Neurodevelopmental Disorders (NDs) are a heterogeneous group of disorders and are considered multifactorial diseases with both genetic and environmental components. Epigenetic dysregulation driven by adverse environmental factors has recently been documented in neurodevelopmental disorders as the possible etiological agent for their onset. However, most studies have focused on the epigenomes of the probands rather than on a possible epigenetic dysregulation arising in their mothers and influencing neurodevelopment during pregnancy. The aim of this research was to analyze the methylation profile of four well-known genes involved in neurodevelopment (BDNF, RELN, MTHFR and HTR1A) in the mothers of forty-five age-matched AS (Asperger Syndrome), ADHD (Attention Deficit Hyperactivity Disorder) and typically developing children. We found a significant increase of methylation at the promoter of the RELN and HTR1A genes in AS mothers compared to ADHD and healthy control mothers. For the MTHFR gene, promoter methylation was significantly higher in AS mothers compared to healthy control mothers only. The observed dysregulation in AS mothers could potentially contribute to the affected condition in their children deserving further investigation.

INTRODUCTION:Neoangiogenesis has a crucial role in multiple myeloma (MM), and circulating endothelial cells (CECs) contribute to neovascularization by inducing tumor progression and metastasis and by repairing damage to bone marrow vasculature after stem cell transplantation (HSC). We recently proved in a national multicenter study the possibility to reach a high-level standardization in CEC count and analysis based on a polychromatic flow cytometry Lyotube (BD). Our study aimed at assessing the kinetics of CECs in patients with MM undergoing autologous hematopoietic stem cell transplantation (Au-HSCT).METHODS:Blood samples for analysis were collected at different time points before (T0, T1) and after (T2, T3, T4) Au-HSCT. For each sample, 20 × 106 leukocytes were processed as already described (Lanuti 2016 e 2018) through a multistep procedure. CECs were eventually identified as 7-ADDneg/Syto16pos/CD45neg/CD34pos/CD146pos.RESULTS:Twenty-six MM patients were enrolled in the study. Overall, we observed a constant increase of CECs values from T0 to T3 (day of neutrophil engraftment) followed by decrease at T4 (100 days after transplantation). Using the median value of CECs at T3, we could define a cut-off concentration of 618/mL, with patients with more infective complications having CECs above that value (9/13 vs. 2/13; p = 0.005).CONCLUSION:CECs value may be a function of endothelial damage caused by conditioning regimen, as suggested by the increase of their level during the engraftment period. A more severe endothelial damage is reflected by the increase of infective complications in patients with higher CECs value at T3.

BACKGROUND:Surgical reconstruction of chronic wounds is often infeasible due to infection, comorbidities, or poor viability of local tissues. The aim of this study was to describe the authors' technique for improving the regenerative and antimicrobial potential of a combination of modified nanofat and platelet-rich plasma (PRP) in nonhealing infected wounds.METHODS:Fourteen patients met the inclusion criteria. Fat tissue was harvested from the lower abdomen following infiltration of a solution of 1,000 mL of NaCl solution, 225 mg of ropivacaine, and 1 mg of epinephrine. Aspiration was performed using a 3-mm cannula with 1-mm holes. The obtained solution was decanted and mechanically emulsified, but was not filtered. Non-activated leukocyte-rich PRP (naLR-PRP) was added to the solution before injection. Patients underwent three sessions of injection of 8-mL naLR-PRP performed at 2-week intervals.RESULTS:Thirteen of 14 patients completed the follow-up. Complete healing was achieved in seven patients (53.8%). Four patients (30.8%) showed improvement, with a mean ulcer width reduction of 57.5%±13.8%. Clinical improvements in perilesional skin quality were reported in all patients, with reduced erythema, increased thickness, and increased pliability. An overall wound depth reduction of 76.6%±40.8% was found. Pain was fully alleviated in all patients who underwent re-epithelization. A mean pain reduction of 42%±33.3% (as indicated by visual analog scale score) was found in non-re-epithelized patients at a 3-month follow-up.CONCLUSIONS:The discussed technique facilitated improvement of both the regenerative and the antimicrobial potential of fat grafting. It proved effective in surgically-untreatable infected chronic wounds unresponsive to conventional therapies.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

The use of granulocyte colony-stimulating factor (G-CSF) primed bone marrow (G-BM) has been recently considered as an alternative to mobilized hematopoietic stem cells from peripheral blood (G-PB), especially in the haploidentical transplant setting. The purpose of this study was to compare the effect of in vivo G-CSF priming on BM and PB hematopoietic, mesenchymal (MSC), and immune cells. Forty healthy donors undergoing BM harvest for haploidentical transplant were given subcutaneous recombinant human G-CSF for 7 days. BM and PB samples were harvested on days-7 and 0. The hematopoietic stem/progenitor cells increased significantly after G-CSF priming in both BM and PB with a selective rise of BM CD34(+)CD38(-) cell subset. A striking enhancement of the mesenchymal progenitors was detected in G-BM. CD3(+), CD4(+), CD8(+), and CD19(+) cell fractions; the naive CD4(+) and CD8(+) subpopulations; and natural killer and regulatory T cells increased in G-BM, whereas only slight changes were detected in PB. Myeloid dendritic cells (DC1) were significantly up regulated in both G-BM and G-PB, whereas DC2 increased only in G-BM. In conclusion, our results show substantial differences in the biologic effects exerted by G-CSF at BM and PB levels on hematopoietic cells and immune cell fractions. Furthermore, the impressive rise of MSC progenitors in G-BM might also be relevant to provide MSCs for several clinical use. (C) 2016 American Society for Blood and Marrow Transplantation.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Glyoxalase I (GLO1) is a homodimeric Zn(2+)-dependent isomerase involved in the detoxification of methylglyoxal and in limiting the formation of advanced glycation end-products (AGE). We previously found the rs4746 A332 (Glu111) allele of the GLO1 gene, which encodes for glyoxalase I, associated with "unaffected sibling" status in families with one or more children affected by Autism Spectrum Disorder (ASD). To identify and characterize this protective allele, we sequenced GLO1 exons and exon-intron junctions, detecting two additional SNPs (rs1049346, rs1130534) in linkage disequilibrium with rs4746. A family-based association study involving 385 simplex and 20 multiplex Italian families yielded a significant association with autism driven only by the rs4746 C332 (Ala111) allele itself (P < 0.05 and P < 0.001 under additive and dominant/recessive models, respectively). Glyoxalase enzymatic activity was significantly reduced both in leukocytes and in post-mortem temporocortical tissue (N = 38 and 13, respectively) of typically developing C332 allele carriers (P < 0.05 and <0.01), with no difference in Glo1 protein levels. Conversely, AGE amounts were significantly higher in the same C332 post-mortem brains (P = 0.001), with a strong negative correlation between glyoxalase activity and AGE levels (τ = -0.588, P < 0.01). Instead, 19 autistic brains show a dysregulation of the glyoxalase-AGE axis (τ = -0.209, P = 0.260), with significant blunting of glyoxalase activity and AGE amounts compared to controls (P < 0.05), and loss of rs4746 genotype effects. In summary, the GLO1 C332 (Ala111) allele confers autism vulnerability by reducing brain glyoxalase activity and enhancing AGE formation, but years after an autism diagnosis the glyoxalase-AGE axis appears profoundly disrupted, with loss of C332 allelic effects.

Electrostimulation represents a well-known trophic factor for different tissues. In vitro electrostimulation of non-stem and stem cells induces myogenic predifferentiation and may be a powerful tool to generate cells with the capacity to respond to local areas of injury. We evaluated the effects of in vivo electrostimulation on infarcted myocardium using a miniaturized multiparameter implantable stimulator in rats. Parameters of electrostimulation were organized to avoid a direct driving or pacing of native heart rhythm. Electrical stimuli were delivered for 14 days across the scar site. In situ electrostimulation used as a cell-free, cytokine-free stimulation system, improved myocardial function, and increased angiogenesis through endothelial progenitor cell migration and production of vascular endothelial growth factor (VEGF). In situ electrostimulation represents a novel means to stimulate repair of the heart and other organs, as well as to precondition tissues for treatment with cell-based therapies.

Purpose: Published data demonstrated that zoledronic acid (ZOL) exhibits antiangiogenetic effects. A promising tool for monitoring antiangiogenic therapies is the measurement of circulating endothelial cells (CECs) and circulating endothelial precursor cells (CEPs) in the peripheral blood of patients. Our aim was to investigate the effects of ZOL on levels of CECs and CEPs in localized prostate cancer. Methods: Ten consecutive patients with a histologic diagnosis of low-risk prostate adenocarcinoma were enrolled and received an intravenous infusion of ZOL at baseline (T0), 28 days (T28) and 56 days (T56). Blood samples were collected at the following times: T0 (before the first infusion of ZOL), T3 (72 h after the first dose), T28, T56 (both just before the ZOL infusion) and T84 (28 days after the last infusion of ZOL) and CEC/CEP levels were directly quantified by flow cytometry at all these time points. Results: Our analyses highlighted a significant reduction of mean percentage of CECs and CEPs after initiation of ZOL treatment [p = 0.014 (at day 3) and p = 0.012 (at day 84), respectively]. Conclusion: These preliminary results demonstrate that ZOL could exert an antiangiogenic effect in early prostate cancer through CEP and CEC modulation.

Leukemia 184 molecular therapies, especially in resistant/refractory cases.Controversies on and potential benefits of novel approaches based on STP inhibitors will be discussed.Epigenetic changes will be examined to address their importance in association with post-translational modifications.Future perspectives will encompass review of Reverse Phase Protein Arrays (RPPA) as a promising new tool for translational studies in clinical sample series, helping define the functional proteomic profile at steady-state; this approach may lead to the identification of aberrant molecular target(s), possibly guiding the choice of specific molecular therapies, on one hand, and leading to the elucidation of post-treatment changes, on the other, with the overall objective of improving the strategies currently employed to counteract leukemia chemo-and targeted drug-resistance. Multidrug resistanceChemo-resistance has been one of the major problems in cancer treatment, contributing to therapeutic failure, particularly in leukemia patients treated with regimens containing drugs such as anthracyclines, vinca alkaloids, or epipodophyllins, all modulated in their intracellular retention by the P-glycoprotein 170 (P-gp) product of the mdr-1 gene (Longley et al., 2005).An expanding number of proteins with a prominent role in MDR-dependent mechanisms of accelerated drug efflux and potentially implicated in leukemia drug resistance have been described: the multidrug resistance-associated protein 1 (MRP1), the lung-resistance protein (LRP) and the transporter breast cancer resistance protein (BCRP) (Chen, 2010).MRP1 is a protein distinct from MDR1, with different substrate specificity (amphophilic anions or glutathione-conjugated) and different susceptibility to drugs that can restore chemotherapy sensitivity (butathionine, which depletes intracellular glutathione content).LRP is a component of the major vault protein complex that appears to be involved in nuclear-cytoplasmic trafficking and in promoting drug redistribution away from the nucleus.BCRP is another member of the ATP-binding cassette protein involved in the MDR phenotype.In our previous studies (Tafuri et al., 2002) we have established the frequency and biologic-clinical significance of MDR1 expression and function and of MDR-associated proteins in de novo adult ALL.Our analysis was performed in the context of the ALL0496 study of the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) cooperative group.This prospective multicenter study enrolled a large series of uniformly treated de novo adult ALL patients, providing a unique opportunity to investigate the prognostic influence of MDR1 on CR achievement and clinical outcome, in context with other clinical and biological factors.Our results indicate that MDR1 is expressed in approximately 20% of patients enrolled in the study, playing a statistically significant unfavorable prognostic role on CR achievement at both univariate and multivariate analysis; in addition, together with age and CD34 expression, MDR1 expression significantly predicted response failure.Since MDR1 expression was described as an independent predictor of CR in patients with de novo adult ALL, additional studies (Gregorj et al., 2005) were performed by our group to investigate the prognostic role of other proteins implicated in drug efflux mechanisms in ALL.Analysis of primary ALL samples evaluated for the expression of MRP1 and BCRP found these proteins expressed in 51.3% and 72.9% of the cases, respectively.BCRP expression was associated with age (P=0.027),but not with MDR1 expression.Neither BCRP nor MRP1 individually influenced CR achievement.In contrast, when their simultaneous coexpression was analyzed, the majority of double negative patients achieved CR (P=0.034).Alterations in drug-efflux due to aberrant ABC transporter protein expression in cancer cells www.intechopen.comAberrant Proliferative and Apoptotic Pathways in Acute Lymphoblastic Leukemia (ALL): Molecular Therapies to Overcome Chemo-Resistance 185 led to the development of P-gp inhibitors, tested in clinical trials with discouraging results.In fact, the combined use of the MDR-reversal agent cyclosporine was associated with unacceptable toxicity, while a second generation-drug, vaslapodar (PSC-833), had unpredictable pharmacokinetic interactions.Third-generation inhibitors (tariquidar XR9576, zosuquidar LY335979, laniquidar R101933, and ONT-093) have high potency and specificity for P-gp but no appreciable impact on cytochrome P450 3A4 drug metabolism and no clinically significant drug interactions with common chemotherapy agents.Overall, the proposed chemo-sensitizing activity of MDR-reversal agents evaluated in clinical trials failed to improve either response rate or survival in leukemia patients (Molnár et al., 2010).Since advances in microarray and proteomic technologies, in conjunction with the development of novel small molecules therapeutically targeting molecular determinants of resistance in cancer cells, have opened new opportunities to combat chemo-resistance, studies have explored and characterized the signaling pathways involved in the regulation of tumor cell response to chemotherapy, mostly those involved in aberrant proliferation and prolonged survival. Signal transduction pathwaysLeukemic cells are frequently characterized by the constitutive activation of multiple signaling cascades, which ensures a proliferative and survival advantage over normal hematopoietic cells.In fact, several studies have shown that the PI3K/PTEN/AKT/mTOR, RAS/RAF/MEK/ERK, Notch1, and JAK/STAT pathways are frequently up-regulated in ALL (Brown et al., 2008).Herein, some of these key signaling are reviewed as potential molecular targets for specific therapeutic interventions in ALL (Fig. 1). PI3K/PTEN/AKT/mTOR pathwayThe phosphoinositide 3-kinase (PI3K)/PTEN/AKT/mTOR pathway is a crucial signaling cascade involved in the control of cell growth, survival, proliferation, motility, apoptosis and autophagy (Fig. 1).This pathway plays a pivotal role in maintaining numerous physiological processes, such as nutrient uptake, ribosomal biogenesis and cellular metabolism (Brown et al., 2008).PI3K is activated by a variety of extracellular stimuli, including receptor tyrosine kinases (i.e.FLT3, EGFR, HER-2/neu) (Fathi et al., 2010).The activation of PI3K catalyzes the phosphatidylinositol-4,5-bisphosphate (PIP2) conversion to the phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the inner surface of the cell membrane, allowing for the recruitment of PDK1 and AKT to the membrane.AKT is an important mediator of the intracellular cascade, acting on multiple downstream targets to regulate proliferation and apoptotic signals.Downstream of AKT, a key protein is represented by mTOR, which regulates translation in response to nutrient levels (Fig. 1).The major negative regulator of the PI3K/AKT/mTOR pathway is the lipid phosphatase PTEN (Phosphatase and tensin homologue deleted on chromosome 10).The PI3K/PTEN/AKT/mTOR pathway is deregulated in several human cancers, including ALL.First, Barata et al. (2004) demonstrated that the PI3K pathway is essential for IL-7mediated survival, activation, proliferation, and growth of T-ALL cells indicating the existence of a functional IL-7-mediated PI3K signaling cascade in T-ALL cells.Additional reports showed that the constitutive activation of PI3K/PTEN/AKT/mTOR pathway occurs frequently in childhood ALL and is crucial for blast survival (Avellino et al., 2005;Jotta et al., 2010).Aberrant activation of this signaling cascade can be induced by overproduction of

In spite of improvements in the last decade, relapses still occurs in the majority of ALL patients, with a long-term survival rate of only 30–40%. Recently, small-molecule inhibitors have been developed for targeting deregulated signal transduction pathways involved in proliferation and apoptosis. Current evidence identifies the Raf/MEK/ERK, the PI3K/AKT/mTOR, and the Bcl-2 pathways as potentially relevant targets for therapeutic intervention. We have previously demonstrated that constitutive ERK phosphorylation is an independent predictor of failure to achieve complete remission in adult ALL (Blood 2007; 109:5473) and thus we evaluated the in vitro activity of MEK-inhibitors. However, neither PD98059 nor PD0325901 affected cell growth, cell cycle distribution, and/or apoptosis in ALL cell lines (IC50 >1 μM for PD0325901). These results were confirmed in primary ALL samples, in which PD98059 significantly (P=0.012) inhibited ERK phosphorylation in 8/12 samples, without inducing cell cycle changes or apoptosis. We next investigated the activity of the mTOR inhibitor Temsirolimus. Temsirolimus displayed a biphasic dose-response in a panel of different ALL cell lines, with a flat curve (35–55% of inhibition) at concentrations ranging between 1 and 5,000 nM and more pronounced growth inhibition at concentrations ≥ 10 μM. The CEM cell line was the most sensitive (IC50: 7 nM), Jurkat showed intermediate sensitivity (IC50: 200 nM), while MOLT-4 were resistant (IC50 > 20,000 nM). Cell growth inhibition was associated with inhibition of cell cycle progression, while apoptosis induction was observed in less than 15% of the cells even at the highest concentration of Temsirolimus (20 μM). We then investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories to A.T.), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, in ALL cell lines and in 10 primary samples. ABT-737 showed a potent dose- and time-dependent growth-inhibitory activity in MOLT-4 (IC50: 198 nM), associated with loss of mitochondrial membrane potential, caspase activation, Bcl-2 cleavage, and ultimately apoptosis induction. Conversely, CEM cells proved resistant (IC50: 12,000 nM). We also found that ABT-737 was highly effective in primary adult and childhood ALL, independent of their chromosomal abnormalities, with a significant decrease in viability (p=0.008) and a remarkable induction of apoptosis (from a mean baseline value of 16.8±8.8% to 43.6±22.8%, p=0.04) at 10 nM ABT-737. A dose-dependent down-regulation of Bcl-2 and Bcl-xL expression was observed in sensitive samples, but not in the only resistant one. In summary, our study shows that ALL cells, do not undergo apoptosis in response to single-pathway inhibition, suggesting the presence of multiple, redundant pathways that preserve leukemic cell survival. The only notable exception was the Bcl-2/Bcl-xL inhibitor ABT-737. Studies are ongoing to identify mechanism-driven combinations of agents that would disrupt multiple signal transduction pathways, resulting in synergistic killing and, ultimately, in novel therapeutic strategies for ALL.

Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.

Extracellular signal-regulated kinase-1/2 (ERK1/2) is frequently found constitutively activated (p-ERK1/2) in hematopoietic diseases, suggesting a role in leukemogenesis. The aim of this study was to assess the expression and clinical role of p-ERK1/2 in adult acute lymphoblastic leukemia (ALL). In 131 primary samples from adult de novo ALL patients enrolled in the Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) Leucemia Acute Linfoide (LAL) 2000 protocol and evaluated by flow cytometry, constitutive ERK1/2 activation was found in 34.5% of cases; these results were significantly associated with higher white blood cell (WBC) values (P=.013). In a multivariate analysis, p-ERK1/2 expression was an independent predictor of complete remission achievement (P=.027). Effective approaches toward MEK inhibition need to be explored in order to evaluate whether this may represent a new therapeutic strategy for adult ALL patients.

Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.

The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 > 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 > 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

The aberrant function of transcription factors and/or kinase-based signaling pathways that regulate the ability of hematopoietic cells to proliferate, differentiate, and escape apoptosis accounts for the leukemic transformation of myeloid progenitors. Here, we demonstrate that simultaneous retinoid receptor ligation and blockade of the MEK/ERK signaling module, using the small-molecule inhibitor CI-1040, result in a strikingly synergistic induction of apoptosis in both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells with constitutive ERK activation. This proapoptotic synergism requires functional RAR and RXR retinoid receptors, as demonstrated using RAR- and RXR-selective ligands and RAR-defective cells. In the presence of MEK inhibitors, however, retinoid-induced chromatin remodeling, target-gene transcription, and granulocytic differentiation are strikingly inhibited and apoptosis induction becomes independent of death-inducing ligand/receptor pairs; this suggests that apoptosis induction by combined retinoids and MEK inhibitors is entirely distinct from the classical "postmaturation" apoptosis induced by retinoids alone. Finally, we identify disruption of Bcl-2-dependent mitochondrial homeostasis as a possible point of convergence for the proapoptotic synergism observed with retinoids and MEK inhibitors. Taken together, these results indicate that combined retinoid treatment and MEK blockade exert powerful antileukemic effects and could be developed into a novel therapeutic strategy for both AML and APL.