Increased phospho‐m<scp>TOR</scp> expression in megakaryocytic cells derived from <scp>CD</scp>34+ progenitors of essential thrombocythaemia and myelofibrosis patients

Vicari, Luisa; Martinetti, Daniela; Buccheri, Simona; Colarossi, Cristina; Aiello, Eleonora; Stagno, Fabio; Villari, Loredana; Cavalli, Maide; Raimondo, Francesco Di; Gulisano, Massimo; Maria, Ruggero De; Vigneri, Paolo

doi:10.1111/j.1365-2141.2012.09246.x

Cited by 15 publications

(10 citation statements)

References 10 publications

(9 reference statements)

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“…Data presented indicate that mouse and human cells and cell lines expressing JAK2 V617F are exquisitely sensitive to BEZ235 as single agent showing proliferation arrest, cell cycle blockade and also, at slightly higher concentrations, induction of apoptosis. BEZ235 also inhibited cytokine-induced clonogenic growth of MPN progenitors at concentrations significantly lower than in healthy individuals with particular efficacy against megakaryocytic progenitor probably due to the important role played in this population by PI3K pathway 30, and potently inhibited formation of EEC that are mostly derived from JAK2 V617F mutated progenitors 10–49. The activity of BEZ235 against MPN cell lines and primary cells was synergistically enhanced by combination with the JAK1/JAK2 inhibitor ruxolitinib.…”

Section: Discussionmentioning

confidence: 98%

“…An aberrant activation of the PI3K/Akt pathway has been documented in JAK2 V617F mutated cells 25,26, V617F transgenic 27 or KI 28 mice and primary MPN cells 29,30. Furthermore, a possible therapeutic relevance of targeting the activated PI3K/Akt pathway is supported by the improvement of splenomegaly and constitutional symptoms observed in MF patients enrolled in a clinical trial with everolimus, a rapamycin-derivative inhibitor (rapalog) of mTOR 31.…”

Section: Introductionmentioning

confidence: 95%

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Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Bartalucci

Tozzi

Bogani

et al. 2013

J Cellular Molecular Medi

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Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 95%

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Bartalucci

Tozzi

Bogani

et al. 2013

J Cellular Molecular Medi

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“…Megakaryopoiesis is a process of hematopoietic differentiation previously investigated by the current authors (22). Therefore, the ability of the fresh CD34 + cells and those obtained after thawing to proliferate and differentiate in MKs was compared.…”

Section: Resultsmentioning

confidence: 99%

Effect of trehalose on cryopreservation of pure peripheral blood stem cells

Martinetti¹,

Colarossi²,

Buccheri³

et al. 2017

Biomedical Reports

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Abstract. Stem cells are an important tool for the study of hematopoiesis. Despite developments in cryopreservation, post-thaw cell death remains a considerable problem. Cryopreservation protocol should limit cell damage due to freezing and ensure the recovery of the functional cell characteristics after thawing. Thus, the use of cryoprotectants is essential. In particular, the efficacy of trehalose has been reported for clinical purposes in blood stem cells. The aim of the current study was to establish an efficient method for biological research based on the use of trehalose, to cryopreserve pure peripheral blood stem cells. The efficacy of trehalose was assessed in vitro and the cell viability was evaluated. The data indicate that trehalose improves cell survival after thawing compared with the standard freezing procedure. These findings could suggest the potential for future trehalose application for research purposes in cell cryopreservation. IntroductionStem cells in biological research provide a significant source of information and, thus, contribute to the development of novel therapeutic strategies. In particular, the ability to cultivate stem cells and human hematopoietic progenitor cells in vitro is the fundamental basis for investigating hematopoiesis. An improved understanding of the mechanisms that regulate cell proliferation and differentiation during the stages of hematopoiesis would further elucidate the molecular characteristics of diseases (which are characterized by excessive expansion or a functional defect of certain immature blood components), and facilitate the identification of substances that are able to specifically protect healthy cells from the action of cytotoxic drugs.Hematopoietic stem and progenitor cells (HSPCs) may be isolated from peripheral blood (PB), bone marrow (BM) or umbilical cord blood (CB) (1).Although significant improvements to cell cryopreservation procedures have been achieved (2), the improvement of current cryopreservation protocols that lead to a high mortality rate after thawing for the crystal formations that arise during freezing is a primary goal. Specifically, fast cooling forms intracellular ice crystals, which results in cell destruction and slow cooling forms ice crystals in the extracellular space, with consequent cellular dehydration. Selection of a cryoprotectant, as well as a suitable freezing rate serves to protect cells from these adverse effects (3,4).Cryoprotectants are divided into two classes: Penetrating and nonpenetrating (5). The penetrating cryoprotectants include glycerol and 1,2-propanediol and dimethyl sulfoxide (Me 2 SO); the latter is commonly used for HSPC cryopreservation (6).The non penetrating cryoprotectants comprise polyvinyl pyrrolidone, trehalose, fructose, sucrose and glucose.Trehalose is a non-toxic disaccharide of glucose that preserves the structural integrity of the cells during freezing and thawing (7). Specifically, trehalose is found in numerous organisms, such as nematodes and yeasts, which are capable of surviving du...

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“…Until now, there has been strong evidence supporting the involvement of the PI3K/Akt/mTOR pathway inhibitor in MPNs pathogenesis. Constitutive activation of Akt and mTOR kinases has been observed in JAK2V617F positive cell lines and megakaryocytes of MPNs patients [1,18]. These findings led to the development of different Akt, PI3K or dual mTORC1/mTORC2 complex inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines

et al. 2015

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Background. The JAK2V617F mutation plays a crucial role in the pathogenesis of myeloproliferative neoplasms (MPNs). Inhibition of JAK2 activity by ruxolitinib (RX) results in growth inhibition and apoptosis of cells carrying the JAK2V617F mutation however the exact mechanisms regulating apoptosis have not been fully elucidated. Objectives. This study assessed the potential cytotoxicity of RX against JAK2-positive human cell lines (SET-2 and HEL), either alone or in combination with hydroxyurea, busulphan, rapamycin or LY294002. Material and Methods. Cell viability, the apoptosis rate (annexin-V staining), drop of mitochondrial transmembrane potential (Δψm) and caspase activation, were measured using flow cytometry. Additionally, the expression of several apoptosis-regulating proteins was evaluated. Results. RX showed cytotoxicity against both SET-2 and HEL cell lines. The main mechanism of this action was apoptosis, with significant drop of Δψm, caspase-3 and -9 activation, and moderate activation of caspase-8 (only for SET-2 cells). Corresponding to enhanced apoptosis, the expression levels of some apoptosis-regulating proteins were changed, the most pronounced in both cell lines being up-regulation of Bax and down-regulation of Bcl-2 proteins. Additionally, up-regulation of Bak and Bad (SET-2) and down-regulation of Mcl-1 (HEL) were observed. Of the studied compounds, a combination of RX + LY294002 induced the greatest cytotoxicity in both SET-2 and HEL cell lines, and rapamycin the least. Conclusions. This study shows that the combination of RX and a PI3K kinase inhibitor provokes a significant pro-apoptotic effect in JAK2V617F mutated cells, which may justify the beginning of clinical trials based on the combination of these drugs (Adv Clin Exp Med 2015, 24, 2, 195-202).

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Increased phospho‐mTOR expression in megakaryocytic cells derived from CD34+ progenitors of essential thrombocythaemia and myelofibrosis patients

Cited by 15 publications

References 10 publications

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Effect of trehalose on cryopreservation of pure peripheral blood stem cells

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines

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