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B-type lamins are fundamental components of the nuclear lamina, a complex structure that acts as a scaffold for organization and function of the nucleus. Lamin B1 and B2, the most represented isoforms, are encoded by LMNB1 and LMNB2 gene, respectively. All B-type lamins are synthesized as precursors and undergo sequential post-translational modifications to generate the mature protein. B-type lamins are involved in a wide range of nuclear functions, including DNA replication and repair, regulation of chromatin and nuclear stiffness. Moreover, lamins B1 and B2 regulate several cellular processes, such as tissue development, cell cycle, cellular proliferation, senescence, and DNA damage response. During embryogenesis, B-type lamins are essential for organogenesis, in particular for brain development. As expected from the numerous and pivotal functions of B-type lamins, mutations in their genes or fluctuations in their expression levels are critical for the onset of several diseases. Indeed, a growing range of human disorders have been linked to lamin B1 or B2, increasing the complexity of the group of diseases collectively known as laminopathies. This review highlights the recent findings on the biological role of B-type lamins under physiological or pathological conditions, with a particular emphasis on brain disorders and cancer. Graphical abstract

The University of Bologna School of Medicine in 2003 adopted a near-peer teaching (NPT) program with senior medical students teaching and assisting younger students in human anatomy laboratories. This study aimed to evaluate the effectiveness and outcomes of this program—unique on the Italian academic panorama—from the tutors’ perspective. An anonymous online survey was administered to all those who acted as peer tutors in the period from 2003 to 2021; it evaluated tutors’ perceptions regarding the influence of the tutoring experience on their skillset gains, academic performance, and professional career. Furthermore, tutors were asked to express their views on the value of cadaver dissection in medical education and professional development. The overall perception of the NPT program was overwhelmingly positive and the main reported benefits were improved long-term knowledge retention and academic performance, improved communication, team-working and time management skills, and enhanced self-confidence and motivation. Most tutors strongly believed that cadaver dissection was an invaluable learning tool in medical education, helped them to develop professionalism and human values, and positively influenced the caring of their future patients. Nearly all the participants highlighted the importance of voluntary body donation for medical education and research. The present results supported the thesis that tutors themselves benefited from the act of teaching peers; this impactful experience equipped them with a wide range of transferable skills that they could draw on as future educators and healthcare professionals.

Glioblastoma represents the most lethal brain tumor in adults. Several studies have shown the key role of phospholipase C β1 (PLCβ1) in the regulation of many mechanisms within the central nervous system suggesting PLCβ1 as a novel signature gene in the molecular classification of high-grade gliomas. This study aims to determine the pathological impact of PLCβ1 in glioblastoma, confirming that PLCβ1 gene expression correlates with glioma’s grade, and it is lower in 50 glioblastoma samples compared to 20 healthy individuals. PLCβ1 silencing in cell lines and primary astrocytes, leads to increased cell migration and invasion, with the increment of mesenchymal transcription factors and markers, as Slug and N-Cadherin and metalloproteinases. Cell proliferation, through increased Ki-67 expression, and the main survival pathways, as β-catenin, ERK1/2 and Stat3 pathways, are also affected by PLCβ1 silencing. These data suggest a potential role of PLCβ1 in maintaining a normal or less aggressive glioma phenotype.

Renal failure is a worldwide disease with a continuously increasing prevalence and involving a rising need for long-term treatment, mainly by haemodialysis. Arteriovenous fistula (AVF) is the favourite type of vascular access for haemodialysis; however, the lasting success of this therapy depends on its maturation, which is directly influenced by many concomitant processes such as vein wall thickening or inflammation. Understanding the molecular mechanisms that drive AVF maturation and failure can highlight new or combinatorial drugs for more personalized therapy. In this review we analysed the relevance of critical enzymes such as PI3K, AKT and mTOR in processes such as wall thickening remodelling, immune system activation and inflammation reduction. We focused on these enzymes due to their involvement in the modulation of numerous cellular activities such as proliferation, differentiation and motility, and their impairment is related to many diseases such as cancer, metabolic syndrome and neurodegenerative disorders. In addition, these enzymes are highly druggable targets, with several inhibitors already being used in patient treatment for cancer and with encouraging results for AVF. Finally, we delineate how these enzymes may be targeted to control specific aspects of AVF in an effort to propose a more specialized therapy with fewer side effects.

Polyphosphoinositides (PPIns) and their modulating enzymes are involved in regulating many important cellular functions including proliferation, differentiation or gene expression, and their deregulation is involved in human diseases such as metabolic syndromes, neurodegenerative disorders and cancer, including Acute Myeloid Leukemia (AML). Given that PPIns regulating enzymes are highly druggable targets, several studies have recently highlighted the potential of targeting them in AML. For instance many inhibitors targeting the PI3K pathway are in various stages of clinical development and more recently other novel enzymes such as PIP4K2A have been implicated as AML targets. PPIns have distinct subcellular organelle profiles, in part driven by the specific localisation of enzymes that metabolise them. In particular, in the nucleus, PPIns are regulated in response to various extracellular and intracellular pathways and interact with specific nuclear proteins to control epigenetic cell state. While AML does not normally manifest with as many mutations as other cancers, it does appear in large part to be a disease of dysregulation of epigenetic signalling and many novel therapeutics are aimed at reprogramming AML cells toward a differentiated cell state or to one that is responsive to alternative successful but limited AML therapies such as ATRA. Here, we propose that by combining bioinformatic analysis with inhibition of PPIns pathways, especially within the nucleus, we might discover new combination therapies aimed at reprogramming transcriptional output to attenuate uncontrolled AML cell growth. Furthermore, we outline how different part of a PPIns signalling unit might be targeted to control selective outputs that might engender more specific and therefore less toxic inhibitory outcomes.

Phosphoinositide-specific phospholipases C (PI-PLCs) are a class of enzymes involved in the phosphatidylinositol metabolism, which is implicated in the activation of several signaling pathways and which controls several cellular processes. The scientific community has long accepted the existence of a nuclear phosphoinositide (PI) metabolism, independent from the cytoplasmic one, critical in nuclear function control. Indeed, nuclear PIs are involved in many activities, such as cell cycle regulation, cell proliferation, cell differentiation, membrane transport, gene expression and cytoskeletal dynamics. There are several types of PIs and enzymes implicated in brain activities and among these enzymes, PI-PLCs contribute to a specific and complex network in the developing nervous system. Moreover, considering the abundant presence of PI-PLCβ1, PI-PLCγ1 and PI-PLCβ4 in the brain, a specific role for each PLC subtype has been suggested in the control of neuronal activity, which is important for synapse function, development and other mechanisms. The focus of this review is to describe the latest research about the involvement of PI-PLC signaling in the nervous system, both physiologically and in pathological conditions. Indeed, PI-PLC signaling imbalance seems to be also linked to several brain disorders including epilepsy, movement and behavior disorders, neurodegenerative diseases and, in addition, some PI-PLC subtypes could become potential novel signature genes for high-grade gliomas.

Human body dissection is fundamental in medical education, as it allows future physicians to learn about the body's morphology in three dimensions, to recognize anatomical variations and to develop and increase the essential qualities of respect, compassion and empathy for patients. It is equally important in clinical training as it allows surgeons to improve their manual dexterity and practical skills and to test innovative surgical techniques and devices. In Italy prior to 2020, body acquisition and use for study and research purposes were regulated by a generic set of old directives and national decrees which dealt only marginally with these issues. However, in 2013, a whole body donation program was officially set up at the Institute of Human Anatomy of the University of Bologna. Completely free and voluntary informed consent has always been regarded as a core prerequisite and, since its inception, the program exclusively accepted bequeathed bodies. On February 10, 2020, a specific law governing the disposition of post mortem human body and tissues for study, training and scientific research purposes was definitively enacted. The present work traces the University of Bologna's experience leading to the whole body donation program and the brand new dissecting room. It describes the program of Bologna as an example of "good practice" in body donation, aimed at ensuring education and clinical training by means of both traditional gross anatomy and innovative technology. Moreover, it analyzes the results achieved in terms of increased donor enrollment and improved teaching/training quality and the strengths of this program in light of the provisions enshrined in the new law.

Autosomal-dominant leukodystrophy (ADLD) is a rare fatal neurodegenerative disorder with overexpression of the nuclear lamina component, Lamin B1 due to LMNB1 gene duplication or deletions upstream of the gene. The molecular mechanisms responsible for driving the onset and development of this pathology are not clear yet. Vacuolar demyelination seems to be one of the most significant histopathological observations of ADLD. Considering the role of oligodendrocytes, astrocytes, and leukemia inhibitory factor (LIF)-activated signaling pathways in the myelination processes, this work aims to analyze the specific alterations in different cell populations from patients with LMNB1 duplications and engineered cellular models overexpressing Lamin B1 protein. Our results point out, for the first time, that astrocytes may be pivotal in the evolution of the disease. Indeed, cells from ADLD patients and astrocytes overexpressing LMNB1 show severe ultrastructural nuclear alterations, not present in oligodendrocytes overexpressing LMNB1. Moreover, the accumulation of Lamin B1 in astrocytes induces a reduction in LIF and in LIF-Receptor (LIF-R) levels with a consequential decrease in LIF secretion. Therefore, in both our cellular models, Jak/Stat3 and PI3K/Akt axes, downstream of LIF/LIF-R, are downregulated. Significantly, the administration of exogenous LIF can partially reverse the toxic effects induced by Lamin B1 accumulation with differences between astrocytes and oligodendrocytes, highlighting that LMNB1 overexpression drastically affects astrocytic function reducing their fundamental support to oligodendrocytes in the myelination process. In addition, inflammation has also been investigated, showing an increased activation in ADLD patients’ cells.

An increasing number of reports suggests a significant involvement of the phosphoinositide (PI) cycle in cancer development and progression. Diacylglycerol kinases (DGKs) are very active in the PI cycle. They are a family of ten members that convert diacylglycerol (DAG) into phosphatidic acid (PA), two-second messengers with versatile cellular functions. Notably, some DGK isoforms, such as DGKα, have been reported to possess promising therapeutic potential in cancer therapy. However, further studies are needed in order to better comprehend their involvement in cancer. In this review, we highlight that DGKs are an essential component of the PI cycle that localize within several subcellular compartments, including the nucleus and plasma membrane, together with their PI substrates and that they are involved in mediating major cancer cell mechanisms such as growth and metastasis. DGKs control cancer cell survival, proliferation, and angiogenesis by regulating Akt/mTOR and MAPK/ERK pathways. In addition, some DGKs control cancer cell migration by regulating the activities of the Rho GTPases Rac1 and RhoA.

The immune system is a complex network that acts to protect vertebrates from foreign microorganisms and carries out immunosurveillance to combat cancer. In order to avoid hyper-activation of the immune system leading to collateral damage tissues and organs and to prevent self-attack, the network has the intrinsic control mechanisms that negatively regulate immune responses. Central to this negative regulation are regulatory T (T-Reg) cells, which through cytokine secretion and cell interaction limit uncontrolled clonal expansion and functions of activated immune cells. Given that positive or negative manipulation of T-Regs activity could be utilised to therapeutically treat host versus graft rejection or cancer respectively, understanding how signaling pathways impact on T-Regs function should reveal potential targets with which to intervene. The phosphatidylinositol-3-kinase (PI3K) pathway controls a vast array of cellular processes and is critical in T cell activation. Here we focus on phosphoinositide 3-kinases (PI3Ks) and their ability to regulate T-Regs cell differentiation and function.

Nuclear inositides have a specific subcellular distribution that is linked to specific functions; thus their regulation is fundamental both in health and disease. Emerging evidence shows that alterations in multiple inositide signalling pathways are involved in pathophysiology, not only in cancer but also in other diseases. Here, we give an overview of the main features of inositides in the cell, and we discuss their potential as new molecular therapeutic targets.

Phosphoinositide-specific phospholipases C (PLCs) are a class of enzymes involved in several cell activities, such as cell cycle regulation, proliferation, differentiation and cytoskeletal dynamics. Among these enzymes, PLCγ1 is one of the most expressed PLCs in the brain, contributing to a complex network in the developing nervous system. Several studies have shown that PLCγ1 signaling imbalance is linked to several brain disorders, including glioblastoma, the most aggressive brain tumor in adults. Indeed, it has been demonstrated a link between PLCγ1 inhibition and the arrest of glioma cell motility of fetal rat brain aggregates and the impairment of cell invasion abilities following its down-regulation. This study aims to determine the pathological influence of PLCγ1 in glioblastoma, through a translational study which combines in silico data, data from glioblastoma patients' samples and data on engineered cell lines. We found out that PLCγ1 gene expression correlates with the pathological grade of gliomas, and it is higher in fifty patients' glioblastoma tissue samples compared to twenty healthy controls. Moreover, it was demonstrated that PLCγ1 silencing in U87-MG leads to a reduction in cell migration and invasion abilities. The opposite trend was observed following PLCγ1 overexpression, suggesting an interesting possible involvement of PLCγ1 in gliomas' aggressiveness.

Background and Rationale. miRNAs are small non-coding RNAs that regulate gene expression by acting on the epigenetic machinery and are themselves controlled by epigenetic mechanisms. The expression of miRNAs is linked to cancer development and miRNA profiles are studied as new prognostic factors or therapeutic new perspectives (Jiang X et al. Nat Commun 2016). High-risk MDS are now treated with hypomethylating agents, like Azacitidine (AZA), alone or in combination with other drugs, such as Lenalidomide (LEN). Recent data showed that the concurrent acquisition of specific point mutations on PI3KCD, PLCG2 and AKT3 genes is associated with loss of response to AZA+LEN therapy (Follo MY et al. Leukemia 2019). Inositide signalling regulated by Phospholipase C (PLC) and PI3K/AKT is indeed involved in epigenetic processes and in MDS progression to AML, through the regulation of proliferation, differentiation and apoptosis. Patients and Methods. This study included 26 high-risk MDS patients treated with AZA (75 mg/m2/day, days 1-5, sc) and LEN (10 mg/day, days 1-21 or 8-21, orally) every 4 weeks. Patients showing complete remission (CR), partial remission (PR), any hematologic improvement (HI) or marrow CR+HI following IWG response criteria were considered as responders, while patients showing stable disease or disease progression were considered as non-responders. miRNAs expression was assessed using an Affymetrix miRNA 4.0 array on patients' cells extracted at baseline and during the therapy, at the 4th (T4) and 8th (T8) cycle of therapy. Results were then validated by Real-Time PCR and miRNA targets were studied by dual Luciferase assay. Real-Time PCR was also used to examine the expression of PLC genes. Results. All patients included in this study were considered evaluable for response. According to the revised IWG criteria (14), the overall response rate (ORR) was 76.9% (20/26 cases): CR (5/26, 19.2%), PR (1/26, 3.8%), marrow CR (mCR, 2/26, 7.7%), HI (6/26, 23.1%), mCR+HI (6/26, 23.1%), whereas 6/26 patients (23.1%) had a stable disease. For our analyses, we considered 10 patients as responders (R, showing response within T4 and maintaining it at T8), 10 losing response (LR, showing response within T4 and losing it at T8) and 6 non-responders (NR, never showing a response). Paired analysis between R and NR patients showed a statistically significant up-regulation of miR-192-5p and miR-21-5p between T0 and T4, as well as a down-regulation of miR-224-5p between T4 and T8, hinting at a relevant role for these miRNAs during AZA+LEN response. Real-Time PCR analyses confirmed the modulation of miR-192-5p and an altered expression of PLC genes during AZA+LEN therapy in all patients' subgroups, as well as an involvement of BCL-2 (possible target of miR-192-5p) that was also proven in vitro by dual Luciferase assays. Furthermore, as miR-192-5p expression seemed to be correlated with response, we performed Kaplan-Meier analyses and found out an association between high levels of miR-192-5p at T4 and OS (p=0.08) or LFS (p=0.04) in our MDS cases. More interestingly, this correlation was stronger (p=0.03) in R, as compared with LR and NR. Conclusions. This study shows that AZA+LEN therapy in MDS affects the expression of miR-192-5p, whose high level at T4 is associated with higher OS and LFS in responder patients. Moreover, we showed that miR-192-5p specifically targets and inhibits BCL-2, hinting at a regulation of MDS proliferation and apoptosis. Additional studies, to be performed in a larger cohort of MDS patients, are needed to confirm these data, as well as better understand the molecular mechanisms and the prognostic relevance of miR-192-5p in AZA+LEN therapy. Disclosures Cavo: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy, Honoraria; Novartis: Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Finelli: Celgene BMS: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy; Novartis: Consultancy, Speakers Bureau.

Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In several cancer types, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] play significant roles in regulating survival, proliferation, invasion, and growth of cancer cells. Phosphoinositide-specific phospholipase C (PLC) catalyze the generation of the essential second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP3) by hydrolyzing PtdIns(4,5)P2. DAG and InsP3 regulate Protein Kinase C (PKC) activation and the release of calcium ions (Ca2+) into the cytosol, respectively. This event leads to the control of several important biological processes implicated in cancer. PLCs have been extensively studied in cancer but their regulatory roles in the oncogenic process are not fully understood. This review aims to provide up-to-date knowledge on the involvement of PLCs in cancer. We focus specifically on PLCβ, PLCγ, PLCδ, and PLCε isoforms due to the numerous evidence of their involvement in various cancer types.

Introduction: An accurate diagnostic and prognostic evaluation of patients with myelodysplastic syndromes (MDS) is essential: treatments are different for patients at lower or higher risk of evolut...

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases. In this prospect we analyze the role of PI-PLCβ1 nuclear isoform in relation to the cell cycle regulation, the cell differentiation, and different physiopathological pathways focusing on the importance of the nuclear localization from both molecular and clinical point of view. PI-PLCβ1 is essential for G1/S transition through DAG and Cyclin D3 and plays also a central role in G2/M progression through Cyclin B1 and PKCα. In the differentiation process of C2C12 cells PI-PLCβ1 increases in both myogenic differentiation and osteogenic differentiation. PI-PLCβ1 and Cyclin D3 reduction has been observed in Myotonic Dystrophy (DM) suggesting a pivotal role of these enzymes in DM physiopathology. PI-PLCβ1 is also involved in adipogenesis through a double phase mechanism. Moreover, PI-PLCβ1 plays a key role in the normal hematopoietic differentiation where it seems to decrease in erythroid differentiation and increase in myeloid differentiation. In Myelodysplastic Syndromes (MDS) PI-PLCβ1 has a genetic and epigenetic relevance and it is related to MDS patients' risk of Acute Myeloid Leukemia (AML) evolution. In MDS patients PI-PLCβ1 seems to be also a therapeutic predictive outcome marker. In the central nervous system, PI-PLCβ1 seems to be involved in different pathways in both brain cortex development and synaptic plasticity related to different diseases. Another PI-PLC isozyme that could be related to nuclear activities is PI-PLCζ that is involved in infertility processes. J. Cell. Biochem. 118: 1969-1978, 2017. © 2017 Wiley Periodicals, Inc.

Background and Rationale. Inositide signalling regulated by Phospholipase C (PLC) and AKT is involved in epigenetic processes and in MDS progression to AML. miRNAs are small non-coding RNAs that regulate gene expression by acting on the epigenetic machinery and are themselves controlled by epigenetic mechanisms. The expression of miRNAs has been definitively linked to cancer development and miRNA profiles are studied as new prognostic factors or therapeutic new perspectives (Jiang X et al. Nat Commun 2016). Azacitidine (AZA) is a standard first-line therapy in high-risk MDS. Its combination with Lenalidomide (LEN) has been tested, but its molecular effect is still under investigation, although the concurrent acquisition of specific point mutations on PI3KCD, PLCG2 and AKT3 genes has recently been associated with loss of response to this combination therapy (Follo MY et al. Leukemia 2019). Here we further analyzed the effect of AZA+LEN therapy on epigenetic processes and inositide regulation, focusing on miRNA expression in MDS patients. Patients and Methods. This study included 12 high-risk MDS patients treated with AZA (75 mg/m2/day, days 1-5, sc) and LEN (10 mg/day, days 1-21, orally) every 4 weeks. Patients showing complete remission (CR), partial remission (PR) or any hematologic improvement were considered as responders, while patients showing stable disease or disease progression were considered as non responders. miRNAs expression was assessed using an Affymetrix miRNA 4.0 array on patients' cells extracted at baseline and during the therapy, at the 4th (T4) and 8th (T8) cycle of therapy. Results. All patients included in this study were considered evaluable for response. 2 patients never responded, while 10 patients showed a positive response within T4: 9 of them maintained it at T8, whereas the remaining patient lost response at T8. These clinical results do not mirror the expected clinical outcomes, but this is due to our small population. However, our analyses could be relevant to test the molecular effect of the therapy in a time course, as well as comparing different phases (baseline vs T4; baseline vs T8; T4 vs T8). Paired analysis within the same patients during treatment course showed 61 miRNAs up- or down-regulated (p Conclusions. This preliminary study shows that AZA+LEN therapy affects the expression of specific clusters of miRNAs that target the PI3K signalling and, more specifically, three inositide genes that are mutated and associated with loss of response to this combination therapy, i.e. PI3KCD, AKT3 and PLCG2. Additional studies are warranted to confirm these data and to further analyze the role of these clusters, especially to test their pathogenetic or therapeutic relevance in MDS. Disclosures Cavo: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Honoraria, Speakers Bureau; Karyopharm: Honoraria. Finelli:Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Recurrent cytogenetic aberrations, genetic mutations and variable gene expression have been consistently recognized in solid cancers and in leukaemia, including in Myelodysplastic Syndromes (MDS). Besides conventional cytogenetics, the growing accessibility of new techniques has led to a deeper analysis of the molecular significance of genetic variations. Indeed, gene mutations affecting splicing genes, as well as genes implicated in essential signalling pathways, play a pivotal role in MDS physiology and pathophysiology, representing potential new molecular targets for innovative therapeutic strategies.

Specific myeloid-related and inositide-specific gene mutations can be linked to myelodysplastic syndromes (MDS) pathogenesis and therapy. Here, 44 higher-risk MDS patients were treated with azacitidine and lenalidomide and mutations analyses were performed at baseline and during the therapy. Results were then correlated to clinical outcome, overall survival (OS), leukemia-free-survival (LFS) and response to therapy. Collectively, 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases): 17 patients showed a durable response, 9 patients early lost response and 8 patients never responded. The most frequently mutated genes were ASXL1, TET2, RUNX1, and SRSF2. All patients early losing response, as well as cases never responding, acquired the same 3 point mutations during therapy, affecting respectively PIK3CD (D133E), AKT3 (D280G), and PLCG2 (Q548R) genes, that regulate cell proliferation and differentiation. Moreover, Kaplan–Meier analyses revealed that this mutated cluster was significantly associated with a shorter OS, LFS, and duration of response. All in all, a common mutated cluster affecting 3 inositide-specific genes is significantly associated with loss of response to azacitidine and lenalidomide therapy in higher risk MDS. Further studies are warranted to confirm these data and to further analyze the functional role of this 3-gene cluster.

Nuclear inositides have a specific subcellular distribution that is linked to specific functions; thus their regulation is fundamental both in health and disease. Emerging evidence shows that alterations in multiple inositide signalling pathways are involved in pathophysiology, not only in cancer but also in other diseases. Here, we give an overview of the main features of inositides in the cell, and we discuss their potential as new molecular therapeutic targets.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections. The evidence of a clinical correlation between the presence of a monoallelic gene deletion of Phospholipase Cβ1 (PLCβ1) and the progression of MDS to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments. Patients affected by MDS with a higher risk of AML evolution have a reduction in the expression of the nuclear PLCβ1, which is also epigenetically relevant in MDS. This strengthens the importance of PLCβ1 localization. In fact, PLCβ1 is a molecular target for hypomethylating agents, such Azacitidine (AZA)(1). High-risk MDS patients that respond to the drug showed an increased expression of nuclear PLCβ1 and its downstream target Cyclin D3 (CCND3), an induction of normal myeloid differentiation, and a better prognosis. Stemming from these data, our goal was to analyze the correlation between CCND3, PLCβ1 and AZA treatment. Firstly, we treated two different cellular lines, AML HL60 and histiocytic lymphoma U937, with AZA 5μM (Ec50 for HL60 cells) for 24 hours. Then, we used Real-Time PCR and Western blot to quantify both gene and protein expression. Moreover, we showed that CCND3 promoter has one CpG island. For this reason, it is possible that AZA could directly affect both PLCβ1 and CCND3 promoters. Therefore, we studied PLCβ1 binding to CCND3 promoter by chromatin immunoprecipitation (CHIP), before and after AZA treatment. Our results evidenced that the recruitment of PLCβ1 to CCND3 promoter is specifically increased after AZA treatment, leading to suppose that PLCβ1 could have a pivotal role in MDS with either a direct or indirect effect on cell cycle, proliferation and differentiation. These complicate relations need future deepening in order to demonstrate how PLCβ1 binding actually regulates CCND3 expression and how much this expression depends on CCND3 direct promoter demethylation and PLCβ1 control., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Introduction. The association of Azacitidine (AZA) and Lenalidomide (LEN), either administered concurrently or sequentially, has proven effective in Myelodysplastic Syndromes (MDS), however the optimum dose and schedule remains unknown. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in higher-risk MDS pts. Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the IWG criteria (Cheson, 2006). Moreover, the aim of this analysisis is to enucleate the clinical and biological features of pts who showed long-lasting (≥ 20 cycles) responses. Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS with IPSS risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles. For responder pts the same treatment was continued until disease progression or unacceptable toxicity. Results. From March 2013, 44 pts (27 males), median age: 72 (48-83 yrs) were enrolled, from 13 hematologic Centers. 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. Treatment was given for a median of 8.5 (1-68) cycles; in ARM 1: 9 (1-68) cycles; in ARM 2: 8 (1-63) cycles, respectively. Median follow-up: 15 (2-77) months. 10/44 pts (22.7%) did not complete at least 6 cycles of treatment for causes other than disease progression, and were not considered evaluable for response. Among the 34/44 pts (77.3%) evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. Intention-to-treat (ITT) analysis: ORR: 59.1%. First response was observed after a median of 2 (1-8) cycles. The Best Response achieved was: CR: 8 pts (23.5%) (ITT: 18.1%), PR: 1 pt (2.9%) (ITT: 2.2%), mCR: 3 pts (8.8%) (ITT: 6.8%), HI: 8 pts (23.5%) (ITT: 18.1%), mCR+HI: 6 pts (17.6%) (ITT: 13.6%). Median duration of hematologic response: 10.5 months. 37 pts (84.1%) died , and 20 pts (45.4%) showed progression to AML. Grade >2 non haematological toxicity: 54.5%. Median OS: 15 months. OS was significantly longer in responder pts as compared to the other pts (28 vs 7 months, p2 non haematological toxicity (ARM 1: 66.7%; ARM 2: 43.5%), AML incidence (ARM 1: 33.3%; ARM 2: 56.5%; p=0.2150) and OS (ARM 1: 14 months; ARM 2: 16 months). However, among responder pts, sequential treatment showed a longer clinical benefit, as compared to combined treatment. Responder pts of ARM 2 showed a significantly longer median duration of response (18 vs 6 months, p=0.0481), a longer median duration of therapy (28 vs 10 months, p=0.0870; 20 vs 10 cycles, p=0.1181), more long-lasting (≥ 20 cycles) responses (34.8% vs 9.5%, p=0.1017) and a longer OS (35 vs 26 months, p=0.3868), as compared to responder pts of ARM 1. Overall, 10/44 long-responder pts (22.7%) received ≥ 20 cycles; 5/10 pts (50%) achieved CR. IPSS risk: Intermediate-2 (8 pts); High (2 pts); IPSS-R risk: Intermediate (2 pts); High (6 pts); Very High (2 pts); IPSS cytogenetic risk: Good (5 pts); Intermediate (3 pts); Poor (2 pts); IPSS-R cytogenetic risk: Good (5 pts); Intermediate (4 pts); Very Poor (1 pt); 4/6 patients with altered karyotype achieved cytogenetic remission; it is noteworthy that the only 3 pts of the entire series who showed no gene mutations at baseline are included in this subset of long-responders pts, while 5/10 pts showed at baseline ≥ 1 prognostically unfavorable gene mutations (none with TP53 mutations), with variable VAFs during treatment. Moreover all long-responder pts showed a common gene mutation on SOD2 gene, and mutations on PLCG2 gene. Conclusions. Our results confirm the efficacy of both AZA+LEN treatment regimens in higher-risk MDS pts, in terms of ORR and OS, although sequential treatment was associated with a longer clinical benefit among responder pts. A subset of pts (22,7 %) with less unfavorable cytogenetic and molecular characteristics showed a long-lasting response to treatment. Disclosures Finelli: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Crugnola:BMS: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Rigolin:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Jannsen, BMS, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, AbbVie, Karyopharm, Adaptive: Consultancy, Honoraria.

The immune system is a complex network that acts to protect vertebrates from foreign microorganisms and carries out immunosurveillance to combat cancer. In order to avoid hyper-activation of the immune system leading to collateral damage tissues and organs and to prevent self-attack, the network has the intrinsic control mechanisms that negatively regulate immune responses. Central to this negative regulation are regulatory T (T-Reg) cells, which through cytokine secretion and cell interaction limit uncontrolled clonal expansion and functions of activated immune cells. Given that positive or negative manipulation of T-Regs activity could be utilised to therapeutically treat host versus graft rejection or cancer respectively, understanding how signaling pathways impact on T-Regs function should reveal potential targets with which to intervene. The phosphatidylinositol-3-kinase (PI3K) pathway controls a vast array of cellular processes and is critical in T cell activation. Here we focus on phosphoinositide 3-kinases (PI3Ks) and their ability to regulate T-Regs cell differentiation and function.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by peripheral blood cytopenia and abnormal myeloproliferation, as well as a variable risk of evolution into acute myeloid leukemia (AML). The nucleus is a highly organized organelle with several distinct domains where nuclear inositides localize to mediate essential cellular events. Nuclear inositides play a critical role in the modulation of erythropoiesis or myelopoiesis. Here, we briefly review the nuclear structure, the localization of inositides and their metabolic enzymes in subnuclear compartments, and the molecular aspects of nuclear inositides in MDS.

PI-PLCβ1 is involved in cell proliferation, differentiation, and myelodysplastic syndrome (MDS) path-ogenesis. Moreover, the increased activity of PI-PLCβ1 reduces the expression of PKC-α, which, in turn, delays the cell proliferation and is linked to erythropoiesis. Lenalidomide is currently used in low-risk patients with MDS and del(5q), where it can suppress the del(5q) clone and restore normal erythropoiesis. In this study, we analyzed the effect of lenalidomide on 16 patients with low-risk del(5q) MDS, as well as del(5q) and non-del(5q) hematopoietic cell lines, mainly focusing on erythropoiesis, cell cycle, and PI-PLCβ1/PKC-α signaling. Overall, 11 patients were evaluated clinically, and 10 (90%) had favorable responses; the remaining case had a stable disease. At a molecular level, both responder patients and del(5q) cells showed a specific induction of erythropoiesis, with a reduced γβ-globin ratio, an increase in glycophorin A, and a nuclear translocation of PKC-α. Moreover, lenalidomide could induce a selective G0/G1arrest of the cell cycle in del(5q) cells, slowing down the rate proliferation in those cells. Altogether, our results could not only better explain the role of PI-PLCβ1/PKC-a signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies.-Poli, A., Ratti, S., Finelli, C., Mongiorgi, S., Clissa, C., Lonetti, A., Cappellini, A., Catozzi, A., Barraco, M., Suh, P.-G., Manzoli, L., McCubrey, J. A., Cocco, L., Follo, M. Y. Nuclear translocation of PKC-a is associated with cell cycle arrest and erythroid differentiation in myelodysplastic syndromes (MDSs). FASEB J. 32, 681-692 (2018). www.fasebj.org.

Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis, and membrane trafficking. An array of kinases, phosphatases, and lipases acts on PI creating an important number of second messengers involved in different cellular processes. Although, commonly, PI signaling was described to take place at the plasma membrane, many evidences indicated the existence of a PI cycle residing in the nuclear compartment of eukaryotic cells. The discovery of this mechanism shed new light on many nuclear functions, such as gene transcription, DNA modifications, and RNA expression. As these two PI cycles take place independently of one another, understanding how nuclear lipid signaling functions and modulates nuclear output is fundamental in the study of many cellular processes. J. Cell. Physiol. 231: 1645-1655, 2016. © 2015 Wiley Periodicals, Inc.

Phosphatidylinositols (PIs) are responsible for several signaling pathways related to many cellular functions, such as cell cycle regulation at different check-points, cell proliferation, cell differentiation, membrane trafficking and gene expression. PI metabolism is not only present at the cytoplasmic level, but also at the nuclear one, where different signaling pathways affect essential nuclear mechanisms in eukaryotic cells. In this review we focus on nuclear inositide signaling in relation to cell cycle regulation. Many evidences underline the pivotal role of nuclear inositide signaling in cell cycle regulation and cell proliferation associated to different strategic physiopathological mechanisms in several cell systems and diseases.

Background: Several clinical studies demonstrated the efficacy and safety of combining azacitidine with lenalidomide in Myelodysplastic Syndromes (MDS), but the molecular implications of this therapy (i.e. gene mutations and microRNA profiling) are still under investigation. Moreover, phosphoinositide-specific phospholipase C (PI-PLC) beta1 is modulated in MDS cells treated with azacitidine, whereas lenalidomide, restoring a normal erythropoiesis, can affect PI-PLCgamma1 pathways. Purpose: This study firstly aimed to assess the effect of azacitidine and lenalidomide combination therapy on the expression of inositide-dependent signalling, focusing particularly on PI-PLC isoenzymes. Moreover, an extensive analysis on gene mutations and microRNA expression profiling was also performed. Patients and Methods: This study included 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Paired samples, before and during treatment, were analyzed. Firstly, we quantified the expression of PI-PLC isoenzymes by Real-Time PCR and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In addition, the frequency of recurrent gene mutations associated with myeloid malignancies was evaluated, using an Illumina Cancer Myeloid Panel, and the expression of microRNAs was assessed using an Affimetrix array. Results: In our case series, 34/44 patients completed at least 6 cycles of treatment and were clinically evaluable, with an overall response rate of 76.5% (26/34 cases). Firstly, the therapy affectedthe MDS cell cycle. Moreover, the expression of PI-PLCs and Beta-Globin could be associated with a favourable clinical response to the combination therapy, hinting at a specific contribution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. At baseline, the most frequent gene mutations were ASXL1 (59%), TET2 (41%), RUNX1 (35%) and SRSF2 (29%). Interestingly, all patients showing SRSF2 mutations evolved into AML. Moreover, all patients maintaining a stable disease during the therapy had increasing variant allele frequencies, showing a genetic instability. Finally, microRNA analysis revealed specific clusters associated with the treatment: 14 microRNAs were overexpressed and 28 were under-expressed as compared to baseline levels. Interestingly, down-regulation of 5 microRNAs was significantly associated with the lack of response. Conclusions: Our results show that the combination of azacitidine and lenalidomide can affect PI-PLC signalling, possibly regulating MDS cell cycle, myeloid and erythroid differentiation. Moreover, the therapy can induce a change in the specific gene mutation and microRNA profile that, if confirmed by larger studies, could be important to better evaluate the response to this therapy.

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.

Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation and osteogenesis. During myogenic differentiation of murine C2C12 myoblasts, PLC-β1 signaling pathway involves the Inositol Polyphosphate Multikinase (IPMK) and β-catenin as downstream effectors. By means of c-jun binding to cyclin D3 promoter, the activation of PLC-β1 pathway determines cyclin D3 accumulation. However, osteogenesis requires PLC-β1 expression and up-regulation but it does not affect cyclin D3 levels, suggesting that the two processes require the activation of different mediators.

Nuclear phosphoinositide-phospholipase C (PI-PLC) beta1 plays a crucial role in the molecular steps that regulate cell proliferation and differentiation in several experimental models, such as myoblasts and hematopoietic cells, via interaction with other important molecular players. Indeed, PI-PLCbeta1 and its related molecules are definitely involved in hematopoiesis, and particularly in drug-induced myeloid or erythroid differentiation. Here, we review the role of nuclear PI-PLCbeta1 signalling in normal hematopoiesis, in pathogenesis and in drug-related induction of hematopoietic differentiation, with particular reference to the current therapy of Myelodysplastic Syndromes (MDS).

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic diseases, mainly affecting the elderly, characterized by unilinear or multilinear peripheral cytopenia, bone marrow ineffective haemopoiesis, and a varying risk of progression to acute myeloid leukemia (AML). On the basis of the prognostic score systems currently used, MDS patients are generally classified as having higher risk (HR) or lower risk (LR) MDS. Two drugs, azacitidine (AZA) and decitabine (DAC), defined, because of their proven mechanism of action, as DNA methyltransferase inhibitors (DNMTIs), or hypomethylating agents (HMAs), have proven effective in improving peripheral cytopenias and quality of life, reducing or eliminating transfusion need, delaying leukemic evolution, and (only for AZA) prolonging overall survival (OS). HMAs are currently the first therapeutic choice for MDS patients who are not candidates for allogeneic hematopoietic stem cell transplantation (allo-HSCT). HMAs have also been used before and after allo-HSCT, but their role in this setting needs to be confirmed by larger studies. Although data from several clinical and biological studies might help to identify patients with a higher probability to respond to HMAs, to date this treatment should not be denied to any HR MDS patient. Several attempts have been made to combine HMAs with other therapeutic agents, and these results await confirmation by further studies.

Introduction: Nuclear inositide signaling pathways specifically regulate cell proliferation and differentiation. Interestingly, the modulation of nuclear inositides in hematological malignancies can differentially affect erythropoiesis or myelopoiesis. This is particularly important in patients with myelodysplastic syndromes (MDS), who show both defective erythroid and myeloid differentiation, as well as an increased risk of evolution into acute myeloid leukemia (AML). Areas covered: This review focuses on the structure and function of specific nuclear inositide enzymes, whose impairment could be linked with disease pathogenesis and cancer. The authors, stemming from literature and published data, discuss and describe the role of nuclear inositides, focusing on specific enzymes and demonstrating that targeting these molecules could be important to develop innovative therapeutic approaches, with particular reference to MDS treatment. Expert opinion: Demethylating therapy, alone or in combination with other drugs, is the most common and current therapy for MDS patients. Nuclear inositide signaling molecules have been demonstrated to be important in hematopoietic differentiation and are promising new targets for developing a personalized MDS therapy. Indeed, these enzymes can be ideal targets for drug design and their modulation can have several important downstream effects to regulate MDS pathogenesis and prevent MDS progression to AML.

Background and Rationale. Azacitidine (AZA) is a standard first-line therapy in high-risk MDS. Also its combination with Lenalidomide (LEN) has been tested, but its molecular effect is still under investigation. Here we analyzed the effect of AZA+LEN therapy on gene mutations and microRNA expression in MDS patients. Patients and Methods. This study included 44 high-risk MDS patients treated with AZA (75 mg/m2/day, days 1-5, sc) and LEN (10 mg/day, days 1-21, orally) every 4 weeks. Patients showing complete remission (CR), partial remission (PR) or any hematologic improvement were considered as responders, while patients showing stable disease or disease progression were considered as non responders. Molecular analyses were performed at baseline and during the therapy. Gene mutations were studied by an Illumina Cancer Myeloid Panel and an Ion Torrent specific panel, whereas microRNAs expression was assessed using an Affymetrix miRNA 4.0 array. Results. 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases). 13 patients showed a positive response within the 4th cycle (T4) and maintained it at T8; 9 patients showed a positive response within T4 and lost response at T8; 4 patients responded after T4 and maintained the response at T8; 8 patients never responded. Molecular analyses were performed on serial samples (baseline, T4 and T8) available for 30 patients. Results from the Illumina analysis on cancer myeloid genes showed that 3/30 cases had no mutations at all, all other cases showed mutations both at baseline and during the therapy. The most frequently mutated genes were ASXL1 (14 cases = 47%), TET2 (11 cases = 37%), RUNX1 (8 cases = 27%) and SRSF2 (5 cases = 17%). All samples with a decreasing variant allele frequency (VAF) had a favourable response at T8 (CR, marrow CR or PR), while none of the non responders showed a decreasing VAF. Ion Torrent analysis of 24 inositide-specific genes showed that all patients had mutations both at baseline and during the therapy. Interestingly, all patients responding at T4 and losing response at T8, as well as cases that did not respond, acquired the same 3 point mutations at T8, affecting respectively PIK3CD (D133E), AKT3 (D280G) and PLCG2 (Q548R) genes. Patients responding at T4 and losing response at T8 showed these mutations even at T4. Kaplan-Meier analyses revealed that the presence of these mutations was significantly associated with a decreased duration of therapy (39.5 vs 8.5 months; p As for microRNA profiling, paired analysis between responders and non responders showed specific clusters of up- or down-regulated microRNAs. Interestingly, unpaired analysis on patients responding at T4 and losing response at T8 showed 18 up- and 11 down-regulated microRNAs, like miR-3613-3p and miR-6757-5p, whose predicted targets are our 3 genes among the others. Also in patients never responding to the therapy there was a specific cluster of 3 up- and 12 down-regulated microRNAs and, interestingly, 7 of these microRNAs, like miR-4786-5p or miR-6853-3p, targeting our 3-gene cluster among the others, were altered also in patients losing response. Conclusions. Our results show that the presence of a common cluster of point mutations affecting 3 inositide-specific genes (PI3KCD, AKT3, PLCG2, all regulating cell proliferation), is significantly associated with loss of response to AZA+LEN therapy. Moreover, also a cluster of 7 microRNAs, targeting our 3 genes among the others, is associated with unfavourable outcome. Further studies are warranted to confirm these data, to further analyze the role of this 3-gene cluster and to identify the specific targets for the dysregulated microRNAs identified. Disclosures Gobbi: Janssen: Consultancy; Amgen: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfister: Membership on an entity's Board of Directors or advisory committees. Cavo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Finelli:Janssen: Consultancy, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

Introduction. The association of Azacitidine (AZA) and Lenalidomide (LEN), either administered concurrently (Sekeres, 2010; 2012; 2017), or sequentially (Platzbecker, 2013; Di Nardo 2015; Mittelman 2016; Narayan 2016) has proven effective in Myelodysplastic Syndromes (MDS), however the optimum dose and schedule remains unknown. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in higher-risk MDS pts. Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the IWG criteria (Cheson, 2006). Secondary endpoints: a) rate of CR; b) duration of responses; c) overall survival (OS). Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS with IPSS risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles. For responder patients the same treatment was continued until disease progression or unacceptable toxicity. Results. From March 2013, 44 pts (27 males), median age: 72 (48-83 yrs) were enrolled, from 13 hematologic Centers. At baseline, IPSS risk was: Intermediate-2: 31 pts; High: 9 pts; not determined (N.D.) (because of lack of cytogenetic data): 2 pts. (all with RAEB-2). In 2 pts IPSS risk was Intermediate-1, but they were enrolled because of severe thrombocytopenia and neutropenia, respectively. IPSS-R risk was: intermediate: 8 pts; High: 16 pts; Very-High: 18 pts; N.D.: 2 pts. In 5 pts (11.4%) del(5q) was present (additional cytogenetic alterations: 1 in 1 pt, and > 1 in 4 pts , respectively). 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. Treatment was given for a median of 8.5 (1-52) cycles; in ARM 1: 9 (1-51) cycles; in ARM 2: 8 (1-52) cycles, respectively. Median follow-up: 15 (2-54) months; 47 (37-54) months for survivors. 10/44 pts (22.7%) did not complete at least 6 cycles of treatment for causes other than disease progression (6 pts for adverse events, 2 pts for consent withdrawal and 2 pts for medical decision), and were not considered evaluable for response. Among the 34/44 pts (77.3%) evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. Intention-to-treat (ITT) analysis: ORR: 59.1%. First response was observed after a median of 2 (1-8) cycles. The Best Response achieved was: CR: 8 pts (23.5%) (ITT: 18.1%), PR: 1 pt (2.9%) (ITT: 2.2%), mCR: 3 pts (8.8%) (ITT: 6.8%), HI: 8 pts (23.5%) (ITT: 18.1%), mCR+HI: 6 pts (17.6%) (ITT: 13.6%). The remaining 8 pts showed either Stable Disease (SD) (6 pts, 17.6%) or Disease Progression (DP) (2 pts, 5.9%). Among the 27 pts (21 evaluable for response) with an abnormal karyotype at baseline, ORR was 66.7% (ITT: 51.8%) and 4 pts achieved complete cytogenetic response. Median duration of hematologic response: 10.5 months. 34 pts (77,3%) died , and 17 pts (38.6%) showed progression to AML. Grade >2 non haematological toxicity: 54.5%. Median OS: 15 months. No significant differences between the 2 arms were observed, in terms of ORR (ARM 1: 76.5%, ITT: 61.9%; ARM 2: 76.5%, ITT: 56.5%), CR rate (ARM 1: 17.6%, ITT: 14.3%; ARM 2: 29.4%, ITT: 21.7%), grade >2 non haematological toxicity (ARM 1: 66.7%; ARM 2: 43.5%), AML incidence (ARM 1: 28.6%; ARM 2: 47.8%) and OS (ARM 1: 14 months; ARM 2: 16 months), but the median response duration was significantly longer in ARM 2 (18 months) as compared to ARM 1 (6 months) (p=0.0459). At the time of last analysis, 5/44 (11.4%) patients, 1/21 (4.8%) in ARM 1, and 4/23 (17.4%) in ARM 2, were still maintaining the haematological response, and were still in treatment, after 54, 54, 52, 51 and 37 months, and after 52, 51, 33, 48 and 35 cycles of therapy, respectively. The changes observed during treatment in mutational status of inositide-specific genes and microRNA expression profiling were related to clinical outcome, predicting a negative response to therapy. Conclusions. Our results confirm the efficacy of both AZA+LEN treatment regimens in higher-risk MDS pts, in terms of ORR and OS, although pts treated with the sequential regimen showed a significantly longer duration of haematological response. Disclosures Finelli: Celgene: Other: speaker fees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: speaker fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fees. Candoni:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Merck SD: Honoraria, Speakers Bureau. Gobbi:Novartis: Consultancy; Janssen: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfister: Membership on an entity's Board of Directors or advisory committees. Rigolin:Gilead: Research Funding. Cavo:GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction. Azacitidine (AZA) is able to induce hematologic responses in 50-60 % of patients (pts) with Myelodysplastic Syndromes (MDS) and moreover to prolong survival in higher risk MDS pts. Recently, several studies have evaluated the efficacy and safety of combining, in high-risk MDS pts, AZA with Lenalidomide (LEN), either administered concurrently (Sekeres, 2010; 2012), or sequentially (Platzbecker, 2013), in both cases showing promising results, although in a limited number of pts. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in high-risk MDS pts (IPSS score risk: High or INT-2). Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the International Working Group (IWG) criteria (Cheson, 2006). Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS (WHO 2008 classification) with International Prognostic Scoring System (IPSS) risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles (32 weeks). For responder patients (CR, PR, mCR, or HI) the same treatment was continued until disease progression or unacceptable toxicity. A sample size of 44 pts was planned. Results. From March 2013, 44 pts (27 males), with a median age of 72 (48-83 yrs) were enrolled, from 13 hematologic italian Centers. At baseline, WHO diagnosis was: RCMD: 2 pts; RCMD-RS: 1 pt ; RAEB-1: 11 pts; RAEB-2: 30 pts; IPSS risk was: Intermediate-2: 31 pts; High: 9 pts; not determined (N.D.) (because of lack of cytogenetic data): 2 pts. (all with RAEB-2). In 2 pts IPSS risk was Intermediate-1, but they were enrolled because of severe thrombocytopenia and neutropenia, respectively. IPSS-R risk was: intermediate: 8 pts; High: 16 pts; Very-High: 18 pts; N.D.: 2 pts; IPSS cytogenetic risk was: Good: 17 pts; Intermediate: 11 pts; Poor: 14 pts; N.D.: 2 pts. 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. At the time of this analysis, enrolment of the planned 44 pts was completed. 34/44 pts (77.3%) completed ≥ 6 cycles of treatment, and are evaluable for response. The remaining 10 pts (4 in ARM 1 and 6 in ARM 2) are not evaluable for response, as they discontinued treatment before completing the 6th cycle because of adverse events (6 pts, 13.6%), consent withdrawal (2 pts, 4.5%) or medical decision (2 pts, 4.5%), respectively. Treatment was given for a median of 8 (1-28) cycles; in ARM 1: 9 (1-22) cycles, in ARM 2: 8 (1-28) cycles, respectively. Among the 34 pts evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. The Best Response achieved was: CR: 8 pts (23.5%), PR: 1 pt (2.9%), mCR: 3 pts (8.8%), HI: 8 pts (23.5%), mCR+HI: 6 pts (17.6%). The remaining 8 pts showed either Stable Disease (SD) (6 pts, 17.6%) or Disease Progression (DP) (2 pts, 5.9%). First Response was detected after a median of 2 (1-8) cycles. Responder pts were: 13/17 (ORR: 76.5 %) in ARM 1 (3 CR; 1 PR; 1 mCR; 4 HI, 4 mCR+HI), and 13/17 (ORR: 76.5 %) in ARM 2 (5 CR; 2 mCR; 4 HI; 2 mCR+HI), respectively. Overall, the median duration of response was 8.5 (2-23) months: 6 (2-19) months in ARM 1; 16 (2-23) months in ARM 2. A grade > 2 non hematologic toxicity was observed in 24/44 (54.5 %) pts (ARM 1: 66.7%; ARM 2: 43.5%). 27/44 pts (61.4 %) (ARM 1: 61.9%; ARM 2: 60.9%) had a dose reduction of LEN because of hematologic or non-hematologic toxicity. 22 pts (50%) died (ARM 1: 47.6%; ARM 2: 52.2%). 14 pts (31.8%) (ARM 1: 23.8%; ARM 2: 39.1%) showed progression to AML. Overall, median survival was 13 (1-28) months; ARM 1: 13 (1-25) months; ARM 2: 14 (2-28) months. Conclusions. Our results confirm the efficacy of both AZA + LEN treatment regimens in high-risk MDS pts. Moreover, at a molecular level, a significant increase of phosphoinositide-specific phospholipase C (PI-PLC) beta1 and/or PI-PLCgamma1 expression was associated with a favourable clinical response to treatment. Responder cases also showed an increase of Beta-globin expression, hinting at a specific contribution of LEN on erythroid activation Disclosures Finelli: Janssen: Other: Speaker; Novartis: Other: Speaker; Celgene: Other: Speaker, Research Funding. Visani:Celgene: Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Among cellular second messengers inositides play key roles in signal transduction pathways. Indeed, nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta 1 and Akt are involved in cell cycle progression and apoptosis. Nuclear lipid metabolism has raised interest in the last years, mainly because of its link with haematopoietic progenitor cells. Myelodysplastic syndromes (MDS) are stem-cell clonal diseases characterized by an impaired hempoiesis and a differentiation defect in one or more of the bone marrow lineages, often leading to progression to acute myeloid leukaemia (AML). The MDS evolution to AML is not completely understood but, at a molecular level, the nuclear inositide signalling pathways can play an important role in this process.

Abstract 1289 Introduction. Azacitidine (AZA) is a DNA methyltransferase inhibitor currently approved for the treatment of high-risk MDS patients, which has been demonstrated to be feasible and effective also in low-risk MDS (Fenaux P et al, Lancet Oncol 2009; Musto P et al, Cancer 2010). However, at least 4 or 6 cycles of therapy are required for assessing the hematologic response, and predictive markers of responsiveness are still lacking. PI-PLCbeta1 plays a role in the MDS progression to AML and is a specific target for AZA therapy (Follo MY et al, PNAS 2009). Indeed, PI-PLCbeta1 has been demonstrated to be a dynamic marker for responsiveness to demethylating therapy, in that PI-PLCbeta1 mRNA increase or decrease could be associated with favourable response or failure, respectively. Stemming from these data, in this study we further investigated the role of PI-PLCbeta1 in MDS patients during AZA therapy. Methods. The study included 60 patients, 22 low-risk MDS (WHO: RA, RARS, RCMD, RAEB-1, and IPSS risk Low or Int-1), and 38 high-risk MDS (WHO: RCMD, RAEB-1, RAEB-2, and IPSS risk Int-1 or High). All the patients received a minimum of 6 cycles, in the absence of disease progression or unacceptable toxicity. Hematologic response was defined according to the revised IWG criteria (Cheson et al, Blood 2006). Positive clinical responses were defined as: Complete Remission (CR), Partial Remission (PR) or Hematologic Improvement (HI). At a molecular level, for each patient we quantified the amount of PI-PLCbeta1 mRNA at baseline and before each cycle of AZA therapy. PI-PLCbeta1 ratio was calculated as the mean expression of PI-PLCbeta1 at cycles 1 to 3, as compared with the baseline level within the same subject. In case the mean value of PI-PLCbeta1 gene expression during the cycles 1 to 3 was above the baseline level, we defined it as a “PI-PLCbeta1 early increase”. On the contrary, a “stable PI-PLCbeta1” expression was observed when subjects did not show any increase during the first three cycles of therapy, as compared with baseline. Results. Patients' median age was 69 years (range 37–85) and the median follow-up was 23 months (range 1–103). The median number of AZA cycles was 11 (range 3–59) for high-risk MDS, and 8 (range 1–8) for low-risk MDS. Positive clinical responses were observed in 37/60 (62%) of the MDS patients (7 CR, 1 PR, 29 HI). In particular, 13/22 (59%) of our low-risk MDS and 24/38 (63%) of our high-risk MDS patients showed a positive clinical response to AZA, with 4 CR, 1 PR, and 19 HI in high-risk MDS, and 3 CR and 10 HI in low-risk MDS. Overall survival (OS), Progression-Free Survival (PFS), and Overall Response Rate (ORR) were analyzed using a Kaplan-Meier method, considering p-values Conclusions. Taken together, our data confirm the role of PI-PLCbeta1 as a dynamic marker of response to AZA and show that the detection of an increase in PI-PLCbeta1 gene expression within the first three cycles of AZA therapy is associated with a better clinical outcome and a longer hematological response. Further analyses are needed to confirm in a larger group of patients the predictive role of PI-PLCbeta1 mRNA detection during AZA therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 2384 Poster Board II-361 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 Disclosures: Finelli: Celgene: Consultancy.

This study tested the hypothesis that PI-PLCβ1 is associated with myeloid differentiation and that its expression could be useful for predicting the response of MDS patients to azacitidine, as the clinical effect of epigenetic treatments is often detectable only after several cycles of therapy. To this end, PI-PLCβ1 was quantified on 70 MDS patients (IPSS risk: 13 Low, 20 Int-1, 31 Int-2, 6 High) at baseline and during the first 3 cycles of azacitidine. Results were then compared with the hematologic response, as assessed after the sixth cycle of azacitidine therapy. Overall, 60 patients completed 6 cycles of azacitidine, and for them, a clinical and molecular evaluation was possible: 37 of these patients (62%) showed a specific increase of PI-PLCβ1 mRNA within the first 3 cycles, which was associated with a longer duration of response and with an increased myeloid differentiation, as evidenced by PI-PLCγ2 induction and the recruitment of specific myeloid-associated transcription factors to the PI-PLCβ1 promoter during azacitidine response. Moreover, the increase of cyclin D3 gene expression throughout all of the therapy showed that PI-PLCβ1-dependent signaling is indeed activated in azacitidine responder patients. Taken together, our results show that PI-PLCβ1 quantification in MDS predicts the response to azacitidine and is associated with an increased myeloid differentiation.