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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.

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.

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.

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.