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Francesca CHIARINI

Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze,sede Istituti Anatomici (area Policlinico)


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Pubblicazioni

2022 - Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma [Articolo su rivista]
Chiarini, Francesca; Paganelli, Francesca; Balestra, Tommaso; Capanni, Cristina; Fazio, Antonietta; Manara, Maria Cristina; Landuzzi, Lorena; Petrini, Stefania; Evangelisti, Camilla; Lollini, Pier-Luigi; Martelli, Alberto M; Lattanzi, Giovanna; Scotlandi, Katia
abstract

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.


2022 - Pathobiology and Therapeutic Relevance of GSK-3 in Chronic Hematological Malignancies [Articolo su rivista]
Martelli, Alberto M; Paganelli, Francesca; Evangelisti, Camilla; Chiarini, Francesca; Mccubrey, James A
abstract

Glycogen synthase kinase-3 (GSK-3) is an evolutionarily conserved, ubiquitously expressed, multifunctional serine/threonine protein kinase involved in the regulation of a variety of physiological processes. GSK-3 comprises two isoforms (alpha and beta) which were originally discovered in 1980 as enzymes involved in glucose metabolism via inhibitory phosphorylation of glycogen synthase. Differently from other proteins kinases, GSK-3 isoforms are constitutively active in resting cells, and their modulation mainly involves inhibition through upstream regulatory networks. In the early 1990s, GSK-3 isoforms were implicated as key players in cancer cell pathobiology. Active GSK-3 facilitates the destruction of multiple oncogenic proteins which include beta-catenin and Master regulator of cell cycle entry and proliferative metabolism (c-Myc). Therefore, GSK-3 was initially considered to be a tumor suppressor. Consistently, GSK-3 is often inactivated in cancer cells through dysregulated upstream signaling pathways. However, over the past 10-15 years, a growing number of studies highlighted that in some cancer settings GSK-3 isoforms inhibit tumor suppressing pathways and therefore act as tumor promoters. In this article, we will discuss the multiple and often enigmatic roles played by GSK-3 isoforms in some chronic hematological malignancies (chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, and B-cell non-Hodgkin's lymphomas) which are among the most common blood cancer cell types. We will also summarize possible novel strategies targeting GSK-3 for innovative therapies of these disorders.


2021 - Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine [Articolo su rivista]
Carano, Francesco; Teti, Gabriella; Ruggeri, Alessandra; Chiarini, Francesca; Giorgetti, Arianna; Mazzotti, Maria C; Fais, Paolo; Falconi, Mirella
abstract

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.


2021 - Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm [Articolo su rivista]
Teti, G; Chiarini, F; Mazzotti, E; Ruggeri, A; Carano, F; Falconi, M.
abstract


2021 - GSK-3: a multifaceted player in acute leukemias [Articolo su rivista]
Martelli, Am; Evangelisti, C; Paganelli, F; Chiarini, F; Mccubrey, Ja.
abstract


2021 - The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine [Articolo su rivista]
Paganelli, F.; Chiarini, F.; Palmieri, A.; Martinelli, M.; Sena, P.; Bertacchini, J.; Roncucci, L.; Cappellini, A.; Martelli, A. M.; Bonucci, M.; Fiorentini, C.; Ferri, I. H.
abstract

The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with im-munomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer.


2020 - B-ALL Complexity: Is Targeted Therapy Still A Valuable Approach for Pediatric Patients? [Articolo su rivista]
Ratti, S; Lonetti, A; Follo, My; Paganelli, F; Martelli, Am; Chiarini, F; Evangelisti, C
abstract


2020 - Crosstalks of GSK3 signaling with the mTOR network and effects on targeted therapy of cancer [Articolo su rivista]
Evangelisti, C; Chiarini, F; Paganelli, F; Marmiroli, S; Martelli, Am
abstract

Abstract The introduction of therapeutics targeting specific tumor-promoting oncogenic or non-oncogenic signaling pathways has revolutionized cancer treatment. Mechanistic (previously mammalian) target of rapamycin (mTOR), a highly conserved Ser/Thr kinase, is a central hub of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR network, one of the most frequently deregulated signaling pathways in cancer, that makes it an attractive target for therapy. Numerous mTOR inhibitors have progressed to clinical trials and two of them have been officially approved as anticancer therapeutics. However, mTOR-targeting drugs have met with a very limited success in cancer patients. Frequently, the primary impediment to a successful targeted therapy in cancer is drug-resistance, either from the very beginning of the therapy (innate resistance) or after an initial response and upon repeated drug treatment (evasive or acquired resistance). Drug-resistance leads to treatment failure and relapse/progression of the disease. Resistance to mTOR inhibitors depends, among other reasons, on activation/deactivation of several signaling pathways, included those regulated by glycogen synthase kinase-3 (GSK3), a protein that targets a vast number of substrates in its repertoire, thereby orchestrating many processes that include cell proliferation and survival, metabolism, differentiation, and stemness. A detailed knowledge of the rewiring of signaling pathways triggered by exposure to mTOR inhibitors is critical to our understanding of the consequences such perturbations cause in tumors, including the emergence of drug-resistant cells. Here, we provide the reader with an updated overview of intricate circuitries that connect mTOR and GSK3 and we relate them to the efficacy (or lack of efficacy) of mTOR inhibitors in cancer cells.


2020 - Inhibition of Methyltransferase DOT1L Sensitizes to Sorafenib Treatment AML Cells Irrespective of MLL-Rearrangements: A Novel Therapeutic Strategy for Pediatric AML [Articolo su rivista]
Lonetti, A; Indio, V; Laginestra, Ma; Tarantino, G; Chiarini, F; Astolfi, A; Bertuccio, Sn; Martelli, Am; Locatelli, F; Pession, A; Masetti, R
abstract


2020 - Lamin A and Prelamin A Counteract Migration of Osteosarcoma Cells [Articolo su rivista]
Evangelisti, C; Paganelli, F; Giuntini, G; Mattioli, E; Cappellini, A; Ramazzotti, G; Faenza, I; Maltarello, Mc; Martelli, Am; Scotlandi, K; Chiarini, F; Lattanzi, G
abstract


2020 - Targeting Wnt/β-catenin and PI3K/Akt/mTOR pathways in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Evangelisti, C; Chiarini, F; Cappellini, A; Paganelli, F; Fini, M; Santi, S; Martelli, Am; Neri, Lm; Evangelisti, C.
abstract


2020 - The Role Played by Wnt/β-Catenin Signaling Pathway in Acute Lymphoblastic Leukemia [Articolo su rivista]
Chiarini, F; Paganelli, F; Martelli, Am; Evangelisti, C
abstract


2020 - The Unfolded Protein Response: A Novel Therapeutic Target in Acute Leukemias [Articolo su rivista]
Martelli, Am; Paganelli, F; Chiarini, F; Evangelisti, C; Mccubrey, Ja
abstract


2019 - Advances in understanding the mechanisms of evasive and innate resistance to mTOR inhibition in cancer cells [Articolo su rivista]
Chiarini, F; Evangelisti, C; Lattanzi, G; Mccubrey, Ja; Martelli, Am.
abstract


2019 - Age-Related Alterations Affecting the Chondrogenic Differentiation of Synovial Fluid Mesenchymal Stromal Cells in an Equine Model [Articolo su rivista]
Mazzotti, E; Teti, G; Falconi, M; Chiarini, F; Barboni, B; Mazzotti, A; Muttini, A.
abstract


2019 - New advances in targeting aberrant signaling pathways in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Paganelli, F; Lonetti, A; Anselmi, L; Martelli, Am; Evangelisti, C; Chiarini, F
abstract


2019 - Phospholipase C-β1 interacts with cyclin E in adipose- derived stem cells osteogenic differentiation [Articolo su rivista]
Ramazzotti, G; Fiume, R; Chiarini, F; Campana, G; Ratti, S; Billi, Am; Manzoli, L; Follo, My; Suh, Pg; Mccubrey, J; Cocco, L; Faenza, I
abstract


2019 - Targeting metabolic vulnerabilities and aberrant signal transduction pathways in paediatric T-cell Acute Lymphoblastic Leukaemia (T-ALL) [Abstract in Atti di Convegno]
Anselmi, Laura; Accordi, Benedetta; Serafin, Valentina; Zavatti, Manuela; Braglia, Luca; Paganin, Maddalena; Chiarini, Francesca; Bresolin, Silvia; Patterson, Shaun; Ruzzene, Maria; Maria Martelli, Alberto; Basso, Giuseppe; Huang, Xu; Marmiroli, Sandra
abstract


2019 - The Cutting Edge: The Role of mTOR Signaling in Laminopathies [Articolo su rivista]
Chiarini, F; Evangelisti, C; Cenni, V; Fazio, A; Paganelli, F; Martelli, Am; Lattanzi, G.
abstract


2018 - Dual inhibition of PI3K/mTOR signaling in chemoresistant AMLprimary cells. [Articolo su rivista]
Bertacchini, Jessika; Frasson, Chiara; Chiarini, Francesca; D'Avella, Daniele; Accordi, Benedetta; Anselmi, Laura; Barozzi, Patrizia; Foghieri, Fabio; Luppi, Mario; Martelli, Alberto M.; Basso, Giuseppe; Najmaldin, Saki; Khosravi, Abbas; Rahim, Fakher; Marmiroli, Sandra
abstract

A main cause of treatment failure for AML patients is resistance to chemotherapy. Survival of AML cells may depend on mechanisms that elude conventional drugs action and/or on the presence of leukemia initiating cells at diagnosis, and their persistence after therapy. MDR1 gene is an ATP-dependent drug efflux pump known to be a risk factor for the emergence of resistance, when combined to unstable cytogenetic profile of AML patients. In the present study, we analyzed the sensitivity to conventional chemotherapeutic drugs of 26 samples of primary blasts collected from AML patients at diagnosis. Detection of cell viability and apoptosis allowed to identify two group of samples, one resistant and one sensitive to in vitro treatment. The cells were then analyzed for the presence and the activity of P-glycoprotein. A comparative analysis showed that resistant samples exhibited a high level of MDR1 mRNA as well as of P-glycoprotein content and activity. Moreover, they also displayed high PI3K signaling. Therefore, we checked whether the association with signaling inhibitors might resensitize resistant samples to chemo-drugs. The combination showed a very potent cytotoxic effect, possibly through down modulation of MDR1, which was maintained also when primary blasts were co-cultured with human stromal cells. Remarkably, dual PI3K/mTOR inactivation was cytotoxic also to leukemia initiating cells. All together, our findings indicate that signaling activation profiling associated to gene expression can be very useful to stratify patients and improve therapy.


2018 - Phosphatidylinositol 3-kinase inhibition potentiates glucocorticoid response in B-cell acute lymphoblastic leukemia [Articolo su rivista]
Evangelisti, C; Cappellini, A; Oliveira, M; Fragoso, R; Barata, Jt; Bertaina, A; Locatelli, F; Simioni, C; Neri, Lm; Chiarini, F; Lonetti, A; Buontempo, F; Orsini, E; Pession, A; Manzoli, L; Martelli, Am; Evangelisti, C.
abstract

Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance.


2018 - Therapeutic targeting of CK2 in acute and chronic leukemias [Articolo su rivista]
Buontempo, F; Mccubrey, Ja; Orsini, E; Ruzzene, M; Cappellini, A; Lonetti, A; Evangelisti, C; Chiarini, F; Evangelisti, C; Barata, Jt; Martelli, Am.
abstract

CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.


2018 - Therapeutic Targeting of mTOR in T-Cell Acute Lymphoblastic Leukemia: An Update [Articolo su rivista]
Evangelisti, C; Chiarini, F; Mccubrey, Ja; Martelli, Am.
abstract


2017 - Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene [Articolo su rivista]
Masetti, R; Bertuccio, Sn; Astolfi, A; Chiarini, F; Lonetti, A; Indio, V; De Luca, M; Bandini, J; Serravalle, S; Franzoni, M; Pigazzi, M; Martelli, Am; Basso, G; Locatelli, F; Pession, A
abstract

CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. METHODS: We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. RESULTS: As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. CONCLUSIONS: Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.


2016 - Advances in understanding the acute lymphoblastic leukemia bone marrow microenvironment: From biology to therapeutic targeting [Articolo su rivista]
Chiarini, F; Lonetti, A; Evangelisti, C; Buontempo, F; Orsini, E; Evangelisti, C; Cappellini, A; Neri, Lm; Mccubrey, Ja; Martelli, Am
abstract


2016 - Improving nelarabine efficacy in T cell acute lymphoblastic leukemia by targeting aberrant PI3K/AKT/mTOR signaling pathway [Articolo su rivista]
Lonetti, A; Cappellini, A; Bertaina, A; Locatelli, F; Pession, A; Buontempo, F; Evangelisti, C; Evangelisti, C; Orsini, E; Zambonin, L; Neri, Lm; Martelli, Am; Chiarini, F
abstract

BACKGROUND: Although in recent years, the introduction of novel chemotherapy protocols has improved the outcome of T cell acute lymphoblastic leukemia (T-ALL) patients, refractory and/or relapsing disease remains a foremost concern. In this context, a major contribution was provided by the introduction of the nucleoside analog nelarabine, approved for salvage treatment of T-ALL patients with refractory/relapsed disease. However, nelarabine could induce a life-threatening, dose-dependent neurotoxicity. To improve nelarabine efficacy, we have analyzed its molecular targets, testing selective inhibitors of such targets in combination with nelarabine. METHODS: The effectiveness of nelarabine as single agent or in combination with PI3K, Bcl2, and MEK inhibitors was evaluated on human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts. The efficacy of signal modulators in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed by flow cytometry, western blotting, and quantitative real-time PCR in T-ALL settings. RESULTS: Treatment with nelarabine as a single agent identified two groups of T-ALL cell lines, one sensitive and one resistant to the drug. Whereas sensitive T-ALL cells showed a significant increase of apoptosis and a strong down-modulation of PI3K signaling, resistant T-ALL cells showed a hyperactivation of AKT and MEK/ERK1/2 signaling pathways, not caused by differences in the expression of nelarabine transporters or metabolic activators. We then studied the combination of nelarabine with the PI3K inhibitors (both pan and dual γ/δ inhibitors), with the Bcl2 specific inhibitor ABT199, and with the MEK inhibitor trametinib on both T-ALL cell lines and patient samples at relapse, which displayed constitutive activation of PI3K signaling and resistance to nelarabine alone. The combination with the pan PI3K inhibitor ZSTK-474 was the most effective in inhibiting the growth of T-ALL cells and was synergistic in decreasing cell survival and inducing apoptosis in nelarabine-resistant T-ALL cells. The drug combination caused AKT dephosphorylation and a downregulation of Bcl2, while nelarabine alone induced an increase in p-AKT and Bcl2 signaling in the resistant T-ALL cells and relapsed patient samples. CONCLUSIONS: These findings indicate that nelarabine in combination with PI3K inhibitors may be a promising therapeutic strategy for the treatment of T-ALL relapsed patients.


2016 - Synergistic cytotoxic effects of bortezomib and CK2 inhibitor CX-4945 in acute lymphoblastic leukemia: Turning offthe prosurvival ER chaperone BIP/Grp78 and turning on the proapoptotic NF-κB [Articolo su rivista]
Buontempo, Francesca; Orsini, Ester; Lonetti, Annalisa; Cappellini, Alessandra; Chiarini, Francesca; Evangelisti, Camilla; Evangelisti, Cecilia; Melchionda, Fraia; Pession, Andrea; Bertaina, Alice; Locatelli, Franco; Bertacchini, Jessika; Neri, Luca Maria; Mccubrey, James A.; Martelli, Alberto Maria
abstract

The proteasome inhibitor bortezomib is a new targeted treatment option for refractory or relapsed acute lymphoblastic leukemia (ALL) patients. However, a limited efficacy of bortezomib alone has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. Recently, it has been documented that UPR disruption could be considered a selective anti-leukemia therapy. CX- 4945, a potent casein kinase (CK) 2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. In this study, we analyzed in T- and B-ALL preclinical settings, the molecular mechanisms of synergistic apoptotic effects observed after bortezomib/CX-4945 combined treatment. We demonstrated that, adding CX-4945 after bortezomib treatment, prevented leukemic cells from engaging a functional UPR in order to buffer the bortezomibmediated proteotoxic stress in ER lumen. We documented that the combined treatment decreased pro-survival ER chaperon BIP/Grp78 expression, via reduction of chaperoning activity of Hsp90. Bortezomib/CX-4945 treatment inhibited NF-κB signaling in T-ALL cell lines and primary cells from T-ALL patients, but, intriguingly, in B-ALL cells the drug combination activated NF-κB p65 pro-apoptotic functions. In fact in B-cells, the combined treatment induced p65-HDAC1 association with consequent repression of the anti-apoptotic target genes, Bcl-xL and XIAP. Exposure to NEMO (IKKγ)-binding domain inhibitor peptide reduced the cytotoxic effects of bortezomib/CX-4945 treatment. Overall, our findings demonstrated that CK2 inhibition could be useful in combination with bortezomib as a novel therapeutic strategy in both T- and B-ALL.


2016 - Therapeutic potential of targeting sphingosine kinases and sphingosine 1-phosphate in hematological malignancies [Articolo su rivista]
Evangelisti, C; Evangelisti, C; Buontempo, F; Lonetti, A; Orsini, E; Chiarini, F; Barata, Jt; Pyne, S; Pyne, Nj; Martelli, Am.
abstract

Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.


2015 - Autophagy in acute leukemias: a double-edged sword with important therapeutic implications [Articolo su rivista]
Evangelisti, C; Evangelisti, C; Chiarini, F; Lonetti, A; Buontempo, F; Neri, Lm; Mccubrey, Ja; Martelli, Am
abstract


2015 - Current treatment strategies for inhibiting mTOR in cancer [Articolo su rivista]
Chiarini, F; Evangelisti, C; Mccubrey, Ja; Martelli, Am
abstract


2015 - PI3K pan-inhibition impairs more efficiently proliferation and survival of T-cell acute lymphoblastic leukemia cell lines when compared to isoform-selective PI3K inhibitors [Articolo su rivista]
Lonetti, A; Cappellini, A; Spartà, Am; Chiarini, F; Buontempo, F; Evangelisti, C; Evangelisti, C; Orsini, E; Mccubrey, Ja; Martelli, Am
abstract


2015 - 13. PLCβ1a and PLCβ1b selective regulation and cyclin D3 modulation reduced by kinamycin F during k562 cell differentiation [Articolo su rivista]
Bavelloni, A; Dmitrienko, Gi; Goodfellow, Vj; Ghavami, A; Piazzi, M; Blalock, W; Chiarini, F; Cocco, L; Faenza, I
abstract


2014 - A COMBINATION OF BORTEZOMIB WITH CX-4945, A CASEIN KINASE 2 (CK2) INHIBITOR, HAS SYNERGISTIC CYTOTOXIC EFFECTS IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) CELL LINES [Abstract in Atti di Convegno]
Buontempo, Francesca; Orsini, E; Cappellini, Alessandra; Lonetti, Annalisa; Evangelisti, C; Chiarini, Francesca; Spartà, Am; Bressanin, D; Evangelisti, C; Martelli, Am
abstract

Introduction. The proteasome inhibitor bortezomib is a new treatment option for patients with refractory or relapsed ALL, particularly when used in combination with conventional chemotherapy or other targeted agents. Indeed, a limited efficacy of bortezomib alone in ALL patients has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. CX-4945, a potent CK2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. Here, we have explored the cytotoxic effects of a bortezomib/CX-4945 combination in a panel of B- and T-ALL cell lines. Methods. B- (KOPN-8, NALM-6, RS4;11) and T- (MOLT-4, Jurkat, CEM-R) ALL cell lines were pretreated with bortezomib (Selleck Chemicals, Houston TX, USA), for six hours and then with CX-4945 (Selleck Chemicals, Houston TX, USA), for 16/24 hours. MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining and flow cytometric analysis. Protein expression was studied by western blot. Results. Cells were cultured in the presence of bortezomib or CX-4945, either alone or in combination at a fixed ratio. The combined treatment was more effective in reducing cell viability in all B-ALL cell lines and in MOLT-4 cells. Annexin V/PI staining was performed; interestingly, no synergism was detected if the two drugs were administered together since the beginning of treatment. In response to treatment with 2.5 nM bortezomib followed by 5 μM CX-4945, we detected an increase in apoptosis in B-ALL cell lines and in MOLT-4 cells after 24 h. Western blot analysis for the cleaved forms of caspase-3 and PARP, confirmed a higher apoptosis induction by the combined treatment. A reduction in anti-apoptotic Bcl2 concomitant with an increase in proapoptotic Bax, suggested that bortezomib/CX-4945 treatment caused a mitochondrial apoptosis. IRE1a and CHOP (established markers of ER stress/UPR-mediated apoptosis) levels increased in response to the combined treatment, in contrast the expression of GRP78/BIP (a marker of UPR activation) decreased, suggesting that a potential mechanism by which the drug combination induced cell death, involved ER stress induction by bortezomib and the inability to respond by adequate activation of the UPR signaling which was blocked by CX-4945. Conclusions. Here, we demonstrated that the proteasome inhibitor bortezomib and the CK2 inhibitor CX-4945 interact in a synergistic manner to induce apoptosis both in B- and in T-ALL cell lines. Drug cytotoxicity was associated with modulation of the ER stress/UPR signaling pathway. Importantly, the synergism was observed only when bortezomib treatment preceded CX-4945 administration. Therefore, our findings support clinical application of bortezomib in combination with CX- 4945 in B- and T-ALL treatment.


2014 - Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Lonetti, A; Antunes, Il; Chiarini, F; Orsini, E; Buontempo, F; Ricci, F; Tazzari, Pl; Pagliaro, P; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; Mccubrey, Ja; Barata, Jt; Martelli, Am.
abstract


2014 - ASSESSMENT OF THE EFFECT OF SPHINGOSINE KINASE INHIBITORS ON APOPTOSIS, UNFOLDED PROTEIN RESPONSE AND AUTOPHAGY OF T-ACUTE LYMPHOBLASTIC LEUKEMIA CELLS: INDICATIONS FOR NOVEL THERAPEUTICS [Abstract in Atti di Convegno]
Evangelisti, Cecilia; Evangelisti, Camilla; Teti, Gabriella; Falconi, Mirella; Cappellini, Alessandra; Chiarini, Francesca; Buontempo, Francesca; Bressanin, D; Lonetti, Annalisa; Spartà, A; Bittman, R; Pyne, S; Pyne, N; Martelli, Am
abstract

Introduction. Sphingosine 1-phosphate (S1P) is involved in many processes such as cell survival, growth, migration, and cancer. S1P is formed by the phosphorylation of sphingosine by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). While the role of SK1 in cancer is well established, the role of SK2 in regulating apoptosis is still a matter of debate, even though emerging evidence has highlighted the importance of SK2 in cancer. Therefore, SKs represent a promising target for cancer therapy. Recently, the importance of S1P in hematological malignancies has been described. Here, we analyze the therapeutic effects of 2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), an SK1/2 inhibitor, and (R)-FTY720 methyl ether (ROMe), a SK2-selective inhibitor, in T-ALL cell lines. Methods. T- ALL cell lines (Molt-4, Jurkat, CEM-R) were treated with SKi and ROMe for 40 h, then MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining. Protein expression was studied by western blot (WB). Results. SKi and ROMe induced a decrease in cell viability, as demonstrated by MTT assays. Annexin-V/PI staining and flow cytometric analysis of cells treated with a SKi concentration equivalent to the IC50 documented the occurrence of apoptotic cell death. This was confirmed by WB analysis for caspase and PARP cleavage in all the cell lines. However, we also observed a SKi-induced autophagy by means of WB and transmission electron microscopy analysis in Jurkat and CEM-R cells. We then analyzed the expression of ER stress/UPR hallmarks. SKi activated the ER stress/UPR pathway and this occurred following apoptosis and correlated with autophagy. The combination of SKi and chloroquine (an autophagy inhibitor) induced a decrease in cell viability, indicating that the UPR/autophagic response is likely to be a protective mechanism. In Molt-4 cells, we detected an increase in SK1 expression after 40 h of treatment with SKi, that could represent an attempt of cells to escape SKi-induced apoptosis, as high SK1 expression is known to enhance cell growth and survival. Notably, SKi synergized with vincristine at concentrations of SKi that were much below its respective IC50, suggesting that vincristine sensitized T-ALL cells to SKi and that combining chemotherapeutic agents with SK inhibitors could be feasible in the treatment of T-ALL. Finally, ROMe treatment induced an autophagic type of cell death. Conclusions. We report herein that SKi and ROMe affect T-ALL cell viability, but they exert their effects through different mechanisms. While ROMe induces an autophagic cell death, SKi induces apoptosis. Moreover, for the first time, we demonstrated that SKi activates an ER stress/UPR pathway in T-ALL cells and this is linked with a protective autophagic response. Thus, our findings indicate that SK1 or SK2 may represent potential targets for treating T-ALL, thereby enabling better management of this cancer.


2014 - Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics [Articolo su rivista]
Evangelisti, C; Evangelisti, C; Teti, G; Chiarini, F; Falconi, M; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; Mccubrey, Ja; Beak, Dj; Bittman, R; Pyne, S; Pyne, Nj; Martelli, Am
abstract

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.


2014 - Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling [Articolo su rivista]
Buontempo, F; Orsini, E; Martins, L R; Antunes, I; Lonetti, A; Chiarini, F; Tabellini, G; Evangelisti, C; Evangelisti, C; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; Mccubrey, J A; Cappellini, A; Barata, J T; Martelli, A M
abstract

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2 alpha. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1 alpha and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2 alpha/PI3K/Akt/mTOR signaling.


2014 - MYCN is a novel oncogenic target in pediatric T-cell acute lymphoblastic leukemia [Articolo su rivista]
Astolfi, A; Vendemini, F; Urbini, M; Melchionda, F; Masetti, R; Franzoni, M; Libri, V; Serravalle, S; Togni, M; Paone, G; Montemurro, L; Bressanin, D; Chiarini, F; Martelli, Am; Tonelli, R; Pession, A
abstract

MYCN is an oncogene frequently overexpressed in pediatric solid tumors whereas few evidences suggest his involvement in the pathogenesis of haematologic malignancies. Here we show that MYCN is overexpressed in a relevant proportion (40 to 50%) of adult and pediatric T-cell acute lymphoblastic leukemias (T-ALL). Focusing on pediatric T-ALL, MYCN-expressing samples were found almost exclusively in the TAL1-positive subgroup. Moreover, TAL1 knockdown in T-ALL cell lines resulted in a reduction of MYCN expression, and TAL1 directly binds to MYCN promoter region, suggesting that TAL1 pathway activation could sustain the up-regulation of MYCN. The role of MYCN in T-ALL was investigated by peptide nucleic acid (PNA-MYCN)-mediated transcriptional silencing of MYCN and by siRNAs. MYCN knockdown in T-ALL cell lines resulted in a reduction of cell viability, up to 50%, while no effect was elicited with a mismatch PNA. The inhibitory effect of PNA-MYCN on cell viability was due to a significant increase in apoptosis. PNA-MYCN treatment in pediatric T-ALL samples reduced cell viability of leukemic cells from patients with high MYCN expression, while no effect was obtained in MYCN-negative blast cells. These results showed that MYCN is frequently overexpressed in pediatric T-ALL and suggested his role as a candidate for molecularly-directed therapies.


2014 - Targeting Signaling Pathways in T-cell acute lymphoblastic leukemia initiating cells [Capitolo/Saggio]
Martelli, Am; Lonetti, A; Buontempo, F; Ricci, F; Tazzari, Pl; Evangelisti, C; Bressanin, D; Cappellini, A; Orsini, E; Chiarini, F
abstract

Leukemia initiating cells (LICs) represent a reservoir that is believed to drive relapse and resistance to chemotherapy in blood malignant disorders. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder of immature hematopoietic precursors committed to the T-cell lineage. T-ALL comprises about 15% of pediatric and 25% of adult ALL cases and is prone to early relapse. Although the prognosis of T-ALL has improved especially in children due to the use of new intensified treatment protocols, the outcome of relapsed T-ALL cases is still poor. Putative LICs have been identified also in T-ALL. LICs are mostly quiescent and for this reason highly resistant to chemotherapy. Therefore, they evade treatment and give rise to disease relapse. At present great interest surrounds the development of targeted therapies against signaling networks aberrantly activated in LICs and important for their survival and drug-resistance. Both the Notch1 pathway and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) network are involved in T-ALL LIC survival and drug-resistance and could be targeted by small molecules. Thus, Notch1 and PI3K/Akt/mTOR inhibitors are currently being developed for clinical use either as single agents or in combination with conventional chemotherapy for T-ALL patient treatment. In this review, we summarize the existing knowledge of the relevance of Notch1 and PI3K/Akt/mTOR signaling in T-ALL LICs and we examine the rationale for targeting these key signal transduction networks by means of selective pharmacological inhibitors.


2014 - Therapeutic potential of targeting mTOR in T-cell acute lymphoblastic leukemia (Review) [Articolo su rivista]
Evangelisti, C; Evangelisti, C; Chiarini, F; Lonetti, A; Buontempo, F; Bressanin, D; Cappellini, A; Orsini, E; Mccubrey, Ja; Martelli, Am.
abstract


2014 - Therapeutic targeting of Polo-like kinase-1 and Aurora kinases in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Spartà, Am; Bressanin, D; Chiarini, F; Lonetti, A; Cappellini, A; Evangelisti, C; Evangelisti, C; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; Mccubrey, Ja; Martelli, Am.
abstract


2013 - Enhancing the effectiveness of nucleoside analogs with mTORC1 blockers to treat acute myeloid leukemia patients [Articolo su rivista]
Martelli, Am; Lonetti, A; Amadori, S; Mccubrey, Ja; Chiarini, F
abstract


2013 - Increased NGAL (Lnc2) expression after chemotherapeutic drug treatment [Capitolo/Saggio]
Chappell, Wh; Abrams, Sl; Stadelman, Km; Lahair, Mm; Franklin, Ra; Cocco, L; Evangelisti, C; Chiarini, F; Martelli, Am; Steelman, Ls; Mccubrey, Ja.
abstract


2013 - K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway [Articolo su rivista]
Poli, A; Faenza, I; Chiarini, F; Matteucci, A; Mccubrey, Ja; Cocco, L.
abstract


2013 - Targeting phosphatidylinositol 3-kinase signaling in acute myelogenous leukemia [Articolo su rivista]
Evangelisti, C; Evangelisti, C; Bressanin, D; Buontempo, F; Chiarini, F; Lonetti, A; Soncin, M; Spartà, A; Mccubrey, Ja; Martelli, Am.
abstract


2012 - A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia [Articolo su rivista]
Chiarini, F; Lonetti, A; Teti, G; Orsini, E; Bressanin, D; Cappellini, A; Ricci, F; Tazzari, Pl; Ognibene, A; Falconi, M; Pagliaro, P; Iacobucci, I; Martinelli, G; Amadori, S; Mccubrey, Ja; Martelli, Am.
abstract

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G₀/G₁-phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34⁺/CD38⁻/CD123⁺), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.


2012 - A role of PI-PLCb1 in G1/S transition in human erythroleukemia k562 cells [Poster]
Poli, Alessandro; Faenza, Irene; Matteucci, Alessandro; Chiarini, Francesca; Cocco, LUCIO ILDEBRANDO
abstract


2012 - Activity of the selective IκB kinase inhibitor BMS-345541 against T-cell acute lymphoblastic leukemia: involvement of FOXO3a [Articolo su rivista]
Buontempo, F; Chiarini, F; Bressanin, D; Tabellini, G; Melchionda, F; Pession, A; Fini, M; Neri, Lm; Mccubrey, Ja; Martelli, Am.
abstract

Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G 2/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21(Cip1) expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types. It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.


2012 - Advances in Targeting Signal Transduction Pathways [Articolo su rivista]
Mccubrey, Ja; Steelman, Ls; Chappell, Wh; Sun, L; Davis, Nm; Abrams, Sl; Franklin, Ra; Cocco, L; Evangelisti, C; Chiarini, F; Martelli, Am; Libra, M; Candido, S; Ligresti, G; Malaponte, G; Mazzarino, Mc; Fagone, P; Donia, M; Nicoletti, F; Polesel, J; Talamini, R; Bäsecke, J; Mijatovic, S; Maksimovic-Ivanic, D; Michele, M; Tafuri, A; Dulińska-Litewka, J; Laidler, P; D'Assoro, Ab; Drobot, L; Umezawa, D; Montalto, G; Cervello, M; Demidenko, Zn.
abstract


2012 - AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications [Articolo su rivista]
Grimaldi, Cecilia; Chiarini, Francesca; Tabellini, G.; Ricci, F.; Tazzari, P. L.; Battistelli, M.; Falcieri, E.; Bortul, R.; Melchionda, F.; Iacobucci, Ilaria; Pagliaro, P.; Martinelli, Giovanni; Pession, Andrea; Barata, J. T.; Mccubrey, J. A.; Martelli, ALBERTO MARIA
abstract

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be down-modulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4(+) T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34(+)/CD7(-)/CD4(+)) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.


2012 - Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Simioni, C; Neri, Lm; Tabellini, G; Ricci, F; Bressanin, D; Chiarini, F; Evangelisti, C; Cani, A; Tazzari, Pl; Melchionda, F; Pagliaro, P; Pession, A; Mccubrey, Ja; Capitani, S; Martelli, Am.
abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.


2012 - Dual Inhibition of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin: a Therapeutic Strategy for Acute Leukemias [Articolo su rivista]
Chiarini, F; Evangelisti, C; Buontempo, F; Bressanin, D; Fini, M; Cocco, L; Cappellini, A; Mccubrey, Ja; Martelli, Am.
abstract

The phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) are two major signaling molecules in the PI3K/Akt/mTOR signal transduction cascade. This pathway is a key regulator of a wide range of physiological cell processes which include proliferation, differentiation, survival, metabolism, exocytosis, motility, and autophagy. However, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences response to therapeutic treatments. Therefore, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome. The PI3K/Akt/mTOR signaling network is activated in acute leukemias of both myelogenous and lymphoid lineage, where it correlates with poor prognosis and enhanced drug-resistance. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds that targeted the catalytic site of both PI3K and mTOR (e.g. PI-103, NVP-BEZ235). In preclinical settings, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against leukemic cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin and its derivatives (rapalogs). At variance with rapamycin/rapalogs, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenetic protein translation in leukemic cells. Hence, they strongly reduced the proliferation rate and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors represent a promising option for future targeted therapies of leukemic patients.


2012 - Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels [Articolo su rivista]
Bressanin, D; Evangelisti, C; Ricci, F; Tabellini, G; Chiarini, F; Tazzari, Pl; Melchionda, F; Buontempo, F; Pagliaro, P; Pession, A; Mccubrey, Ja; Martelli, Am.
abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.


2012 - Mutations and deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades which alter therapy response [Articolo su rivista]
Mccubrey, Ja; Steelman, Ls; Chappell, Wh; Abrams, Sl; Montalto, G; Cervello, M; Nicoletti, F; Fagone, P; Malaponte, G; Mazzarino, Mc; Candido, S; Libra, M; Bäsecke, J; Mijatovic, S; Maksimovic-Ivanic, D; Milella, M; Tafuri, A; Cocco, L; Evangelisti, C; Chiarini, F; Martelli, Am.
abstract


2012 - PI3K/AKT/mTORC1 and MEK/ERK signaling in T-cell acute lymphoblastic leukemia: new options for targeted therapy [Articolo su rivista]
Martelli, Alberto M; Tabellini, Giovanna; Ricci, Francesca; Evangelisti, Camilla; Chiarini, Francesca; Bortul, Roberta; Mccubrey, James A; Manzoli, Francesco Antonio
abstract


2012 - Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance [Articolo su rivista]
Mccubrey, Ja; Steelman, Ls; Chappell, Wh; Abrams, Sl; Franklin, Ra; Montalto, G; Cervello, M; Libra, M; Candido, S; Malaponte, G; Mazzarino, Mc; Fagone, P; Nicoleti, F; Bäsecke, J; Mijatovic, S; Maksimovic-Ivanic, D; Milella, M; Tafuri, A; Chiarini, F; Evangelisti, C; Cocco, L; Martelli, Am.
abstract


2012 - Targeting the liver kinase B1/AMP-activated protein kinase pathway as a therapeutic strategy for hematological malignancies [Articolo su rivista]
Martelli, Am; Chiarini, F; Evangelisti, C; Ognibene, A; Bressanin, D; Billi, Am; Manzoli, L; Cappellini, A; Mccubrey, Ja.
abstract


2012 - Temsirolimus, an mTOR inhibitor, in combination with lower-dose clofarabine as salvage therapy for older patients with acute myeloid leukaemia: results of a phase II GIMEMA study (AML-1107) [Articolo su rivista]
Amadori, S.; Stasi, R.; Martelli, ALBERTO MARIA; Venditti, A.; Meloni, G.; Pane, F.; Martinelli, Giovanni; Lunghi, M.; Pagano, L.; Cilloni, D.; Rossetti, E.; Di Raimondo, F.; Fozza, C.; Annino, L.; Chiarini, Francesca; Ricci, F.; Ammatuna, E.; La Sala, E.; Fazi, P.; Vignetti, M.
abstract

The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20 mg/m2 on days 15 and temsirolimus 25 mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3.5 months, and median overall survival was 4 months (9.1 months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; P = 0.0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen.


2012 - Two hits are better than one: targeting both phosphatidylinositol 3-kinase and mammalian target of rapamycin as a therapeutic strategy for acute leukemia treatment [Articolo su rivista]
Martelli, Am; Chiarini, F; Evangelisti, C; Cappellini, A; Buontempo, F; Bressanin, D; Fini, M; Mccubrey, Ja.
abstract

Phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) are two key components of the PI3K/Akt/mTOR signaling pathway. This signal transduction cascade regulates a wide range of physiological cell processes, that include differentiation, proliferation, apoptosis, autophagy, metabolism, motility, and exocytosis. However, constitutively active PI3K/Akt/mTOR signaling characterizes many types of tumors where it negatively influences response to therapeutic treatments. Hence, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors may improve cancer patient outcome. The PI3K/Akt/mTOR signaling cascade is overactive in acute leukemias, where it correlates with enhanced drug-resistance and poor prognosis. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds targeting the catalytic site of both kinases. In preclinical models, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against acute leukemia cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin. At variance with rapamycin, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenic protein translation. Therefore, they strongly reduced cell proliferation and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors may represent a promising option for future targeted therapies of acute leukemia patients.


2011 - Effect of Lenalidomide Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients [Relazione in Atti di Convegno]
Follo, MATILDE YUNG; Clissa, Cristina; Mongiorgi, Sara; Chiarini, Francesca; Baccarani, Michele; Martinelli, Giovanni; Manzoli, Lucia; Martelli, ALBERTO MARIA; Finelli, Carlo; Cocco, LUCIO ILDEBRANDO
abstract

Nuclear inositide signalling pathways are involved in the MDS progression to AML. Indeed, in the last few years our group demonstrated not only that MDS cells can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Lenalidomide has proven effectiveness in 70–80% low-risk MDS cases with del(5q), resulting in transfusion-independence with a rise in hemoglobin levels, suppression of the 5q clone, and improvement of bone marrow morphologic features. In particular, in del(5q) MDS, Lenalidomide probably acts by directly suppressing the dysplastic clone, while in non-del(5q) it might enhance an effective erythropoiesis, possibly via activation of the EPO signalling, which in turn is associated with PI-PLCgamma1 pathways. However, the exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still not completely clarified. Interestingly, Lenalidomide might inhibit the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS cells, which display a marked apoptosis and a low proliferation rate. In this study we examined four MDS patients treated with Lenalidomide, and compared them with four low-risk MDS patients (IPSS: Low or Int-1) who only received best supportive care. In our study, all of the patients treated with Lenalidomide were affected by del(5q) low-risk MDS (IPSS: Low or Int-1), with transfusion-dependent anemia, and had only received supportive care before undergoing Lenalidomide treatment. Clinically, all of the patients responded to Lenalidomide: one patient reached Complete Remission, whilst the other three patients showed erythroid Hematologic Improvement. In contrast, all of the patients who were treated only with best supportive care maintained a Stable Disease. As for the molecular effects of Lenalidomide on lipid signalling pathways, we analyzed the expression of critical molecules involved in both cell proliferation and differentiation, that is PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling. Ongoing analyses are also trying to examine the effect of Lenalidomide on Akt phosphorylation and Globin genes, which are specifically associated with erythropoiesis. So far, our results indicate that, in our responder patients, both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Lenalidomide, whereas PI-PLCgamma1 is specifically induced, as compared with both healthy subjects and low-risk MDS patients treated with supportive care. Overall, these findings hint at a specific activation of PI-PLCgamma1 signalling following Lenalidomide treatment, and possibly pave the way to larger investigations aiming to better understand the role of these pathways in the mechanism of action of Lenalidomide in del(5q) MDS.


2011 - Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Evangelisti, Camilla; Ricci, Francesca; Tazzari, Pierluigi; Chiarini, Francesca; Battistelli, Michela; Falcieri, Elisabetta; Ognibene, Andrea; Pagliaro, Pasqualepaolo; Cocco, Lucio; Mccubrey, James A; Martelli, Alberto M
abstract

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival. J. Cell. Physiol. 226: 822-831, 2011. (C) 2010 Wiley-Liss, Inc.


2011 - Synergistic induction of PI-PLCbeta1 signaling by azacitidine and valproic acid in high-risk myelodysplastic syndromes [Articolo su rivista]
Follo, MATILDE YUNG; Finelli, Carlo; Mongiorgi, Sara; Clissa, C; Chiarini, Francesca; Ramazzotti, Giulia; Paolini, Stefania; Martinelli, Giovanni; Martelli, ALBERTO MARIA; Cocco, LUCIO ILDEBRANDO
abstract

The association between azacitidine (AZA) and valproic acid (VPA) has shown high response rates in high-risk myelodysplastic syndromes (MDS) cases with unfavorable prognosis. However, little is known about the molecular mechanisms underlying this therapy, and molecular markers useful to monitor the disease and the effect of the treatment are needed. Phosphoinositide-phospholipase C (PI-PLC) Β1 is involved in both genetic and epigenetic mechanisms of MDS progression to acute myeloid leukemia. Indeed, AZA as a single agent was able to induce PI-PLCΒ1 expression, therefore providing a promising new tool in the evaluation of response to demethylating therapies. In this study, we assessed the efficacy of the combination of AZA and VPA on inducing PI-PLCΒ1 expression in high-risk MDS patients. Furthermore, we observed an increase in Cyclin D3 expression, a downstream target of PI-PLCΒ1 signaling, therefore suggesting a potential combined activity of AZA and VPA in high-risk MDS in activating PI-PLCΒ1 signaling, thus affecting cell proliferation and differentiation. Taken together, our findings might open up new lines of investigations aiming at evaluating the role of the activation of PI-PLCΒ1 signaling in the epigenetic therapy, which may also lead to the identification of innovative targets for the epigenetic therapy of high-risk MDS.


2011 - Targeted inhibition of mTORC1 and mTORC2 by active-site mTOR inhibitors has cytotoxic effects in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Evangelisti, Camilla; Ricci, F.; Tazzari, P.; Tabellini, G.; Battistelli, M.; Falcieri, E.; Chiarini, Francesca; Bortul, R.; Melchionda, F.; Pagliaro, P.; Pession, Andrea; Mccubrey, J. A.; Martelli, ALBERTO MARIA
abstract

The mammalian Target Of Rapamycin (mTOR) serine/threonine kinase belongs to two multi-protein complexes, referred to as mTORC1 and mTORC2. mTOR-generated signals have critical roles in leukemic cell biology by controlling mRNA translation of genes that promote proliferation and survival. However, allosteric inhibition of mTORC1 by rapamycin has only modest effects in T-cell acute lymphoblastic leukemia (T-ALL). Recently, ATP-competitive inhibitors specific for the mTOR kinase active site have been developed. In this study, we have explored the therapeutic potential of active-site mTOR inhibitors against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. The inhibitors affected T-ALL cell viability by inducing cell-cycle arrest in G(0)/G(1) phase, apoptosis and autophagy. Western blot analysis demonstrated a Ser 473 Akt dephosphorylation (indicative of mTORC2 inhibition) and a dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors. The inhibitors strongly synergized with both vincristine and the Bcl-2 inhibitor, ABT-263. Remarkably, the drugs targeted a putative leukemia-initiating cell sub-population (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, the inhibitors displayed remarkable anti-leukemic activity, which emphasizes their future development as clinical candidates for therapy in T-ALL.


2011 - Temsirolimus, An Allosteric mTORC1 Inhibitor, Is Synergistic with Clofarabine in AML and AML Leukemia Initiating Cells [Abstract in Atti di Convegno]
Chiarini, Francesca; Grimaldi, C; Ricci, F; Tazzari, Pl; Iacobucci, Ilaria; Martinelli, Giovanni; Pagliaro, P; Mccubrey, J; Amadori, S; Martelli, ALBERTO MARIA
abstract


2010 - Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235, against T-cell acute lymphoblastic leukemia [Articolo su rivista]
Chiarini, Francesca; Grimaldi, Cecilia; F., Ricci; P. L., Tazzari; Evangelisti, Camilla; A., Ognibene; M., Battistelli; E., Falcieri; F., Melchionda; Pession, Andrea; P., Pagliaro; J. A., Mccubrey; Martelli, ALBERTO MARIA
abstract


2010 - Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms [Articolo su rivista]
Martelli, ALBERTO MARIA; V., Papa; P. L., Tazzari; F., Ricci; Evangelisti, Camilla; Chiarini, Francesca; Grimaldi, Cecilia; A., Cappellini; Martinelli, Giovanni; Ottaviani, Emanuela; P., Pagliaro; S., Horn; J., Bäsecke; L. H., Lindner; H., Eibl; J. A., Mccubrey
abstract


2010 - Pediatric early T-cell precursor leukemia with NF1 deletion and high-sensitivity in vitro to tipifarnib [Articolo su rivista]
Biagi, C; Astolfi, A; Masetti, R; Serravalle, S; Franzoni, M; Chiarini, F; Melchionda, F; Pession, A.
abstract


2010 - The Emerging Role of the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Network in Cancer Stem Cell Biology [Articolo su rivista]
Martelli, ALBERTO MARIA; Evangelisti, Camilla; Chiarini, Francesca; Grimaldi, Cecilia; J. A., Mccubrey
abstract

The cancer stem cell theory entails the existence of a hierarchically organized, rare population of cells which are responsible for tumor initiation, self-renewal/maintenance, and mutation accumulation. The cancer stem cell proposition could explain the high frequency of cancer relapse and resistance to currently available therapies. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway regulates a wide array of physiological cell functions which include differentiation, proliferation, survival, metabolism, autophagy, and motility. Dysregulated PI3K/Akt/mTOR signaling has been documented in many types of neoplasias. It is now emerging that this signaling network plays a key role in cancer stem cell biology. Interestingly, cancer stem cells displayed preferential sensitivity to pathway inhibition when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling pathways between neoplastic stem cells and healthy stem cells could be identified. In this review, we present the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of cancer stem cells, both in solid and hematological tumors. We then highlight how targeting PI3K/Akt/mTOR signaling with small molecules could improve cancer patient outcome.


2010 - The emerging role of the phosphatidylinositol 3-kinase/Akt/mTOR signaling network in normal myelopoieis and leukemogenesis [Articolo su rivista]
Martelli, ALBERTO MARIA; Evangelisti, Camilla; Chiarini, Francesca; Grimaldi, Cecilia; A., Cappellini; A., Ognibene; J. A., Mccubrey
abstract


2010 - The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling network and the control of normal myelopoiesis [Articolo su rivista]
Martelli, ALBERTO MARIA; Chiarini, Francesca; Evangelisti, Camilla; Grimaldi, Cecilia; Ognibene, A; Manzoli, Lucia; Billi, ANNA MARIA; Mccubrey, Ja
abstract


2010 - The phosphatidylinositol 3-kinase/Akt/mTOR signaling network as a therapeutic target in acute myelogenous leukemia patients [Articolo su rivista]
Martelli, ALBERTO MARIA; Evangelisti, Camilla; Chiarini, Francesca; J. A., Mccubrey
abstract


2009 - Dual inhibition of class IA phoshatidylinositol 3-kinase and mTOR as a new therapeutic option for T-cell acute lymphoblastic leukemia [Articolo su rivista]
Chiarini, Francesca; Fala', Federica; P. L., Tazzari; F., Ricci; Astolfi, Annalisa; Pession, Andrea; P., Pagliaro; J. A., Mccubrey; Martelli, ALBERTO MARIA
abstract

Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/ mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa P-glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors that are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G0-G1 phase cell cycle arrest and apoptosis, which was characterized by activation of caspase-3 and caspase-9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3B. Also, mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multitargeted therapy toward PI3K and mTOR alone or with existing drugs may serve as an efficient treatment toward T-ALL cells, which require up-regulation of PI3K/Akt/mTOR signaling for their survival and growth


2009 - Effect of Erythropoietin Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients [Relazione in Atti di Convegno]
Follo, MATILDE YUNG; Mongiorgi, Sara; Clissa, Cristina; Chiarini, Francesca; Paolini, Stefania; Baccarani, Michele; Martinelli, Giovanni; Manzoli, Lucia; Martelli, ALBERTO MARIA; Finelli, Carlo; Cocco, LUCIO ILDEBRANDO
abstract

Introduction: Phosphoinositide-phospholipase (PI-PLC) C beta1, PI-PLCgamma1 and Akt are key enzymes in nuclear signal transduction pathways, affecting both cell cycle and differentiation in normal physiology and neoplastic transformation. Our group previously showed not only that the Akt/mTOR axis is activated in patients with high-risk MDS (Follo MY et al, Cancer Res 2007), but also that there is an inverse correlation between PI-PLCbeta1 expression and Akt activation (Follo MY et al, Leukemia 2008). Moreover, we recently demonstrated that patients belonging to all of the IPSS risk groups can display a PI-PLCbeta1 mono-allelic deletion, and that this cytogenetic alteration is associated with a higher risk of evolution into Acute Myeloid Leukemia (AML) (Follo MY et al, J Clin Oncol 2009). Erythropoietin (EPO) is an effective treatment of anemia in 40-60% of low-risk MDS, often inducing a prolonged response. Interestingly, the activation of the EPO receptor has been correlated to the PI3K/Akt axis, which in turn is linked to either PI-PLCbeta1 or PI-PLCgamma1 signalling, so that EPO could affect cell proliferation and apoptosis. The aim of this study was therefore to clarify the relationship between EPO treatment and lipid signalling pathways, to investigate their role as molecular targets or predictive factors during EPO therapy. In fact, in patients who are refractory or lose response to EPO there could be a specific activation or inhibition of pathways involved in both cell cycle and differentiation. Patients and Methods: In this study we examined the effect of EPO treatment on lipid signal transduction pathways in MDS patients. The study included 16 patients (IPSS risk: low or intermediate-1), with a favourable response to EPO in 8/16 (50%) of the cases. For each patient we had the opportunity to analyze the expression of PI-PLCbeta1, PI-PLCgamma1, p-Akt and PIP2, which is involved in both PI-PLCbeta1 and PI3K/Akt activation processes, before and during EPO treatment, in order to detect every change in both clinical and biological features. By FISH analysis, we firstly assessed the presence of PI-PLCbeta1 mono-allelic deletion. Then, we quantified PI-PLCbeta1 and PI-PLCgamma1 gene and protein expression, as well as PIP2 and the degree of Akt activation; mRNA levels were quantified by real-time PCR, whereas the protein amount was detected by both a immunocytochemical and a flow cytometric detection approach. Results: The PI-PLCbeta1 mono-allelic deletion was found in 5/16 (31%) low-risk MDS patients: 2 of them showed a rapid evolution into AML, whilst the remaining 3 cases did not respond to EPO treatment. The molecular analyses showed a specific increase in Akt/PI-PLCgamma1 pathway for responder patients, whereas most of the patients refactory to EPO displayed a slight decrease in p-Akt levels and an activation of PI-PLCbeta1 signalling during EPO administration, so that these patients seem to counteract the lack of one PI-PLCbeta1 allele by increasing PI-PLCbeta1 gene and protein expression. Conclusions: Our results, although obtained in a small number of cases, confirm the possible involvement of PI-PLCbeta1 pathways in the EPO signalling. Moreover, our data suggest that the presence of the PI-PLCbeta1 mono-allelic deletion is associated with a worse clinical outcome and with a lack of response to EPO treatment, even in low-risk MDS patients who apparently have a good response profile for EPO (recent diagnosis, absence of long-term transfusion dependence, low or intermediate-1 IPSS risk, serum EPO levels<500 U/L). In fact, in our series, patients with the PI-PLCbeta1 mono-allelic deletion showed an unfavourable outcome (either a rapid evolution into AML or refractoriness to EPO treatment). Moreover, our findings indicate that not only PI-PLCbeta1, but also Akt/PI-PLCgamma1 pathways are critical for cell survival and proliferation in MDS patients treated with EPO. Therefore, these signal transduction pathways could beco


2009 - Involvement of nuclear PLC{beta}1 in lamin B1 phosphorylation and G2/M cell cycle progression [Articolo su rivista]
Fiume, Roberta; Ramazzotti, Giulia; Teti, Gabriella; Chiarini, Francesca; Faenza, Irene; Mazzotti, Giovanni; Billi, ANNA MARIA; Cocco, LUCIO ILDEBRANDO
abstract


2009 - Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS [Articolo su rivista]
Follo, MATILDE YUNG; Finelli, Carlo; Mongiorgi, Sara; Clissa, C; Bosi, C; Testoni, Nicoletta; Chiarini, Francesca; Ramazzotti, Giulia; Baccarani, Michele; Martelli, ALBERTO MARIA; Manzoli, Lucia; Martinelli, Giovanni; Cocco, LUCIO ILDEBRANDO
abstract


2009 - Targeting the phosphatidylinositol 3-kinase/Akt/mTOR signaling network in acute myelogenous leukaemia [Articolo su rivista]
Martelli, A. M.; Evangelisti, C.; Chiarini, F.; Grimaldi, C.; Manzoli, L.; A. McCubrey., J.
abstract

Background: The PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell growth, proliferation and survival not only under physiological conditions but also in a variety of tumor cells. Therefore, the PI3K/Akt/mTOR axis may be a critical target for cancer therapy. Objective: This review discusses how PI3K/Akt/mTOR signaling network is constitutively active in acute myelogenous leukemia (AML), where it strongly influences proliferation, survival and drug-resistance of leukemic cells, and how effective targeting of this pathway with pharmacological inhibitors, used alone or in combination with existing drugs, may result in suppression of leukemic cell growth, including leukemic stem cells. Methods: We searched the literature for articles dealing with activation of this pathway in AML and highlighting the efficacy of small molecules directed against the PI3K/Akt/mTOR signaling cascade. Conclusions: The limit of acceptable toxicity for standard chemotherapy has been reached in AML. Therefore, new therapeutic strategies are needed. Targeting the PI3K/Akt/mTOR signaling network with small molecule inhibitors, alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit selective inhibitors of the PI3K/Akt/mTOR pathway that show effectiveness and safety in the clinical setting are currently underway.


2009 - The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage [Articolo su rivista]
A., Cappellini; Chiarini, Francesca; A., Ognibene; J. A., Mccubrey; Martelli, ALBERTO MARIA
abstract

Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartate residues which could be targeted by caspases other than caspase-3. To test whether other caspases could cleave P-gp in vivo, we investigated the fate of P-gp during roscovitine-and sangivamycin-induced apoptosis in MCF7 human breast cancer cells, as they lack functional caspase-3. MCF7 cells were stably transfected with human cDNA encoding P-gp. P-gp was cleaved in vitro by purified recombinant caspase-3, -6 and -7. However, P-gp cleavage was not detected in vivo in MCF7 cells induced to undergoing apoptosis by either roscovitine or sangivamycin, despite activation of both caspase-6 and -7. Interestingly, P-gp overexpressing MCF7 cells were more sensitive to either roscovitine or sangivamycin than wild-type cells, suggesting a novel potential therapeutic strategy against P-gp overexpressing cells. Taken together, our results support the concept that caspase-3 is the only caspase responsible for in vivo cleavage of P-gp and also highlight small molecules which could be effective in treating P-gp overexpressing cancers


2009 - Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines [Articolo su rivista]
Bregoli, Lisa; Chiarini, Francesca; Gambarelli, A; Sighinolfi, G; Gatti, Am; Santi, Patrizia; Martelli, ALBERTO MARIA; Cocco, LUCIO ILDEBRANDO
abstract


2008 - Alteration of Akt activity increases chemotherapeutic drug and hormonal resistance in breast cancer yet confers an achilles heel by sensitization to targeted therapy [Capitolo/Saggio]
Mccubrey, J. A.; Sokolosky, M. L.; Lehmann, B. D.; Taylor, J. R.; Navolanic, P. M.; Chappell, W. H.; Abrams, S. L.; Stadelman, K. M.; Wong, E. W.; Misaghian, N.; Horn, S.; Bäsecke, J.; Libra, M.; Stivala, F.; Ligresti, G.; Tafuri, A.; Milella, M.; Zarzycki, M.; Dzugaj, A.; Chiarini, Francesca; Evangelisti, Camilla; Martelli, ALBERTO MARIA; Terrian, D. M.; Franklin, R. A.; Steelman, L. S.
abstract

Breast cancer ranks as the second most common cause of cancer death among women in the United States. Only lung cancer, which results primarily from cigarette smoking, induces more cancer deaths in women in the USA. Approximately 1 in 7 women in the United States will be diagnozed with breast cancer during her lifetime (Jemal et al., 2004). Over 210,000 new cases of breast cancer are diagnozed in the United States each year (Centers for Disease Control and Prevention, 2006 Centers for Disease Control and Prevention, Cancer: symptoms of breast cancer, Centers for Disease Control and Prevention, Atlanta, GA (2006) http://www.cdc.gov/cancer/breast/basic_info/symptoms.htm.Centers for Disease Control and Prevention, 2006). Breast cancer is the cause of death of over 40,000 women in the United States each year. Many drugs have been demonstrated to extend survival of breast cancer patients. Anticancer agents frequently used to treat breast cancer include chemotherapeutic drugs such as methotrexate, 5-fluorouracil (5-FU), cyclophosphamide, anthracyclines, taxanes, monoclonal antibodies such as trastuzumab, hormonal based therapeutics such as tamoxifen and aromatase inhibitors. Mechanisms by which these agents inhibit breast cancer progression vary from drug to drug. While these drugs are the mainstay of chemo, immuno and hormonal therapy of breast cancer, a common problem with these treatments is the development of drug resistance. Breast cancer cells can become drug resistant by multiple mechanisms which include: increased expression of membrane transporters which transport the toxic drug out of the cell or modify/detoxify the drug, increased expression of signaling and anti-apoptotic pathways as well as other mechanisms which allow the cells to grow in the presence of the drug. This manuscript will discuss some of the mechanisms by which the altered expression of key signaling and apoptotic pathways may lead to breast cancer drug resistance and how targeting these pathways may result in the suppression of neoplastic growth.


2008 - Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells [Articolo su rivista]
Papa, Veronica; P. L., Tazzari; Chiarini, Francesca; Cappellini, Alessandra; F., Ricci; Billi, ANNA MARIA; Evangelisti, Camilla; E., Ottaviani; Martinelli, Giovanni; Testoni, Nicoletta; J. A., Mccubrey; Martelli, ALBERTO MARIA
abstract


2008 - Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor (2S)-1-(1H-Indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-ami ne (A443654) in T-cell acute lymphoblastic leukemia [Articolo su rivista]
Fala', Federica; Blalock, Wl; Tazzari, Pl; Cappellini, A; Chiarini, Francesca; Martinelli, Giovanni; Tafuri, A; Mccubrey, Ja; Cocco, LUCIO ILDEBRANDO; Martelli, ALBERTO MARIA
abstract


2008 - Synergistic proapoptotic activity of recombinant TRAIL plus the Akt inhibitor perifosine in acute myeloid leukemia cells [Articolo su rivista]
Tazzari, P. L.; Tabellini, Giovanna; Ricci, F.; Papa, Veronica; Bortul, R.; Chiarini, Francesca; Evangelisti, Camilla; Martinelli, Giovanni; Bontadini, A.; Cocco, LUCIO ILDEBRANDO; Mccubrey, J. A.; Martelli, ALBERTO MARIA
abstract


2008 - The novel Akt inhibitor, perifosine, induces caspase-dependent apoptosis and downregulates P-glycoprotein expression in multidrug-resisten human T-acute leukemia cells by a JNK-dependent mechanism [Articolo su rivista]
Chiarini, Francesca; DEL SOLE, Marianna; Mongiorgi, Sara; Gaboardi, GIAN CARLO; Cappellini, A.; Mantovani, Irina; Follo, MATILDE YUNG; Mccubrey, J. A.; Martelli, ALBERTO MARIA
abstract


2007 - Expression of a functional CCR7 chemokine receptor inhibits the post-intravasation steps of metastasis in malignant murine mammary cancer cells [Articolo su rivista]
Croci, S; Nicoletti, G; Landuzzi, L; Palladini, A; Chiarini, F; Nanni, P; Lollini, Pl; DE GIOVANNI, C
abstract


2007 - Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin module for acute myelogenous leukemia therapy: from bench to bedside [Articolo su rivista]
Martelli, ALBERTO MARIA; Tazzari, Pl; Evangelisti, Camilla; Chiarini, Francesca; Blalock, Wl; Billi, ANNA MARIA; Manzoli, Lucia; Mccubrey, Ja; Cocco, LUCIO ILDEBRANDO
abstract


2007 - The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation [Articolo su rivista]
Follo, MATILDE YUNG; Mongiorgi, Sara; Bosi, Costanza; Cappellini, Alessandra; Finelli, Carlo; Chiarini, Francesca; Papa, Veronica; Libra, Massimo; Martinelli, Giovanni; Cocco, LUCIO ILDEBRANDO; Martelli, ALBERTO MARIA
abstract

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is important for both cell growth and survival. In particular, an impaired regulation of the Akt/mTOR axis has been strongly implicated in mechanisms related to neoplastic transformation, through enhancement of cell proliferation and survival. Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by a high risk of evolution into acute myelogenous leukemia (AML). The pathogenesis of the MDS evolution into AML is still unclear, although some recent studies indicate that aberrant activation of survival signaling pathways could be involved. In this investigation, done by means of immunofluorescent staining, we report an activation of the Akt/mTOR pathway in high-risk MDS patients. Interestingly, not only mTOR was activated but also its downstream targets, 4E-binding protein 1 and p70 ribosomal S6 kinase. Treatment with the selective mTOR inhibitor, rapamycin, significantly increased apoptotic cell death of CD33+ (but not CD33-) cells from high-risk MDS patients. Rapamycin was ineffective in cells from healthy donors or low-risk MDS. Moreover, incubation of high-risk MDS patient CD34+ cells with rapamycin decreased the in vitro clonogenic capability of these cells. In contrast, the phosphoinositide 3-kinase inhibitor, LY294002, did not significantly affect the clonogenic activity of high-risk MDS cells. Taken together, our results indicate that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients. Therefore, this signaling network could become an interesting therapeutic target for treating more advanced MDS cases. ©2007 American Association for Cancer Research.