Sara Mongiorgi

The novel Akt inhibitor, perifosine, induces caspase-dependent apoptosis and downregulates P-glycoprotein expression in multidrug-resistant human T-acute leukemia cells by a JNK-dependent mechanism.

A significant impediment to the success of cancer chemotherapy is the occurrence of multidrug resistance, which, in many cases, is attributable to overexpression of membrane transport proteins, such as the 170-kDa P-glycoprotein (P-gp). Also, upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway is known to play an important role in drug resistance, and has been implicated in the aggressiveness of a number of different cancers, including T-acute lymphoblastic leukemia (T-ALL). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine (a synthetic alkylphospholipid), on human T-ALL CEM cells (CEM-R), characterized by both overexpression of P-gp and constitutive upregulation of the PI3K/Akt network. Perifosine treatment induced death by apoptosis in CEM-R cells. Apoptosis was characterized by caspase activation, Bid cleavage and cytochrome c release from mitochondria. The proapoptotic effect of perifosine was in part dependent on the Fas/FasL interactions and c-Jun NH2-terminal kinase (JNK) activation, as well as on the integrity of lipid rafts. Perifosine downregulated the expression of P-gp mRNA and protein and this effect required JNK activity. Our findings indicate that perifosine is a promising therapeutic agent for treatment of T-ALL cases characterized by both upregulation of the PI3K/Akt survival pathway and overexpression of P-gp.

Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS

Lipid signaling pathways are involved in cell growth, differentiation, and apoptosis, and could have a role in the progression of myelodysplastic syndromes (MDS) into acute myeloid leukemia (AML). Indeed, recent studies showed that phosphoinositide-phospholipase (PI-PL)Cbeta1 mono-allelic deletion correlates with a higher risk of AML evolution. Also, a single patient treated with azacitidine, a DNA methyltransferase inhibitor currently used in MDS, displayed a direct correlation between PI-PLCbeta1 gene expression and drug responsiveness. Consequently, we hypothesized that PI-PLCbeta1 could be a target for demethylating therapy. First, we analyzed the structure of PI-PLCbeta1 gene promoter, then quantified the degree of PI-PLCbeta1 promoter methylation and gene expression in MDS patients at baseline and during azacitidine administration. Indeed, PI-PLCbeta1 mRNA increased in responder patients, along with a reduction of PI-PLCbeta1 promoter methylation. Also, the molecular response correlated to and anticipated the clinical outcome, thus suggesting that PI-PLCbeta1 gene reactivation could predict azacitidine responsiveness. Our results demonstrate not only that PI-PLCbeta1 promoter is hypermethylated in high-risk MDS patients, but also that the amount of PI-PLCbeta1 mRNA could predict the clinical response to azacitidine, therefore indicating a promising new therapeutic approach.

The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation.

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.

Phosphoinositide-Phospholipase C β1 Mono-Allelic Deletion Is Associated With Myelodysplastic Syndromes Evolution Into Acute Myeloid Leukemia

PURPOSE To evaluate the association between the presence of phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) mono-allelic deletion with the clinical outcome of myelodysplastic syndromes (MDS) patients. METHODS PI-PLCbeta1, PI-PLCbeta4, and PI-PLCgamma1 cytogenetic investigations were performed on 80 newly diagnosed MDS patients (18 low risk, 26 intermediate 1, 18 intermediate 2, 18 high risk) comparing the results with the clinical outcome of the patients. Moreover, fluorescent in situ hybridization results were validated by real-time polymerase chain reaction (PCR). Finally, PI-PLCbeta1 gene and protein expression were assessed by both real-time PCR and immunocytochemical experiments. RESULTS Collectively, 35 (43.75%) of 80 of the MDS patients showed a specific mono-allelic deletion of PI-PLCbeta1. Kaplan-Meier analysis revealed a significant association (P < .0001) between the PI-PLCbeta1 mono-allelic deletion and a higher risk of evolution into acute myeloid leukemia (AML), since 23 of 35 MDS patients (65.7%) bearing the PI-PLCbeta1 mono-allelic deletion evolved into AML. Even in multivariate analysis, the PI-PLCbeta1 mono-allelic deletion retained a higher significance, with a P < .001, as a prognostic factor of evolution into AML (odds ratio [OR] 1.83; 95% CI, 2.26 to 17.24; P = .00045). Finally, PI-PLCbeta1 deletion was related to an altered gene and protein expression. CONCLUSION PI-PLCbeta1 mono-allelic deletion is associated with a worse clinical outcome in MDS patients, hinting at the identification of a new group at higher risk of AML evolution and representing a reliable prognostic tool. Moreover, targeting PI-PLCbeta1 pathways might emerge as a new therapeutic strategy for MDS.

PI-PLCbeta-1 and activated Akt levels are linked to azacitidine responsiveness in high-risk myelodysplastic syndromes.

PI-PLC β -1 and activated Akt levels are linked to azacitidine responsiveness in high-risk myelodysplastic syndromes

Protein kinase C involvement in cell cycle modulation

Protein kinases C (PKCs) are a family of serine/threonine kinases which act as key regulators in cell cycle progression and differentiation. Studies of the involvement of PKCs in cell proliferation showed that their role is dependent on cell models, cell cycle phases, timing of activation and localization. Indeed, PKCs can positively and negatively act on it, regulating entry, progression and exit from the cell cycle. In particular, the targets of PKCs resulted to be some of the key proteins involved in the cell cycle including cyclins, cyclin-dependent kinases (Cdks), Cip/Kip inhibitors and lamins. Several findings described roles for PKCs in the regulation of G₁/S and G₂/M checkpoints. As a matter of fact, data from independent laboratories demonstrated PKC-related modulations of cyclins D, leading to effects on the G₁/S transition and differentiation of different cell lines. Moreover, interesting data were published on PKC-mediated phosphorylation of lamins. In addition, PKC isoenzymes can accumulate in the nuclei, attracted by different stimuli including diacylglycerol (DAG) fluctuations during cell cycle progression, and target lamins, leading to their disassembly at mitosis. In the present paper, we briefly review how PKCs could regulate cell proliferation and differentiation affecting different molecules related to cell cycle progression.

Synergistic induction of PI-PLCβ1 signaling by azacitidine and valproic acid in high-risk myelodysplastic syndromes

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.

Nuclear phospholipase C beta1 and cellular differentiation.

Phosphoinositides (PI) are the most extensively studied lipids involved in cell signaling pathways. The bulk of PI is found in membranes where they are substrates for enzymes, such as kinases, phosphatases and phospholipases, which respond to the activation by cell-surface receptors. The outcome of the majority of signaling pathways involving lipid second messengers results in nuclear responses finally driving the cell into differentiation, proliferation or apoptosis. Some of these pathways are well established, such as that of PI-specific phospholipase C (PI-PLC), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) into the two second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). Two independent cycles of PI are present inside the cell. One is localized at the plasma membrane, while the most recently discovered PI cycle is found inside the nuclear compartment. The regulation of the nuclear PI pool is totally independent from the plasma membrane counterpart, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. In this report we will focus on the signal transduction-related metabolism of nuclear PI and review the most convincing evidence that the PI cycle is involved in differentiation programs in several cell systems.

Epigenetic regulation of nuclear PI-PLCbeta1 signaling pathway in low-risk MDS patients during azacitidine treatment

Phosphoinositide-phospholipase C (PI-PLC) beta1 can be considered a specific target for demethylating therapy in high-risk myelodysplastic syndrome (MDS) patients, as azacitidine treatment has been associated with a PI-PLCbeta1-specific promoter demethylation, and induction of PI-PLCbeta1 gene and protein expression. However, little is known about the molecular effect of azacitidine in low-risk MDS or the functional mechanisms linked with azacitidine effect on PI-PLCbeta1 promoter. In the present study, we further investigated the role of epigenetic regulation of PI-PLCbeta1, mainly focusing on the structure of the PI-PLCbeta1 promoter. We first examined the effect of azacitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key molecules associated with the nuclear inositide signaling pathways, such as cyclin D3. By applying a chromatin immunoprecipitation method, we also studied the correlation between the demethylating effect of azacitidine and the degree of recruitment to PI-PLCbeta1 promoter of some transcription factors implicated in hematopoietic stem cell proliferation and differentiation, as well as of the methyl-CpG-binding domain proteins, which specifically interact with methylated DNA. Taken together, our results hint at a specific involvement of PI-PLCbeta1 in epigenetic mechanisms, and are particularly consistent with the hypothesis of a role for PI-PLCbeta1 in azacitidine-induced myeloid differentiation.

Nuclear inositide signaling in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are defined as clonal hematopoietic stem‐cell disorders characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. Although distinct morphologic subgroups exist, the natural history of MDS is progression to acute myeloid leukemia (AML). However, the molecular the mechanisms the underlying MDS evolution to AML are not completely understood. Inositides are key cellular second messengers with well‐established roles in signal transduction pathways, and nuclear metabolism elicited by phosphoinositide‐specific phospholipase C (PI‐PLC) β1 and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Recent findings evidenced the role played by nuclear lipid signaling pathways, which could become promising therapeutic targets in MDS. This review will provide a concise and updated revision of the state of art on this topic. J. Cell. Biochem. 109: 1065–1071, 2010.