55% growth inhibition) at concentrations ranging between 1 and 5000 nM and apoptosis induction was not seen until 5000 nM. In CEM-S, CEM-R and JURKAT cell lines, only minor cytostatic effects were observed until 20000 nM. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed at higher concentrations (250–500nM) by induction of apoptosis. In contrast, the CEM-S, CEM-R and JURKAT cells, proved resistant (IC-50 >5 μM), displaying Mcl-1 overexpression. We therefore investigated in these resistant cell lines the combined effects of CCI-779+ABT-737. The JURKAT cells showed a significantly higher (p< 0.01) induction of apoptosis following exposure to ABT-737 and CCI-779 (both at 1000nM), as compared to single agents. Similar effects were observed in CEM-R cells exposed to the drug combination (CCI-779 5000nM and ABT-737 1000nM) (p 40%) was observed in 15/21 treated with ABT-737 (50nM) (Fig.1). The remaining 6/21 samples, proved resistant to ABT-737; in two of these, interestingly, the combined treatment overcame resistance, inducing Mcl-1 down-regulation and apoptosis induction. In summary, the Bcl-2/Bcl-XL (ABT-737) inhibition is an active proapoptotic treatment of ALL, in addition the combined use of mTOR (CCI-779) inhibition may revert ABT-737 resistant phenotypes in a proportion of ALL, via AKT inactivation and Mcl-1 degradation. However, different resistant mechanisms involved in ALL cells (CEM S), prompt further investigation. Disclosures: Foà: Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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.