Ricolinostat, a selective HDAC6 inhibitor, shows anti-lymphoma cell activity alone and in combination with bendamustine

Histone deacetylase inhibitors (HDACis) have emerged as a new class of anticancer agents, targeting the biological process including cell cycle and apoptosis. We investigated and explained the anticancer effects of an HDAC6 inhibitor, ricolinostat alone and in combination with bendamustine in lymphoma cell lines. Cell viability was measured by MTT assay. Apoptosis, reactive oxygen species (ROS) generation, Bcl-2 protein expression, cell cycle progression and tubuline expression were determined by flow cytometry. The effects of ricolinostat alone and in combination on the caspases, PI3K/Akt, Bcl-2 pathways, ER stress and UPR were assessed by immunoblotting. Ricolinostat shows anti lymphoma activity when used as single agent and its capability to induce apoptosis is synergistically potentiated by the bendamustine in lymphoma cell lines. Drug combination reduced the proportion of cells in the G0/G1and S phases and caused an increase of âsub-G0/G1â peak. The synergistic effect accompanied with the increased ROS, activation of caspase-8, -9, and -3, the cleavage of PARP and modulated by Bcl-2 proteins family. In addition, the exposure of ricolinostat induced the acetylation level of α-tubulin, the extend of which was not further modified by bendamustine. Finally, the apoptosis effect of ricolinostat/bendamustine may be mediated by a corresponding effect on microtubule stabilization. Our data suggest that ricolinostat in combination with bendamustine may be a novel combination with potential for use as an antitumor agent in lymphoma

Synergy of NVP-BEZ235 and enzastaurin in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a neoplasm classified as a B-cell malignancy, that accounts for approximately 3 to 8% of Non-Hodgkin's lymphoma (NHL) cases diagnosed annually. MCL is difficult to treat and seldom considered cured. The pathobiology of MCL is complex and includes alteration in the cell cycle, abnormalities in the DNA damage response, and constitutive activation of key antiapoptotic pathways including phosphatidyl-inositol 3-kinase (PI3K)/Akt and nuclear factor-kB. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future MCL therapies. The phosphatidylinositol 3-kinase (PI3K) mammalian target of rapamicin (mTOR) pathway mediates proliferation, survival, and drug resistance in lymphoma cells. NVP-BEZ235 (BEZ235) is a new, orally bioavailable inhibitor of PI3K and mTOR and a representative of a new class of anti-tumour agents.In this study, we analysed the in vitro inhibitory effects of NVP-BEZ235 on mantle lymphoma cell lines (GRANTA-519 and JeKo-1) and its effects in combination with enzastaurin, everolimus and perifosine. Our data suggest that in mantle lymphoma cell lines, BEZ235 in combination with enzastaurin elicits its antitumour effect better than combined with perifosine and everolimus. Our data reveal that the drug combination targets phosphorylation of PI3K/Akt/mTOR pathways and induces both intrinsic and extrinsic apoptosis pathways. Furthermore, inhibition of Bcl-2 anti-apoptosis family members may, in part, explain the efficacy of signalling blockade in lymphoma cells and suggests an additional therapeutic targeting strategy. Therefore, these preclinical data support the potential use of BEZ235 in patients with mantle lymphoma, and in particular provide rationale for combination with enzastaurin

The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines

We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, −9, −3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies

Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+ VSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, Taq- Man® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four Taq- Man® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β- thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week

An Aγ-globin G->A gene polymorphism associated with β(0)39 thalassemia globin gene and high fetal hemoglobin production

Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in β-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify β-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea)

A validated cellular biobank for β-thalassemia

Background: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. Methods: Erythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. Results: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. Conclusion: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results

A Rational Approach to Drug Repositioning in β-thalassemia: Induction of Fetal Hemoglobin by Established Drugs

Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', 'drug rescue', 'drug re-tasking' and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the 'off-label' use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the time needed to bring a drug to market. Finally, patent filing of repurposed drugs is expected to catch the attention of pharmaceutical industries interested in the development of therapeutic protocols for RDs. Repurposed molecules that could be proposed as potential drugs for β-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial

Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for β-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the β+/β+ and β+/β0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24–48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with β-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies. © The Author(s), 2022

Decision quality instrument for treatment of hip and knee osteoarthritis: a psychometric evaluation

Abstract Background A high quality decision requires that patients who meet clinical criteria for surgery are informed about the options (including non-surgical alternatives) and receive treatments that match their goals. The aim of this study was to evaluate the psychometric properties and clinical sensibility of a patient self report instrument, to measure the quality of decisions about total joint replacement for knee or hip osteoarthritis. Methods The performance of the Hip/Knee Osteoarthritis Decision Quality Instrument (HK-DQI) was evaluated in two samples: (1) a cross-sectional mail survey with 489 patients and 77 providers (study 1); and (2) a randomized controlled trial of a patient decision aid with 138 osteoarthritis patients considering total joint replacement (study 2). The HK-DQI results in two scores. Knowledge items are summed to create a total knowledge score, and a set of goals and concerns are used in a logistic regression model to develop a concordance score. The concordance score measures the proportion of patients whose treatment matched their goals. Hypotheses related to acceptability, feasibility, reliability and validity of the knowledge and concordance scores were examined. Results In study 1, the HK-DQI was completed by 382 patients (79%) and 45 providers (58%), and in study 2 by 127 patients (92%), with low rates of missing data. The DQI-knowledge score was reproducible (ICC = 0.81) and demonstrated discriminant validity (68% decision aid vs. 54% control, and 78% providers vs. 61% patients) and content validity. The concordance score demonstrated predictive validity, as patients whose treatments were concordant with their goals had more confidence and less regret with their decision compared to those who did not. Conclusions The HK-DQI is feasible and acceptable to patients. It can be used to assess whether patients with osteoarthritis are making informed decisions about surgery that are concordant with their goals