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Purpose: miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML), namely, DNMTs, CDK6, SP1, KIT, and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and...
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Purpose: miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML), namely, DNMTs, CDK6, SP1, KIT, and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and better outcome in AML. Thus, designing a strategy to increase miR-29b levels in AML blasts may be of therapeutic value. However, free synthetic miRs are easily degraded in bio-fluids and have limited cellular uptake. To overcome these limitations, we developed a novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf-NP-miR-29b). Experimental Design: Delivery efficiency was investigated by flow cytometry, confocal microscopy, and quantitative PCR. The expression of miR-29b targets was measured by immunoblotting. The antileukemic activity of Tf-NP-miR-29b was evaluated by measuring cell proliferation and colony formation ability and in a leukemia mouse model. Results: Tf-NP-miR-29b treatment resulted in more than 200-fold increase of mature miR-29b compared with free miR-29b and was approximately twice as efficient as treatment with non-transferrin- conjugated NP-miR-29b. Tf-NP-miR-29b treatment significantly downregulated DNMTs, CDK6 , SP1, KIT, and FLT3 and decreased AML cell growth by 30% to 50% and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf-NP-miR-29b had significantly longer survival compared with Tf-NP-scramble ( P = 0.015) or free miR-29b (P = 0.003). Furthermore, priming AML cell with Tf-NP-miR-29b before treatment with decitabine resulted in marked decrease in cell viability in vitro and showed improved antileukemic activity compared with decitabine alone (P = 0.001) in vivo. Conclusions: Tf-NP effectively delivered functional miR-29b, resulting in target downregulation and antileukemic activity and warrants further investigation as a novel therapeutic approach in AML.
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Purpose: Older patients with acute myeloid leukemia (AML) and myelodysplastic syndrome have often been excluded from myeloablative-conditioning regimens containing busulfan because of non-disease-related morbidity and mortality. W...
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Purpose: Older patients with acute myeloid leukemia (AML) and myelodysplastic syndrome have often been excluded from myeloablative-conditioning regimens containing busulfan because of non-disease-related morbidity and mortality. We hypothesized that busulfan clearance (BuCL) in older patients (>60 years) would be reduced compared to that in younger patients, potentially explaining observed differences in busulfan tolerability. Methods: AML patients in three CALGB hematopoietic cell transplantation studies were treated with a conditioning regimen using IV busulfan, dosed at 0.8 mg/kg. Plasma busulfan concentrations were determined by LC-MS and analyzed by non-compartmental methods. BuCL was normalized to actual (ABW), ideal (IBW), or corrected (CBW) body weight (kg). Differences in BuCL between age groups were examined using the Wilcoxon rank sum test. Results: One hundred and eighty-five patients were accrued; 174 provided useable pharmacokinetic data. Twenty-nine patients ≥60 years old (median 66; range 60-74) had a significantly higher BuCL versus those <60 years old (median 50; range 18-60): BuCL 236 versus 168 mL/min, p = 0.0002; BuCL/ABW 3.0 versus 2.1 mL/min/kg, p = 0.0001; BuCL/IBW 3.8 versus 2.6 mL/min/kg, p = 0.0035; BuCL/CBW 3.4 versus 2.6 mL/min/kg, p = 0.0005. Inter-patient variability in clearance (CV %) was up to 48 % in both age groups. Phenytoin administration, a potential confounder, did not affect BuCL, regardless of weight normalization (p > 0.34). Conclusions: Contrary to our hypothesis, BuCL was significantly higher in older patients compared to younger patients in these studies and does not explain the previously reported increase in busulfan toxicity observed in older patients.
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Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swel...
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Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca2+ and Na+ ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na+ via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na+, the membrane blebbing was completely inhibited, but Ca2+ influx did not change under these conditions. Na+ influx was modulated by the intracellular Ca2+ concentration ([Ca2+]i). Elevation of [Ca2+]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca2+, capsaicin caused only little increase in [Ca2+]i indicating that the increase in [Ca2+]i observed after capsaicin application is derived essentially from extracellular Ca2+ and not from internal Ca2+ stores. In the absence of extracellular Ca2+ also the process of cell swelling was considerably slower. Calretinin is a Ca2+ buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca2+ signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca2+ and Na+ influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca2+-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.
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Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epith...
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Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epithelioid and mixed (biphasic) mesothelioma; however, little is known about CR's putative functions in tumorigenesis. CR protects against asbestos-induced acute cytotoxicity mediated by the AKT/PI3K pathway, and furthermore, SV40 early region genes are able to upregulate CR in mesothelial cells. However, the precise role of CR in mesothelioma is still unknown. Downregulation of CR via lentiviral-mediated short-hairpin RNA significantly decreased the viability and proliferation of mesothelioma cells in vitro. The effect was strong in epithelioid-dominated cell lines (ZL55 and MSTO-211H). A weaker and delayed effect was observed in mesothelioma cells with prevalent sarcomatoid morphology (SPC111, SPC212 and ZL34). The specificity of the effect was confirmed by stable enhanced green fluorescent protein-CR expression in mesothelioma cell lines and subsequent downregulation. Depletion of CR led these cancer cell lines to enter apoptosis within 72 hr postinfection via strong activation of the intrinsic caspase 9-dependent pathway. Downregulation of CR in immortalized mesothelial cells LP9/TERT-1 strongly blocked proliferation and caused a G1 block without decreasing viability or activating apoptosis pathways. These results demonstrate that downregulation of CR had a strong effect on the viability of MM cells and that CR is essential for cells derived from MM. The authors anticipate these findings to reveal CR as a highly interesting new putative therapeutic target for mesothelioma treatment of especially the epithelioid, as well as of the mixed and sarcomatoid type. What's new? Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms with strong chemotherapy resistance and no effective cure. While the cytosolic Ca2+-buffering protein calretinin (CR) serves as a positive marker for the identification of malignant mesothelioma of the epithelioid and biphasic types, its precise role in mesothelioma remains unknown. The authors demonstrate that down-regulation of CR with lentiviral-mediated shRNA in MM cell lines causes a strong decrease in proliferation/viability and subsequently triggers apoptosis and necrosis in CR-depleted cells. CR-negative fibroblasts are not affected by the shRNA treatment. Calretinin might represent a potential new target for highly needed therapeutic treatments against malignant mesothelioma.
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Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphis...
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Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphism (GHR d3) may account for some of this variability. The authors performed a systematic review (to April 2011), including investigator-only data, to quantify the effects of the GHR fl-d3 and GHR d3-d3 genotypes on rhGH therapy response and used a recently established Bayesian inheritance model-free approach to meta-analyze the data. The primary outcome was the 1-year change-in-height standard-deviation score for the 2 genotypes. Eighteen data sets from 12 studies (1,527 children) were included. After several prior assumptions were tested, the most appropriate inheritance model was codominant (posterior probability = 0.93). Compared with noncarriers, carriers had median differences in 1-year change-in-height standard-deviation score of 0.09 (95% credible interval (CrI): 0.01, 0.17) for GHR fl-d3 and of 0.14 (95% CrI: 0.02, 0.26) for GHR d3-d3. However, the between-study standard deviation of 0.18 (95% CrI: 0.10, 0.33) was considerable. The authors tested by meta-regression for potential modifiers and found no substantial influence. They conclude that 1) the GHR d3 polymorphism inheritance is codominant, contrasting with previous reports; 2) GHR d3 genotypes account for modest increases in rhGH effects in children; and 3) considerable unexplained variability in responsiveness remains.
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5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A ...
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5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphosphate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucleotides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC-induced RRM2 gene expression inhibition involves its direct RNA-incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination.
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Chromosomemaintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (eg, p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overacti...
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Chromosomemaintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (eg, p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC 50 values; 100-500nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.
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The outcome of older (≥ 60 years) acute myeloid leukemia (AML) patients is poor, and novel treatments are needed. In a phase 2 trial for older AML patients, lowdose (20 mg/m 2 per day for 10 days) decitabine, a DNAhypomethylating...
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The outcome of older (≥ 60 years) acute myeloid leukemia (AML) patients is poor, and novel treatments are needed. In a phase 2 trial for older AML patients, lowdose (20 mg/m 2 per day for 10 days) decitabine, a DNAhypomethylating azanucleoside, produced 47% complete response rate with an excellent toxicity profile. To assess the genome-wide activity of decitabine, we profiled pretreatment and post treatment (day 25/course 1) methylomes of marrow samples from patients (n = 16) participating in the trial using deep-sequencing analysis of methylated DNA captured by methyl-binding protein (MBD2). Decitabine significantly reduced global methylation compared with pretreatment baseline (P = .001). Percent marrow blasts did not correlate with global methylation levels, suggesting that hypomethylation was related to the activity of decitabine rather than to a mere decrease in leukemia burden. Hypomethylation occurred predominantly in CpG islands and CpG island-associated regions (P ranged from .03 to .04) A significant concentration (P < .001) of the hypomehtylated CpG islands was found in chromosome subtelomeric regions, suggesting a differential activity of decitabine in distinct chromosome regions. Hypermethylation occurred much less frequently than hypomethylation and was associated with low CpG content regions. Decitabine-related methylation changes were concordant with those previously reported in distinct genes. In summary, our study supports the feasibility of methylome analyses as a pharmacodynamic endpoint for hypomethylating therapies.
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We evaluated the impact of busulfan dose intensity in patients undergoing reduced toxicity/intensity conditioning allogeneic transplantation in a multicenter retrospective study of 112 consecutive patients. Seventy-five patients w...
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We evaluated the impact of busulfan dose intensity in patients undergoing reduced toxicity/intensity conditioning allogeneic transplantation in a multicenter retrospective study of 112 consecutive patients. Seventy-five patients were conditioned with busulfan (0.8mg/kg/dose IV×8 doses), fludarabine (30mg/m 2/day, days -7 to -3), and 6mg/kg of ATG [reduced intensity conditioning (RIC) group], while 37 patients received a more-intense conditioning with busulfan (130mg/m 2/day IV, days -6 to -3), fludarabine (40mg/m 2/day, days -6 to -3) and 6mg/kg of ATG [reduced toxicity conditioning (RTC) group]. At baseline both groups were matched for median age, unrelated donor allografts, and human leukocyte antigen-mismatched allografts. More patients in RIC group had high-risk disease, and higher median comorbidity index. There were no graft rejections. Median time to neutrophil (17 days vs. 15 days; p=0.003) and platelet engraftment (16 days vs. 11 days; p<0.001) was significantly longer in the RIC group. RTC group had significantly more bacterial (62.2% vs. 32%; p=0.004) and fungal infections (13.5% vs. 1.3% p=0.01). For RIC and RTC groups rates of grades II-IV acute GVHD (34% vs. 40%; p-value=0.54), and chronic GVHD (45% vs. 57%; p-value=0.30) were not significantly different. In similar order at 1 year the cumulative-incidence of non-relapse mortality (NRM; 12% vs. 21%; p-value=0.21) and relapse rates (38% vs. 39%; p=0.96) were not significantly different. Patients in RIC and RTC groups had similar 1-year overall survival (61% vs. 50%, p=0.11) and progression-free survival (50% vs. 36%, p-value=0.39). Our data suggest that the merits of higher busulfan dose intensity in the context of fludarabine/busulfan-based RTC may be offset by higher early morbidity.
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This phase I study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) of the heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) in combination with b...
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This phase I study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) of the heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) in combination with bortezomib, and to provide pharmacokinetic data in relapsed or refractory acute myeloid leukemia (AML). Eleven patients were enrolled. The MTD was 17-AAG 150 mg/m2 and bortezomib 0.7 mg/m2. Hepatic toxicity and cardiac toxicity were dose limiting. Co-administration on day 4 led to a decrease in clearance (p = 0.005) and increase in AUC (p = 0.032) of 17-amino-17-demethoxygeldanamycin (17-AG), not observed when 17-AAG was administered alone. Pharmacokinetic parameters of patients who developed toxicities and those who did not were not different. The combination of 17-AAG and bortezomib led to toxicity without measurable response in patients with relapsed or refractory AML. Pharmacokinetic data provide insight for studies of related agents in AML. Next-generation HSP90 inhibitors are appealing for further development in this area.
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