MicroRNAs involvement in fludarabine refractory chronic lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria.</p> <p>Results</p> <p>By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance.</p> <p>Conclusions</p> <p>This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.</p

Employment of Oligodeoxynucleotide plus Interleukin-2 Improves Cytogenetic Analysis in Splenic Marginal Zone Lymphoma

To compare the efficiency of novel mitogenic agents and traditional mitosis inductors, 18 patients with splenic marginal zone lymphoma (SMZL) were studied. Three cultures using oligodeoxynucleotide (ODN) plus interleukin-2 (IL-2), or TPA, or LPS were setup in each patient. Seventeen/18 cases with ODN + IL2 had moderate/good proliferation (94, 4%) as compared with 10/18 cases with TPA and LPS (55%) (P = .015); 14/18 (77, 7%) cases with ODN + IL2 had sufficient good quality of banding as compared with 8/18 cases (44, 4%) with TPA and LPS. The karyotype could be defined from ODN + IL2-stimulated cultures in all 18 patients, 14 of whom (77, 7%) had a cytogenetic aberration, whereas clonal aberrations could be documented in 9 and in 3 cases by stimulation with LPS and TPA, respectively. Recurrent chromosome aberrations in our series were represented by aberrations of chromosome 14q in 5 patients, by trisomy 12 and 7q deletion in 4 cases each, and by abnormalities involving 11q and 13q in two cases each. These findings show that stimulation with ODN + IL2 offers more mitotic figures of better quality and results in an increased rate of clonal aberrations in SMZL, making this method ideal for prospective studies aiming at the definition of the prognostic impact of cytogenetic aberrations in this disorder

Serine metabolism remodeling after platinum-based chemotherapy identifies vulnerabilities in a subgroup of resistant ovarian cancers

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12–15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers

Intracellular evaluation of ER targeting elucidates a mild form of inherited coagulation deficiency

none5Missense mutations reduce protein levels through several molecular mechanisms. Among them, altered targeting to endoplasmic reticulum (ER) and its relationship with clinical phenotypes in patients have been poorly investigated. To address this point, we studied the prepeptide mutations (L-48P, L-42P) associated with mild deficiency of factor VII (FVII), the serine-protease triggering blood coagulation. Mutations were introduced into the native FVII to evaluate secreted and intracellular protein levels, and into a chimeric FVII-GFP to study ER targeting in living cells. In conditioned medium from stably or transiently transfected cells, expression levels of the -48PFVII (9% and 55%, respectively) and particularly those of the -42PFVII (2% and 12%) were decreased compared with those of WtFVII, indicating the causative nature of mutations. Markedly reduced protein levels were observed in cell organelles for -48PFVII (10.5 +/- 4.9 ng/mL; Wt-FVII, 130 +/- 43.4 ng/mL) and -42PFVII (approximately 5 ng/mL), thus suggesting impaired ER targeting. Fluorescence of the -48PFVII-GFP and -42PFVII-GFP was diffuse, covered the nucleus, and declined upon plasma membrane permeabilization with digitonin, which demonstrated mislocalization of variants in the cytosol. Noticeably, the residual fluorescence of -48PFVII-GFP (10%) and -42PFVII-GFP (20%) in organelles was fairly compatible with FVII levels in patients' plasma. The studies with the native and chimeric proteins indicated that both prepeptide mutations were associated with residual expression of normal FVII, which explained the mild form of FVII deficiency in patients. This approach, extendable to other coagulation serine proteases, clearly contributed to elucidate the relationship of genotype with plasma and clinical phenotype.mixedRIZZOTTO L; PINOTTI M; P. PINTON; RIZZUTO R; BERNARDI FRizzotto, Lara; Pinotti, Mirko; Pinton, Paolo; Rizzuto, Rosario; Bernardi, Francesc

U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency

Factor VII (FVII) is the plasma protease triggering coagulation, and its absence is lethal. Life-threatening hemorrhagic symptoms in severe FVII deficiency are prevented by frequent administration of fresh frozen plasma or recombinant FVIIa. Studies in animal and cellular models of human diseases showed that modified small nuclear RNAs (snRNAs) can promote changes in mRNA splicing and thus in gene expression. Splicing mutations in clotting factors, a relatively frequent cause of severe bleeding, represent ideal models to test this strategy, because tiny increases in functional full-length protein levels in patients significantly ameliorate hemorrhagic phenotypes. We explored the snRNA-mediated rescue of coagulation factor VII (FVII) expression impaired by the IVS7+5 g/a mutation, which is associated to life-threatening bleeding in homozygous patients. This change occurs in the first of six homologous 37bp repeats containing cryptic donor splice site (5'ss) identical to the normal one. Expression of extended FVII minigenes in human hepatoma cells (Hep3B) and studies at the mRNA level (RT-PCR, fluorescent labeling and capillary electrophoresis) indicated that the IVS7+5g/a induces exon 7 skipping and activation of the first downstream cryptic 5'ss, thus generating frameshifts. Levels of normal transcripts were barely detectable (<0.2%). To restore correct mRNA processing we engineered the U1-snRNA, the spliceosome components selectively recognizing 5’ss. Vectors for three U1-snRNAs, complementary to the mutated 5’ss (U1+5a) or to neighbouring sequences, were created and co-expressed with FVII minigenes in Hep3B. The U1-snRNAs reduced from 80-40% the exon 7 skipping, thus increasing exon definition. The U1+5a construct also dramatically increased recognition of the correct 5’ss over the 37bp-downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5’ss, a frequent cause of severe defects

The complete impairment of factor VII gene expression by the IVS6+1g/t mutation is compatible with a severe but not lethal bleeding disorder

The complete deficiency of Factor VII (FVII), the protease triggering blood coagulation, is potentially lethal, as suggested by the absence of homozygous large gene deletions in humans and knock-out experiments in mice. We characterized the IVS6+1G>T mutation at the IVS6 donor splice site (5'ss) of FVII gene. The mutation was identified in homozygous condition in two severe FVII deficient patients from Thailand who experienced gastrointestinal bleeding in the first months of life and were subsequently kept under prophylaxis regimen. Plasma FVII activity levels, measured by PT-based and fluorogenic functional assays (FXa and thrombin generation) were strikingly below the commercial FVII deficient/depleted plasma (negative controls). The FVII mRNA was investigated in leukocyte mRNA from a heterozygous subject, which did not reveal any trace of normal transcripts deriving from the mutant IVS6+1T allele. Through the expression of extended FVII minigenes in a human liver cell line (Hep3B), we demonstrated that the mutation primarily induced exon 6 skipping and, to a lower extent, total and partial IVS6 retention, thus producing frameshifts incompatible with FVII biosynthesis and function. The IVS6+1G>T is predicted to impair recognition of the donor splice site by the spliceosomal small nuclear ribonucleoprotein U1 (U1-snRNP), a crucial step in exon definition. To assess whether compensatory U1-snRNP could rescue FVII expression, we developed a modified U1- snRNA+1T complementary to the mutated 5'ss. However, cotransfection with this U1snRNA did not increase, to any appreciable extent, usage of the correct 5'ss. Altogether these findings do not provide experimental evidence even for minimal expression of this FVII gene mutation, affecting the invariable GT dinucleotide of the 5ss and abolishing correct FVII mRNA processing. Intriguingly, our findings also suggest that very low FVII levels, below the detection threshold of recombinant methodology, cause a lifethreatening disorder but can be compatible with life

Rescue of coagulation factor VII mRNA processing and protein function by engineered U1+5A snRNA

Changes affecting mRNA processing represent a frequent cause of severe coagulation factor defects and of all inherited human diseases. We extensively investigated the IVS7+5G/A mutation (9726+5A) in the coagulation factor VII (FVII) gene, occurring in the IVS7 donor splice site (5ss) in the first of six highly homologous 37bp repeats containing several cryptic splice sites. This mutation is the most frequent cause of severe FVII deficiency in Central Italy. A cellular model of this deficiency was created by producing a full length FVII splicing competent construct (pSCFVII-wt). This minigene drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 ±4 ng/mL), which were virtually undetectable upon introduction of the mutation (pSCFVII-9726+5A). At the mRNA level the mutation caused exon 7 skipping and, to a less extent, activation of the 37-bp downstream cryptic site. To attempt rescue of FVII expression, we have engineered the U1-snRNA, the spliceosome component selectively recognizing donor splice sites (5'ss), to re-direct recognition of the mutated donor splice site. Upon cotrasfection of the engineered U1- snRNA (pU1+5A) with pSCFVII-9726+5A, the splicing pattern and protein level were evaluated. At RNA level, the expression of U1+5A reduced from 80% to 40% the exon 7 skipping and increased recognition of the correct 5'ss, resulting in appreciable synthesis of normal transcripts (from hardly detectable to 20%). At protein level, we observed an increase of secreted protein levels in medium (5.0±2.8 ng/mL) and of the FVII coagulant activity, which reached 9.5±3.2% of pSCFVII-wt. The effects of engineered U1-snRNA were dose-dependent. Altogether these results demonstrate for the first time the U1- snRNAmediated rescue at the mRNA and protein levels, thus highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels

U1snRNA-mediated rescue of mRNA processing in severe factor VII deficiency

Small nuclear U1-RNAs (snRNAs), the spliceosome components selectively recognizing donor splice sites (5'ss), were engineered to restore correct mRNA processing in a cellular model of severe coagulation factor VII (FVII) deficiency, caused by the IVS7 9726+5g/a change. Three U1-snRNAs, complementary to the mutated 5’ss (U1+5a) or to neighbouring sequences, were expressed with FVII minigenes in a hepatoma cell line. The U1-snRNAs reduced from 80% to 40% the exon 7 skipping, thus increasing exon definition. The U1+5a construct also dramatically increased recognition of the correct 5'ss over the 37bp-downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5'ss, a frequent cause of severe defects

Establishment, Characterization, and Imaging of a First Platinum-resistant Penile Cancer Patient-derived Xenograft in Nude Mice: A eUROGEN Project

status: publishe

Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency

We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3–5% and 2.7±0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7±0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8±0.9%) and disappeared with rabbit thromboplastin (0.7±0.2%). This suggests that the mutation influences tissue factor/FVII interactions