Activation of nuclear inositide signalling pathways during erythropoietin therapy in low-risk MDS patients.

Inositide signaling pathways can have a role in the Myelodysplastic Syndromes (MDS) progression to acute myeloid leukemia. Erythropoietin (EPO) is currently used in low-risk MDS, where it successfully corrects anemia in 50-70% of patients. However, some MDS patients are refractory to this treatment and little is known about the exact molecular mechanisms underlying the effect of EPO in these subjects. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly focusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, which is activated by the EPO receptor, and PI-PLCbeta1/Cyclin D3 signaling, as Cyclin D3 is associated with hematopoietic proliferation and differentiation. Interestingly, EPO responder patients showed a specific activation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an increase in PI-PLCbeta1 signaling. Moreover, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment.

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.

Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

Synergistic induction of PI-PLCß1 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) ß1 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-PLCß1 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-PLCß1 expression in high-risk MDS patients. Furthermore, we observed an increase in Cyclin D3 expression, a downstream target of PI-PLCß1 signaling, therefore suggesting a potential combined activity of AZA and VPA in high-risk MDS in activating PI-PLCß1 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-PLCß1 signaling in the epigenetic therapy, which may also lead to the identification of innovative targets for the epigenetic therapy of high-risk MDS.

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress.

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

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 NH(2)-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.

Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myeloid leukemia (AML). The progression of myelodysplastic syndromes (MDSs) to AML is thought to be associated with abrogation of apoptotic control mechanisms. However, little is known about signal transduction pathways which may be involved in enhanced survival of MDS cells. In this report, we have performed immunocytochemical and flow cytometric analysis to evaluate the levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt) staining in 90% of the cases (n=22) diagnosed as high-risk MDS, whereas mononuclear cells from normal bone marrow or low-risk MDS patients showed low or absent Ser473 p-Akt staining. Furthermore, all high-risk MDS patients also demonstrated high expression of the Class I PI3K p110delta catalytic subunit and a decreased expression of PTEN. Taken together, our results suggest that Akt activation might be one of the factors contributing to the decreased apoptosis rate observed in patients with high-risk MDS.

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.