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Clinical sequencing across a large prospective cohort of patients with myelodysplasic syndrome uncovers distinct associations between the mono- and biallelic states ofTP53and clinical presentation Tumor protein p53 (TP53) is the most frequently mutated gene in cancer(1,2). In patients with myelodysplastic syndromes (MDS),TP53mutations are associated with high-risk disease(3,4), rapid transformation to acute myeloid leukemia (AML)(5), resistance to conventional therapies(6-8)and dismal outcomes(9). Consistent with the tumor-suppressive role ofTP53, patients harbor both mono- and biallelic mutations(10). However, the biological and clinical implications ofTP53allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS forTP53mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third ofTP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only.TP53multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)(11). Surprisingly, monoallelic patients did not differ fromTP53wild-type patients in outcomes and response to therapy. This study shows that consideration ofTP53allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.

Hypomethylating agents, such as 5-Azacytidine (AZA), significantly modified the therapeutic approach to MDS, primarily in older patients with higher risk disease for whom intensive chemotherapy and allogeneic stem cell transplantation are not an option. In low-risk MDS, 5- AZA aims to reduce transfusion dependency, improve quality of life and hopefully the survival, but it is still unclear if this therapeutic approach would be cost-effective. At a molecular level, the mechanisms underlying the effect of epigenetic therapy are not completely understood, although it is well known that DNA methyltransferase inhibitors can induce the expression of Phosphoinositide-Phospholipase C (PIPLC) beta1 in high-risk MDS. Here, we prospectively investigated the efficacy and safety of AZA in low-risk MDS patients. AZA was administered at a lower intensity schedule, that is 75 mg/sqm/day subcutaneous for 5 days every 28, for a total of 8 cycles, and response was assessed at the 4th and 8th cycle of AZA. Moreover, PI-PLCbeta1 promoter methylation and gene expression levels were quantified before and after each cycle of 5-AZA. The study included 32 patients, and 26 cases completed 8 cycles of AZA. ORR was 47% (15/32) on intention to treat and 58% (15/26) for patients completing the treatment program. In this latter group, 5 (19%) cases achieved CR and 10 (38%) had HI, according to the IWG criteria. Interestingly, three patients have maintained their HI after 37, 34 and 33 months without other treatments. At a molecular level, although baseline PI-PLCbeta1 levels were not correlated to clinical response, 5d-AZA induced a statistically significant decrease in PI-PLCbeta1 promoter methylation in 14/15 responders, which corresponded to a significant increase in PI-PLCbeta1 mRNA. In 9/14 (64%) responsive patients, the first molecular increase in PI-PLCbeta1 level was observed between the 3rd and 4th cycle, therefore anticipating the clinical evaluation. In addition, 8 cases showed a loss of the response after the end of therapy (8th cycle) and these cases displayed a significant reduction of PI-PLCbeta1 levels, below the pre-treatment values, already before the clinical loss of the response. Taken together, our results show that 5d-AZA is safe and effective in a proportion of low risk MDS patients. PI-PLCbeta1 gene expression is a reliable and dynamic marker of response that can be useful to optimize AZA therapy.

Background. The use of hypomethylating agents significantly modified the therapeutic approach to MDS patients, primarily in high-risk MDS patients. In low-risk MDSs the use of AZA hypomethylating agent is less understood. Epigenetic regulation of Phosphoinositide-Phospholipase C (PI-PLC) beta1 promoter and key molecules involved in the nuclear inositide signalling pathways seems to play an important role for the activity of AZA demethylating therapy. Aims. We prospectively evaluated the efficacy and safety of AZA low-dose in Low or Int-1 risk MDS patients who were symptomatic and/or unresponsive to previous treatments. In the same study we evaluated the molecular effects of AZA on PI-PLCbeta1 promoter methylation, in order to investigate a possibly correlation with the response to the drug. Methods. AZA was administered at a dose of 75 mg/mq/daily s.c for 5 consecutive days every 28 days for a total of 8 cycles. Final response was checked at the end of the 8th course. PI-PLC beta1 gene expression was evaluated on peripheral blood samples from patients at baseline, and monthly until the 8th cycle of AZA administration. Results. Between September 2008 and February 2010, 32 MDS patients with IPSS risk Low- or Int-1 were enrolled into the study. Most patients had a normal karyotype (63%) by metaphase cytogenetics, were BRC transfusion-dependent (81%), receiving a median of 4 units/mo, and were previously unresponsive to treatment including ESAs (69%). Twenty-six patients (81%) completed the treatment plan (8 cycles). The Overall Response Rate after the 8th cycle was 58% (15/26 pts) whereas 42% of patients maintained a stable disease; no patient progressed towards a high-risk MDS or AML. Five (19%) patients reached a complete remission whereas 10 (38%) achieved an hematological improvement. Transfusion independent was achieved in 8/26 patients (31%). The median duration of the response was 10 months; five patients maintain their response, that is CR in 2 cases (+24 and +30 months) and HI-E in 3 cases (+14, +25,+26 months) without any treatment or supportive therapy. No clinical or hematologic factor showed a correlation with the response. PI-PLCbeta1 gene expression was quantified in MDS patients at baseline and during AZA treatment. Results were calculated as a percentage ratio of PI-PLCbeta1 expression during AZA treatment compared to baseline. All but one patients (14/15) who achieved an hematologic response during treatment with AZA showed a statistically significant increase in PI-PLCbeta1 mRNA expression (P

Background: Although azacitidine (AZA) has proven effective in myelodysplastic syndromes (MDS), the duration of haematologic response is limited. Introduction: The French Group (Itzykson 2011) identified some clinical and haematologic parameters (poor ECOG performance status, IPSS intermediate and poor risk cytogenetics, circulating blasts, high transfusion need) independently associated with a a poorer outcome, and these 4 criteria were integrated in a 3-group prognostic score. Purpose: These data prompted us to retrospectively analyse our MDS pts treated with AZA who showed a favourable long-lasting response to AZA (i.e: duration of response ≥ 20 months). Materials and Methods: The type of response was defined according to IWG criteria (Cheson 2006): Complete Remission (CR), Marrow CR, Partial Remission (PR), and Hematologic Improvement (HI). Results: Thirty-six pts (M/F: 21/15), from nine Institutions, with a median age of 72 (range 52-84) yrs, showed a response duration ≥ 20 months. At AZA onset, IPSS risk was: low: 3 pts; intermediate- 1: 7 pts; intermediate-2: 21 pts; high: 5 pts. IPSS cytogenetic risk was: low: 23 pts; intermediate: 7 pts; high: 6 pts.Transfusion need was high (≥ 4 RBC units/8 weeks) in 17 pts. Following Itzykson’s AZA prognostic scoring system, the risk was low in 14 pts (38.9%), intermediate in 21 pts (58.3%), and high in 1 pt (2.8%), respectively. The pts received a median of 23.5 cycles of AZA (range: 8-59). The best response achieved was: CR in 21 pts (58.3%); marrow CR: 4 pts (11.1%); PR in 2 pts (5.5%); and HI in 9 pts (25%). Cytogenetic remission was achieved in 7 pts (19.4%). The median duration of response was 24.5 (range: 20-88) months. Twenty-two pts (61.1%) are still maintaining hematologic response, 9 pts (25%) are still alive but discontinued treatment because of disease progression, and 5 pts died (2 for AML). Median OS from the start of AZA was 35.5 (range: 22-120) months. Conclusions: Our data show that a long-lasting hematologic response can be achieved even in a significant fraction of pts presenting one or more poor risk features.

Berberine (BBR) is a common nutraceutical consumed by millions worldwide. BBR has many different effects on human health, e.g., diabetes, diarrhea, inflammation and now more recently it has been proposed to have potent anti-cancer effects. BBR has been shown to suppress the growth of cancer cells more than normal cells. BBR has been proposed to exert its growth-inhibitory effects by many different biochemical mechanisms including: suppression of cell cycle progression, induction of reactive oxygen species, induction of apoptosis and autophagy and interactions with DNA potentially leading to DNA damage, and altered gene expression. Pancreatic cancer is a leading cancer worldwide associated with a poor prognosis. As our population ages, pancreatic cancer has an increasing incidence and will likely become the second leading cause of death from cancer. There are few truly-effective therapeutic options for pancreatic cancer. Surgery and certain chemotherapeutic drugs are used to treat pancreatic cancer patients. Novel approaches to treat pancreatic cancer patients are direly needed as they usually survive for less than a year after being diagnosed. In the following manuscript, we discuss the abilities of BBR and certain chemically- modified BBRs (NAX compounds) to suppress growth of pancreatic cancer cells.

Nuclear inositide signalling is implicated in normal and pathological cell proliferation and differentiation in several distinct models. Among the key molecules of nuclear inositide pathways, phosphoinositide-phospholipase (PI-PLC) C beta1 is essential for regulating hematopoiesis, particularly along myeloid and erythroid lineage. Moreover, Akt activation is associated with protein synthesis, via mTOR pathway, and with erythroid induction, through PI-PLCgamma1 activation. Myelodysplastic syndromes (MDS) are a series of heterogeneous diseases characterized by ineffective hemopoiesis, with a variable risk of evolution into acute myeloid leukemia (AML). Therapeutic approaches for MDS include demethylating agents, such as azacitidine, aiming at reducing cell proliferation, and erythropoietin, useful for sustaining a normal erythropoiesis. In the last few years, a role for nuclear inositide signalling as a therapeutic target in MDS has been disclosed, in that PI-PLCbeta1 increase is associated with azacitidine responsiveness, even when this drug is used in combination with other agents, and Akt is specifically activated in MDS at higher risk of AML evolution. On the other hand, recent data demonstrated that inositide signalling can also be involved in erythroid therapy, given the inhibitory effect of erythropoietin on PI-PLCbeta1 and the activation of Akt/PI-PLCgamma1 pathway, following the administration of erythropoietin. Here, we review the strategic role of nuclear inositide signalling in MDS, in pathogenesis and therapy.

The roles of the epidermal growth factor receptor (EGFR) signaling pathway in various cancers including breast, bladder, brain, colorectal, esophageal, gastric, head and neck, hepatocellular, lung, neuroblastoma, ovarian, pancreatic, prostate, renal and other cancers have been keenly investigated since the 1980's. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about this pathway and how its deregulation can lead to cancer and how it may be differentially regulated in various cell types. Multiple inhibitors to EGFR family members have been developed and many are in clinical use. Current research often focuses on their roles and other associated pathways in cancer stem cells (CSCs), identifying sites where therapeutic resistance may develop and the mechanisms by which microRNAs (miRs) and other RNAs regulate this pathway. This review will focus on recent advances in these fields with a specific focus on breast cancer and breast CSCs. Relatively novel areas of investigation, such as treatments for other diseases (e.g., diabetes, metabolism, and intestinal parasites), have provided new information about therapeutic resistance and CSCs.

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.

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.

Nuclear inositide signaling is nowadays a well-established issue and a growing field of investigation, even though the very first evidence came out at the end of the 1980's. The understanding of its biological role is supported by the recent acquisitions dealing with pathology and namely hematological malignancies. Here, we review this issue highlighting the main achievements in the last years. J. Cell. Physiol. 226: 14–20, 2010. © 2010 Wiley-Liss, Inc.

Background: Lenalidomide is currently used in the treatment of del(5q) lowrisk MDS patients, where it may suppress the del(5q) clone and restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of Lenalidomide in MDS patients are not completely understood, even though it has been demonstrated that in Lenalidomide-sensitive del(5q) cell lines, Akt phosphorylation is inhibited. Interestingly, Akt signalling is activated both in high-risk MDS, where it activates cell proliferation, and in low-risk MDS, where it is associated with response to erythropoietin. Aims: To detect the specific effect of Lenalidomide on clonal del(5q) and normal (5q+) cells, in low-risk MDS patients and in cell lines, mainly focusing on erythropoiesis, cell cycle and signal transduction pathways involved in cell proliferation and differentiation. Methods: This study included 6 patients diagnosed with del(5q) MDS (IPSS: Low or Int-1) treated with Lenalidomide. Given the limited number of cells, we quantified the expression of Akt in bone marrow total mononuclear cells by immunocytochemistry only. In particular, we analyzed Akt and RPS14 co-localization, in order to specifically detect the del(5q) clone. Moreover, by Real-Time PCR analyses, we also assessed the expression of Globin genes, to evaluate the effect of the drug on erythropoiesis. To better discriminate between del(5q) and normal (5q+) cells, we studied the effect of Lenalidomide on Namalwa cells, showing a del(5q) karyotype, and in U937 cells, showing a normal 5q chromosome. In both cell lines, we quantified the expression of Globin genes, cell cycle and inositide signalling molecules, by Real-Time PCR, immunocytochemistry, Western blot and flow cytometry. Results: In our case series, 4/6 del(5q) low-risk MDS patients responded to Lenalidomide, whereas the 2 non responder patients early discontinued Lenalidomide for adverse events, and for these patients neither a clinical assessment of Lenalidomide effect, nor a molecular analysis, were possible. Responder patients showed an activation of erythropoiesis, in that Beta-Globin levels increased, as compared with baseline. Moreover, these subjects displayed a specific phosphorylation of Akt in cells not showing the 5q deletion. As for cell lines, Lenalidomide specifically induced an accumulation of Namalwa cells in G0/G1 phase, which corresponded to a slight decrease of p-Akt, an increase of p27 and a decreased expression of cyclin B1, cyclin E and cyclin D3. On the contrary, in normal (5q+) cells, Lenalidomide did not affect cell cycle, but induced both p-Akt and Gamma-Globin. Summary and Conclusions: Our data show that Lenalidomide can induce a selective arrest of cell cycle in G1 phase of del(5q) cells, therefore slowing the proliferation rate of this clonal cell population. On the contrary, cyclins, Akt and Gamma-Globin are specifically activated in normal (5q+) cells, possibly stimulating a normal cell proliferation and erythroid differentiation. Taken together, these findings could lead to a better comprehension of the effect of Lenalidomide in MDS and pave the way to innovative targeted therapies in low-risk MDS patients without del(5q).

Introduction. Erythropoiesis is associated with the activation of specific inositide-dependent signalling pathways, such as Akt/PLCgamma1. On the other hand, Akt can also be associated with cell cycle and differentiation in MDS, via activation of mTOR pathway. Lenalidomide is currently used in the treatment of del(5q) low-risk MDS patients, to compensate and counteract the MDS ineffective erythropoiesis and selectively inhibit cell cycle in del(5q) cells. The exact molecular mechanisms underlying the effect of Lenalidomide in del(5q) and non-del(5q) MDS cells are still unclear, even though it is clear that Leanlidomide can target signalling molecules playing a role in the maintenance of the balance between apoptosis, proliferation and differentiation, such as Akt. Methods.We studied 6 patients diagnosed with del(5q) Low-Risk MDS (IPSS: Low or Int-1) who were given Lenalidomide. We also analyzed the effect of Lenalidomide on two cell lines: Namalwa CSN.70, bearing a del(5q) karyotype, and U937, with a normal 5q chromosome. In particular, we quantified the expression of several genes implicated in inositide signalling, such as Akt, mTOR and PI-PLCgamma1, as well as Cyclins and Globin genes, in order to assess the effect of Lenalidomide on cell cycle and erythropoiesis. Results. In our case series, 4 out of 6del(5q) Low-Risk MDS patients responded to Lenalidomide and showed an activation of erythropoiesis, in that Beta-Globin levels increased. Moreover, these subjects also displayed a specific phosphorylation of Akt only in non-del(5q) cells, as assessed by co-localization experiments. As for the other 2 cases, patients early discontinued Lenalidomide for adverse events, and for these patients a clinical assessment of Lenalidomide effect was not possible. As for cell lines, our findings hint at a specific activation of cell cycle and erythropoiesis only in U937 cells, whereas cell cycle, but not erythropoiesis, was selectively inhibited in Namalwa CSN.70 cell line, so that in these cells proliferation is slower than in non-del(5q) cells. Conclusions. In our case series, as well as in cell lines, erythropoiesis activation is associated with a response to Lenalidomide, with an induction of Akt/PLCgamma1. Moreover, only in non-del(5q) cells, a normal proliferation is allowed, given that in del(5q) cells our analyses show a cell cycle arrest and an inhibition of Akt/mTOR pathway. Therefore, our data support the hypothesis of a specific activation of both inositide-dependent proliferation and erythroid differentiation pathways in response to Lenalidomide treatment in non-del(5q) cells, whereas in the del(5q) cell clone there is a cell cycle arrest and a slower erythroid differentiation. Taken together, these results point to a specific activation of signalling pathways during Lenalidomide administration and possibly pave the way to a larger investigation aiming to assess the role of these pathways during the therapy.

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

The roles of phosphoinositide-specific phospholipase C (PLC) have been extensively investigated in diverse cell lines and pathological conditions. Among the PLC isozmes, primary PLCs, PLC-β and PLC-γ, are directly activated by receptor activation, unlike other secondary PLCs (PLC-ɛ, PLC-δ1, and PLC-η1). PLC-β isozymes are activated by G protein couple receptor and PLC-γ isozymes are activated by receptor tyrosine kinase (RTK). Primary PLCs are differentially expressed in different tissues, suggesting their specific roles in diverse tissues and regulate a variety of physiological and pathophysiological functions. Thus, dysregulation of phospholipases contributes to a number of human diseases and primary PLCs have been identified as therapeutic targets for prevention and treatment of diseases. Here we review the roles of primary PLCs in physiology and their impact in pathology.

Purpose: This phase II prospective study aimed to evaluate the efficacy and safety of 5-days azacytidine (5d-AZA) in patients with low-risk myelodysplastic syndromes (MDS). Second, single-nucleotide polymorphism (SNP) genetic profile and phosphoinositide-phospholipase C (PI-PLC) β1 levels were studied to evaluate possible biologic markers able to predict the hematologic response. Experimental Design: The study tested a lower intensity schedule of azacytidine. The treatment plan consisted of 75 mg/sqm/d subcutaneous administered for 5 days every 28 days, for a total of 8 cycles. Results: Thirty-two patients were enrolled in the study. The overall response rate was 47% (15 of 32) on intention-to-treat and 58% (15 of 26) for patients completing the treatment program. In this latter group, 5 (19%) achieved complete remission (CR) and 10 (38%) had hematologic improvement, according to the International Working Group (IWG) criteria. Three patients have maintained their hematologic improvement after 37, 34, and 33 months without other treatments. Moreover, 21 and 2 of 26 cases completing 8 cycles were transfusion-dependent for red blood cells and platelets at baseline, respectively. Of these, 7 (33%) and 2 (100%) became transfusion-independent at the end of the treatment program, respectively. Grade 3–4 neutropenia occurred in 28% of patients and 4 patients died early due to infections or hemorrhage. SNP results were not significantly correlated to the clinical outcome, whereas PI-PLCβ1 level anticipated either positive or negative clinical responses. Conclusions: 5d-AZA is safe and effective in a proportion of patients with low-risk MDS. PI-PLCβ1 gene expression is a reliable and dynamic marker of response that can be useful to optimize azacytidine therapy. Clin Cancer Res; 19(12); 3297–308. ©2013 AACR.

Azacitidine (AZA) has proven effective in Myelodysplastic Syndromes (MDS), and the currently approved AZA regimen is 75 mg/sqm/die subcutaneously for 7 days every 28 days. Subsequently, other alternative and more convenient AZA dosing regimens have shown to be effective, in terms of hematologic responses (Lyons, 2009). From September 2004, in our Institution, 57 MDS patients (pts) (43 males), with a median age of 70 (37-84) yrs, were treated with AZA, following 4 different treatment regimens. Group 1 (10 pts), received the currently approved regimen (AZA 7). Group 2 (6 pts), received the AZA 7 regimen with valproic acid and all-trans-retinoic acid. Group 3 (29 pts) received the AZA 5-2-5 regimen: 50 mg/sqm/die SC for 10 days/28 days. Group 4 (12 pts) received the AZA 5 regimen: 75 mg/sqm/die SC for 5 days/28 days. Moreover, we quantified the degree of phosphoinositide-phospholipase C (PI-PLC) beta1 methylation and gene expression before and during AZA administration. At AZA onset, IPSS risk was: low: 3 pts; intermediate-1: 12 pts; intermediate-2: 33 pts; high: 9 pts. R-IPSS risk was: very low: 1 pt; low: 3 pts; intermediate: 7 pts; high: 10 pts; very high: 36 pts. WPSS risk was low: 3 pts; intermediate: 5 pts; high: 9 pts; very high: 40 pts. 9 pts had therapy-related MDS. ECOG-PS was poor (≥2) in 15 pts. Transfusion need was high (≥4 RBC units/8 weeks) in 34 pts. 6 pts presented circulating blasts. Following Itzykson’s AZA prognostic scoring system, the risk was low in 7 pts (12.3 %), intermediate in 48 pts (84.1 %), and high in 2 pts (3.6 %). The pts received a median of 8 cycles of AZA (range: 1-59). 50 pts were considered evaluable for response (at least 6 cycles): 34/50 pts (68%) showed a favourable response following IWG criteria (Cheson, 2006): complete remission (CR) in 7 pts (14% %), hematologic improvement (HI): 27 pts (54%). The median duration of response was 12 (range: 1-88) months. Group 1: 5 responders (62.5%) (1 CR and 4 HI); Group 2: 3 responders (50%) (3 HI); Group 3: 19 responders (73.1%) (5 CR, 14 HI); Group 4: 7 responders (70%) (2 CR, 5 HI). A significant toxicity (grade >2) was observed in 23 (40.4 %) pts. 37 pts died, 13 for AML, 9 for infection, 17 for other causes. Median OS from the start of AZA was 18 (range: 5-108) months. The detection of an increase in PIPLCbeta1 gene expression within the first three cycles of AZA therapy was significantly associated with a better clinical outcome and a longer hematologic response.

Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia.

Introduction. Azacitidine (AZA) has proven effective in myelodysplastic syndromes (MDS). The duration of haematological response is limited (median 13.6 months) (Fenaux, 2009), although some patients (pts) show a prolonged response. These data prompted us to retrospectively analyse our MDS pts treated with AZA, in order to enucleate longresponder pts (duration of response ≥20 months). METHODS. From September 2004, in our Institution, 52 MDS pts (40 males), median age: 70 (37-85) yrs, were treated with AZA, following 4 different treatment regimens: 9 pts received the AZA 7 regimen (AZA 7: 75 mg/sqm/die SC for 7 days/28 days); 6 pts received the combination of AZA 7 with valproic acid and all-trans-retinoic acid ; while 25 and 12 pts respectively received the alternative regimens AZA 5-2-5 and AZA 5 (Lyons, JCO 2009). 37 pts (71%) showed a IPSS high-risk MDS, while 15 pts (29%) with IPSS low-risk MDS received AZA because of refractoriness or ineligibility to erythropoietin, or secondary MDS. Moreover, as our group (Follo, 2009) demonstrated that phosphoinositide-phospholipase C (PIPLC) beta1 may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration. RESULTS. 9 pts (17.3%) showed a prolonged hematologic response (≥ 20 months). Pre-treatment clinical and haematologic features of long-responders: sex (M/F): 4/5; median age: 69 (52- 84); WHO: RCMD-RS: 1 pt; RAEB-1: 1 pt; RAEB-2: 7 pts; IPSS risk: low: 1 pt; int-1: 2 pts; int-2: 5 pts; high: 1 pt; IPSS cytogenetic risk: low: 7 pts; interm: 1 pt; high: 1 pt; ECOG: 0-1: 8 pts, ≥ 2: 1 pt; transfusion need (N° U)/8 weeks: < 4 : 4 pts; ≥ 4 : 5 pts; time from diagnosis (months): < 6 : 6 pts; ≥ 6 : 3 pts. Therapeutic regimen: AZA 7: 3 pts; AZA 5: 3 pts; AZA 5-2-5: 3 pts. Therapeutic response: median number of cycles: 19 (8-59); median time to 1st response: 3 (2-6) months; type of response: Complete Remission (CR): 3 pts; Hematologic Improvement (HI): 6 pts; cytogenetic remission: 1 pt; median duration of response: 30 (24-66) months; doubling of platelet count after 1st cycle: 4 pts; toxicity (grade > 2): 3 pts; 4 pts are still maintaining hematologic response, 3 pts are still alive but discontinued treatment because of disease progression, and 2 pts died (1 for AML and 1 for cachexy). Median survival (from the start of AZA): 38 (25-103) months. All the pts showed an increase in PI-PLCbeta1 expression, that was maintained along with the hematologic response. 15 pts (28.8%) showed a short-lived reponse (< 20 months), 5 pts (9.6%) show a shorter response but are still on treatment, 2 pts underwent allogeneic transplantation after 5 and 10 months. 7 pts (13.4%) are not evaluable for response (< 6 cycles), and 14 pts (26.9%) did not respond to AZA. Conclusions. Our data show that a limited but significant fraction of MDS pts show a long-lasting hematologic and molecular response to AZA.

Introduction. Inositide signalling pathways are involved in cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML. In particular, an altered expression of nuclear PI-PLCbeta1 and activated Akt can lead to a deregulation of cell cycle processes, therefore affecting the survival of primary MDS cells. Moreover, we postulated an inverse correlation between PI-PLCbeta1 expression and Akt activation in MDS. Indeed, these processes are critical especially in low-risk MDS, that usually show a marked apoptosis and a low proliferation rate, which can be rapidly reversed, thus leading to a worse clinical status. Lenalidomide is currently used in the treatment of del(5q) low-risk MDS patients, to compensate and counteract their ineffective erythropoiesis. In fact, this drug has anti-angiogenic activity, suppresses inflammatory cytokine release, induces the erythroid differentiation and enhances the EPO receptor signalling. The exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still unclear, even though it can target signalling pathways playing a role in the maintenance of the balance between apoptosis, proliferation and differentiation, such as PI3K/Akt. Methods. We studied 6 patients diagnosed with del(5q) Low-Risk MDS (IPSS: Low or Int-1) who were given Lenalidomide. We quantified the expression of several genes implicated in inositide signalling, such as PI-PLCbeta1 splicing variants and PIPLCgamma1, as well as Cyclin D3 and Beta-Globin, in order to assess the effect of Lenalidomide on erythropoiesis and cell cycle. Results. In our case series, 4 out of 6 del(5q) Low-Risk MDS patients responded to Lenalidomide and showed an activation of erythropoiesis, in that BetaGlobin levels increased. Moreover, these subjects also displayed an activation of PI-PLCgamma1, which is associated with PI3K/Akt activation. As for the other 2 cases, patients early discontinued Lenalidomide for adverse events, and for these patients a clinical assessment of Lenalidomide effect was not possible. Conclusions. Our data support the hypothesis of a role for PI-PLCgamma1 activation during Lenalidomide treatment, and confirm the activation of erythropoiesis in responder patients. In fact, Lenalidomide increased the expression of genes specifically associated with erythropoiesis, like Globin genes, in our responder patients. Our results also indicate that both PI-PLCbeta1 splicing variants, as well as Cyclin D3, are not significantly affected by Lenalidomide, whereas PIPLCgamma1 is specifically induced. Taken together, these results point to a specific activation of this pathway during the therapy and possibly pave the way to a larger investigation aiming to assess the role of these pathways in Lenalidomide response.

Introduction. Erytropoiesis-stimulating agents (ESAs) are effective in 20-30% of patients (pts) with Myelodysplastic Syndromes (MDS), but response rates are higher when pts are selected on the basis of clinical and hematologic parameters: baseline serum erythropoietin (EPO) level 4 units/8 weeks): 6 pts (17.6%); long interval from diagnosis to the start of EPO ( > 6 months) : 20 pts (58.8%). Starting weekly EPO dose: 80.000 U: 20 pts; 40.000 U: 14 pts. Outcome: median time to response: 8 (4-32) weeks; type of response: Complete Response (CR): 14 pts (44.1%); Haematologic Improvement (HI): 19 pts (55.1%); median duration of response: 47.5 (20-125) months; 15 pts (44.1%) were able to shift to a lower maintenance dose. Relapse occurred in 7 pts (20.6%) ( in 2 pts because of disease progression) after a median of 48 months. 24 pts are still alive, 5 pts died (none of them for AML), and 5 pts were lost at follow-up. Median survival (from the start of EPO): 51 (22-128) months. 13 pts show a shorter response but are still on treatment , 12 pts showed a short-lived reponse (< 20 months), and 10 pts did not respond to EPO. Conclusions. Our data show that, although in MDS the duration of response to EPO is limited, a substantial fraction of pts may show a long-lasting response, and that, unexpectedly, some of them may show unfavourable pre-treatment prognostic features (WPSS risk, high transfusion need).

Introduction. An impaired regulation of the PI3K/Akt axis is often associated with hematologic malignancies. Our group previously demonstrated PI-PLCbeta1 and Akt are inversely correlated in high-risk MDS. EPO treatment is currently used in the therapy of low-risk MDS, to compensate and counteract their ineffective erythropoiesis, although some patients do not respond to this treatment, or lose response. The activation of the EPO receptor has been linked to the activation of the PI3K/Akt/PI-PLCgamma1 axis, so that EPO could affect cell proliferation and apoptosis. Methods. Here we studied 16 MDS patients (IPSS risk: low or intermediate-1) and quantified the expression of several genes implicated in inositide signalling, that is PI-PLCbeta1 splicing variants and its downstream target Cyclin D3, as well as PI-PLCgamma1 and Beta-Globin, in order to assess the effect of EPO therapy. Moreover, we also studied the effect of EPO on Akt activation. Finally, to further investigate the role of PI-PLCbeta1 in erythroid differentiation, we exposed normal human CD34+ cells to EPO and analyzed the effect of PI-PLCbeta1 overexpression on Beta-Globin and PI-PLCgamma1 expression, as well as on Akt phosphorylation. Results. 8 out of 16 patients (50%) showed a favourable response to EPO, which was associated with a specific induction of Beta-Globin gene expression within the first two months of therapy. As for PI-PLCbeta1 and Cyclin D3 gene expression levels, the PIPLCbeta1/Cyclin D3 axis is down-regulated in EPO responder patients after 3-4 months of therapy. Moreover, not only Akt phosphorylation, but also PI-PLCgamma1 gene and protein expression increased during EPO treatment, therefore confirming the activation of the Akt/PIPLCgamma1 pathway in EPO responder patients. Finally, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and BetaGlobin expression. Conclusions. In our EPO responder patients the PIPLCbeta1/Cyclin D3 axis is down-regulated after 3-4 months of therapy. This is consistent with previous findings showing that PI-PLCbeta1, after an early transient increase, is down-regulated in primary human erythroblasts treated with EPO for up to 96 hours, therefore suggesting that PI-PLCbeta1 could be required at the beginning of erythroid differentiation but is dispensable, if not inhibitory, at later stages. These results, along with the fact that EPO responders displayed a specific phosphorylation of Akt, suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of the Akt/PI-PLCgamma1 pathway in EPO signalling, 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.

Since the late 1980s, a growing body of evidence has documented that phosphoinositides and their metabolizing enzymes, which regulate a large variety of cellular functions both in the cytoplasm and at the plasma membrane, are present also within the nucleus, where they are involved in processes such as cell proliferation, differentiation, and survival. Recent studies show that nuclear Phospholipase Cb1 (a key phosphoinositide metabolizing enzyme) could be somehow involved in myelodysplastic syndromes (MDS), i.e. hematopoietic disorders that can evolve into acutemyeloid leukemia (AML). This review aims to highlight the most significant and updated findings about Phospholipase C metabolism in the nucleus under pathological conditions, namely MDS and AML.

Inositol lipids are key regulators of several cellular functions. The identification of an independent nuclear polyphosphoinositides signaling machinery has led the way to find new roles for these molecules. PI-PLC-β1 is the most extensively studied PLC isoform in the nuclear compartment and a key player in the regulation of nuclear lipid signaling. Nuclear PI-PLC-β1 is involved in cell cycle progression and differentiation in response to growth factor stimulation. A growing body of evidence has demonstrated that nuclear phosphoinositides are also involved in cancer cell generation, proliferation, and resistance to apoptosis. Evidence on ex vivo human cancer cells from patients with myelodysplastic syndromes (MDS) confirmed these observations, suggesting the involvement of PI-PLC-β1 both in the pathogenesis of the disease and in the progression of MDS to acute myeloid leukemia. These studies have offered new targets for the development of novel therapeutic strategies as well as new prognostic tools.

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.

The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.

Lenalidomide (Len) has proven effectiveness in 70-80% of low-risk MDS cases with del(5q), resulting in transfusion-independence, and inducing a rise hemoglobin levels, suppression of the 5q clone and improvement of bone marrow morphologic features. In del(5q) MDS, Len might suppress the dysplastic clone, while in non-del(5q) it may promote effective erythropoiesis, via activation of EPO signalling, which in turn is associated with PI-PLCgamma1 pathway. However, the exact molecular mechanisms underlying the effect of Len in MDS cells are still unclear. Interestingly, Len targets 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, which display a marked apoptosis and a low proliferation rate. Recently, our group showed that inositide signalling pathways are involved in the MDS progression to AML. In particular, we demonstrated not only that MDS 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. Here, we report on a patient affected by MDS who was successfully treated with Len. The patient, a 58-year old female, was diagnosed with Refractory Anemia (IPSS: Low) and was given only supportive care before undergoing Len treatment. Shortly after the beginning of the therapy, in 2009, the patient showed a clinical favorable response to Len, and subsequently achieved complete hematologic and cytogenetic remission. To assess the molecular effects of Len on inositide signalling pathways, we analyzed the expression of critical genes involved in cell proliferation and differentiation, i.e. PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling and Akt activation. That is why ongoing analyses are also trying to examine the effect of Len on Akt, and the correlation between Len and the expression of other genes specifically associated with erythropoiesis, like Globin genes. So far, our results indicate that both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Len, whereas PI-PLCgamma1 is specifically induced. Consequently, these findings hint at a specific activation of PI-PLCgamma1 signalling following Len treatment, and possibly pave the way to further investigations aiming to better understand the role of these pathways in the mechanism of action of Len in del(5q) MDS.

Azacitidine (AZA) has proven effective (response rate: 60-80%) in myelodysplastic syndromes (MDS). The currently approved AZA regimen (AZA 7) is 75 mg/sqm/die subcutaneously (SC) for 7 days every 28 days. Recently a different AZA dosing schedule (AZA 5-2-5: 50 mg/m2/d subcutaneously for 5 days, followed by 2 days no treatment, then 50 mg/m2/d for 5 days), which avoids week-end dosing, has shown to induce therapeutic responses consistent with the currently approved schedule, in a population of mainly low risk pts (Lyons, 2009). These data prompted us to investigate the therapeutic effect of the AZA 5-2-5 regimen in high risk MDS pts (IPSS risk: high or intermediate-2). From September 2004, in our Institution, 28 high risk MDS pts. were treated with 2 different AZA dosing schedules. Group 1 (9 pts, 8 males, median age: 68, range 60-84 yrs) received the AZA 7 regimen, while group 2 (19 pts, 13 males, median age: 69, range 37-81 yrs) received the 5-2-5 AZA regimen. Moreover, as our group (Follo, 2009) demonstrated that phosphoinositide-phospholipase C (PI-PLC) beta1 may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration in both groups. Pts of group 1 received a median number of 12 (1-59) AZA cycles. Among the 8 evaluable pts (i.e.: at least 6 cycles) 5 (62.5%) showed a favourable response, following IWG criteria (Cheson, 2006): 1 Complete Remission (CR), 1 Partial Remission (PR) and 3 Hematologic Improvement (HI). 3 pts died because of evolution into Acute Myeloid Leukemia (AML), and 4 pts for other causes. One pts, still alive and under imatinib therapy, developed Ph1+ Chronic Myeloid Leukemia (CML) after 59 courses of AZA. Mean follow-up of group 1 pts: 31 (13-79) months. Pts of group 2 received a median number of 8 (1-17) AZA cycles. Among the 15 evaluable pts, 13 (86.6%) showed a favourable response: 5 CR (33.3%) and 8 HI. 3 pts died because of evolution into AML, and 2 for other causes. 2 pts underwent allogeneic stem cell transplantation, both after achieving CR. 12 pts are alive, 9 of them still under AZA treatment. Mean follow-up of group 2 pts: 14 (1-35) months. PI-PLCbeta1 methylation and gene expression appeared to be related to the therapeutic response, but not to the dose schedule. Our results, although larger studies are required, seem to confirm the effectiveness of the more convenient AZA 5-2-5 regimen, even in high risk MDS.

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) beta1 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.

A inositide-dependent signaling exists in the nucleus. In this review we focused to the nuclear PI-PLC signaling activity, its downstream effects and its role in haematological malignancies. PI-PLC β1 is involved in the physiological control of the cell cycle and by acting on the Cylin D3 promoter plays a crucial role in the process of C2C12 myoblast differentiation. In hematological malignancies recent studies showed that PI-PLC β1 mono-allelic deletion correlates with a higher risk of AML evolution. Moreover it has been shown that PI-PLC β1 promoter is hypermethylated in high-risk MDS patients and that the amount of PI-PLC β1 mRNA could predict the clinical response to azacitidine, a well known demethylating drug. All in all the data reviewed here pave the way to a new prognostic and therapeutic strategy in some haematological malignancies.

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.

Purpose To evaluate the association between the presence of phosphoinositide-phospholipase C β1 (PI-PLCβ1) mono-allelic deletion with the clinical outcome of myelodysplastic syndromes (MDS) patients. Methods PI-PLCβ1, PI-PLCβ4, and PI-PLCγ1 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-PLCβ1 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-PLCβ1. Kaplan-Meier analysis revealed a significant association (P < .0001) between the PI-PLCβ1 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-PLCβ1 mono-allelic deletion evolved into AML. Even in multivariate analysis, the PI-PLCβ1 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-PLCβ1 deletion was related to an altered gene and protein expression. Conclusion PI-PLCβ1 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-PLCβ1 pathways might emerge as a new therapeutic strategy for MDS.

The existence of an inositide-dependent nuclear signaling has been clearly shown. In this review we focused on the nuclear PI-PLC signaling activity and its downstream effects. The main isoform present in the nucleus is PI-PLC β1 and this isoform resides in the nuclear domains called speckles and colocalizes with the splicing factor SC35. PI-PLC β1 is also involved in the physiological control of the cell cycle. Moreover, acting on the cyclin D3 promoter plays a crucial role in the process of C2C12 myoblast differentiation. Finally in hematological malignancies such as high-risk MDS, the deletion of PI-PLC β1 gene has been observed. There is the likelihood that the deletion is a prognostic marker in that 66.7% MDS patients bearing the PI-PLC β1 monoallelic deletion evolved into AML. In addition the expression of nuclear PI-PLC β1 in MDS patients is modulated by the demethylating drug azacytidine. Therefore the analysis of nuclear PI-PLC-β1 appears useful for both MDS prognosis and checking of the epigenetic effect of antileukemic drugs.

PKR is activated in MDS patients and its subcellular localization depends on disease severity

Here we have reviewed the role of nuclear PLCβ1 signaling and namely its down-stream targets as well as the significance of inositide signaling, basically in the nucleus, in MDS blasts. As concerning myogenic differentiation, PLCβ1 along with the γ1 isoform, is essential for C2C12 myoblasts to become myotubes as judged by both molecular and morphological analysis. Both PLCs are capable of inducing, once overexpressed in their physiological compartment, the differentiation of C2C12 myoblasts by targeting specific regions of the cyclin D3 promoter even in the absence of insulin. When dealing with MDS it has been found that nuclear PLCβ1 has a specific role in MDS blasts and this is not only because of the presence of the mono-allelic deletion of its gene in high-risk patients who evolved in AML, but also because of its modulation, namely up-regulation, by means of azacitidine in high-risk responsive patients, which parallels the decrease of activated Akt. Indeed this seems interesting in the light of the fact that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients, and that this signaling network could become an interesting therapeutic target for treating more advanced MDS patients.

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.

Our main goal in this study was to investigate the role of phospholipase C (PLC) beta(1) and PLCgamma(1) in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLCbeta(1) in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLCbeta(1) or PLCgamma(1) cDNA and with small interfering RNAs from regions of the PLCbeta(1) or PLCgamma(1) gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLCbeta(1) and PLCgamma(1) were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLCbeta(1) or PLCgamma(1) expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5'-deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLCbeta(1) or PLCgamma(1) cDNAs and small interfering RNAs, respectively. Our data indicate that PLCbeta(1) or PLCgamma(1) signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLCbeta(1) and PLCgamma(1) during myogenic differentiation in vitro and implies that up-regulation of these enzymes is sufficient to mimic the actions of insulin in this process.

Location impinges on function of some of the main players of nuclear inositol lipid cycle. Here we have discussed the behavior of PI-PLCβ1 in myelodysplastic and cultured leukemia cells. The presence of a cryptic deletion of PI-PLCβ1 gene in high-risk MDS patients is accompanied by altered expression of its mRNA in that the overall decrease of mRNA is characterized by a dramatic decrease of the splicing variant 1a, which is cytosolic and partially nuclear, whilst the splicing variant 1b is still highly represented. This suggests that altered expression of nuclear PI-PLCβ1 could be involved in a disregulation of the cell cycle and have also important effects on cell apoptotic pathways. Moreover, in cultured leukemia cells (Felc) it has been reported by means of a proteomic approach that the splicing factor SRp20 interacts with nuclear PI-PLCβ1 and its expression is modulated by this signaling molecule. All in all, it appears more and more evident that nuclear signaling elicited by PI-PLCβ1 is a key event in the control of cell cycle progression.

Phosphoinositide-specific phospholipase C (PI-PLC) beta 1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLC beta 1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLC beta 1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLC beta 1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCf beta 1. To analyze and quantify the levels of the two different splicing variants of PI-PLC beta 1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PlPLC beta 1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLC beta 1b mRNA compared to PI-PLC beta 1a mRNA. Our data support the contention that the deletion of the PI-PLC beta 1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the la isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.

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.

no abstract available