The bone marrow-mediated protection of myeloproliferative neoplastic cells to Vorinostat and Ruxolitinib relies on the activation of JNK and PI3K signalling pathways

This study was funded by research grants from "Instituto Portugues de Oncologia de Lisboa-Francisco Gentil" (IPOL-FG), "Associacao Portuguesa Contra a Leucemia" (APCL) and "Liga Portuguesa Contra o Cancro" (LPCC). BAC is a recipient of a Post-Doc fellowship from "Fundacao para a Ciencia e Tecnologia" (FCT-SFRH/BPD/79209/2011) and HB from LPCC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The classical BCR-ABL-negative Myeloproliferative Neoplasms (MPN) are a group of heterogeneous haematological diseases characterized by constitutive JAK-STAT pathway activation. Targeted therapy with Ruxolitinib, a JAK1/2-specific inhibitor, achieves symptomatic improvement but does not eliminate the neoplastic clone. Similar effects are seen with histone deacetylase inhibitors (HDACi), albeit with poorer tolerance. Here, we show that bone marrow (BM) stromal cells (HS-5) protected MPN-derived cell lines (SET-2; HEL and UKE-1) and MPN patient-derived BM cells from the cytotoxic effects of Ruxolitinib and the HDACi Vorinostat. This protective effect was mediated, at least in part, by the secretion of soluble factors from the BM stroma. In addition, it correlated with the activation of signalling pathways important for cellular homeostasis, such as JAK-STAT, PI3K, JNK, MEK-ERK and NF-kappa B. Importantly, the pharmacological inhibition of JNK and PI3K pathways completely abrogated the BM protective effect on MPN cell lines and MPN patient samples. Our findings shed light on mechanisms of tumour survival and may indicate novel therapeutic approaches for the treatment of MPN.publishersversionpublishe

Choledochoduodenostomy in the Management of Common Duct Stones or Associated Pathology – An Obsolete Method?

Choledochoduodenostomy (CDD) has been reported as a more effective treatment of CBD stones than T-tube drainage but it is regarded as a last resort or obsolete therapeutic method due to fears of higher mobidity, cholangitis, “sump” syndrome and liver dysfunction. We aimed to assess the aforementioned issues analyzing prospectively our experience from 1976 through Dec.92

Epigenetic alterations in Fanconi Anaemia: Role in pathophysiology and therapeutic potential

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3T beta and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81\% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population.publishersversionpublishe

A lack of diversity, equity, and inclusion in clinical research has direct impact on patient care

info:eu-repo/semantics/publishedVersio

Novel dynamic outcome indicators and clinical endpoints in myelodysplastic syndrome; the European LeukemiaNet MDS Registry and MDS-RIGHT project perspective

Available evidence suggests that in most patients with LR-MDS the risk of death is not related to disease progression but is mainly attributable to non-leukemic death. 2,17 In addition, a proportion of these patients have prolonged survival that precludes the design of clinical trials adopting OS as a primary endpoint. These challenges have resulted in potentially biased assessment of the effectiveness and appropriate use of the available interventions in this patient population. The EUMDS Registry has identified novel meaningful outcome indicators and clinical endpoints, and reliable measures of response to HCI (Figure 4). The results of our analysis indicate that RBCT density is strongly associated with a decreased OS, even at relatively low dose densities. In addition, we observed that an early decrease in platelet count is an independent adverse prognostic indicator in LR-MDS, and combining relative platelet drop and transfusion dependency allows early identification of patients at risk of rapid progression, and may guide early therapeutic interventions, including allogeneic hematopoietic stem cell transplantation or experimental interventions. Taken together, these results indicate that regular RBCT requirement, early platelet count kinetics, and restriction in HRQoL are early independent and meaningful outcome indicators, and reliable measures of effectiveness of therapeutic interventions, evaluated in this set of studies. These findings support the integration of RBCT requirement and HRQoL in the general core outcome sets and in response criteria in patients with LR-MDS, and have important implications for clinical practice and the design of clinical endpoints. Our results strongly support the adoption of freedom from transfusion as a meaningful clinical endpoint in patients with LR-MDS. Anemia is the main determinant of therapeutic intervention in patients with LR-MDS, and ESA are recommended as first-line treatment for patients with symptomatic anemia. 10 The observational studies within the EUMDS Registry showed that the response rate, as well as the capacity of these agents to delay the onset of a regular RBCT need, is most pronounced in RBCT-naïve patients. These results identified early initiation of treatment with ESA as a major treatment response indicator, and indicate that ESA should be recommended in LR-MDS patients with symptomatic anemia before starting regular RBCT. After the onset of RBCT dependency, patients with LR-MDS are prone to long-term accumulation of iron. 1,43 The EUMDS Registry studies provided evidence that elevated LPI levels are associated with reduced survival in RBCT dependent patients, whereas iron chelation therapy normalizes LPI levels. These findings suggest that NTBI and LPI may serve as early indicators of iron toxicity and a means to measure the effectiveness of iron chelation therapy in patients with LR-MDS. However, qualified NTBI and LPI are only currently available in specialized laboratories. 44 Large observational cohorts with detailed clinical and laboratory data, like the EUMDS cohort, are the ideal framework in which to identify well defined MDS subtypes that may benefit from novel targeted treatments. An example of such a subtype is MDS with loss of parts of chromosome 5, namely del5q; these patients have a relatively favorable outcome on lenalidomide treatment. In order to identify homogeneous subsets of patients within MDS, preliminary evidence has suggested that recently identified mutations in splicing factors may recognize distinct disease entities within myeloid neoplasms. 45 Splicing modulators are now in pre-clinical testing, and are very likely to lead to the introduction of effective drugs for specific groups of MDS patients. Luspatercept, a specific inhibitor of growth and differentiation factor-11, a member of the transforming growth factor β superfamily, induced substantial improvement of anemia, especially in patients with ring sideroblasts. 46 Characterization of individual cases by new genetic markers (one of the main objectives of the MDS-RIGHT project) will allow refined classification of patients into biological subgroups that are expected to respond differently to therapeutic interventions to guide discontinuation of those interventions that are less effective or less cost-effective. The main question is whether RCT data and retrospective cohort data in selected tertiary care centers are representative of the 'real world' data of the older patients with LR-MDS in the general population. A careful comparison of the 'real world' data and the RCT data will be needed in order to provide a clear answer to these questions. Meanwhile, the current analyses of data collected over 10 years in the EUMDS Registry provides relevant and important information which could help assess prognosis and response to standard interventions in this older patient group

Vorinostat induces apoptosis and differentiation in myeloid malignancies: genetic and molecular mechanisms.

BACKGROUND: Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action. METHODOLOGY/PRINCIPAL FINDINGS: Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33(+) cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3) and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1) and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1. CONCLUSION: This study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases

Vorinostat effect in myeloid differentiation of CD33⁺ cells from AML patients.

<p>Peripheral blood CD33⁺ cells from AML patients were cultured ex-<i>vivo</i> in the presence of 1 µM vorinostat or vehicle (control) and myeloid differentiation analyzed by flow cytometry with CD11b-PE plus CD13-APC or CD11b-PE plus CD13-APC and CD14-FITC antibodies. Cellular apoptosis was assessed by flow cytometry of annexin-V-FITC/7-AAD co-stained cells and viability by the MTT method. Results are percentage of cells per population subset. nd, not determined.</p

Effect of vorinostat on myeloid differentiation and apoptosis of CD33⁺ cells from AML and AML patients.

<p>CD33⁺ cells from peripheral blood of AML patients (A) and bone marrows of MDS patients (B) were cultured in complete IMDM medium with SCF, IL-3, IL-6, GM-CSF, G-CSF, and EPO in the presence of 1 µM vorinostat or vehicle (Control). After 3 days, myelomonocytic markers, CD11b, CD13 and CD14 and apoptosis were analyzed by flow cytometry. The numbers in CD13/CD11b panels are percentage of CD33 cells on different myeloid maturation stages according the CD13 and CD11b expression levels. I (CD13^hi/CD11b⁻): myeloblasts; II (CD13^lo/int/CD11b⁻): promyelocytes; III (CD13^lo/int/CD11b⁺): myelocytes and metamyelocytes; and IV (CD13^hi/CD11b⁺): band cells and mature neutrophils. Mature monocytes are also CD13^hi/CD11b⁺. Numbers in other panels are percentages of total cells in the respective gates.</p

Effect of GC-rich DNA sequences and SP1 on the transcriptional effect of vorinostat on IER3, cFOX, COX2, p21, Cyclin G2 and CUL1 genes.

<p>A, K562 cells were exposed to Mith.A (100 nM) or vehicle (Control) and not further treated or exposed 30 min later to 5 µM vorinostat for 8 h and gene expression quantified by qPCR. Results are average values ± SEM of at least three independent assays, done in triplicate. B–C, K562 cells were transfected with control (Ctr) or SP1 siRNA and treated with vehicle or vorinostat (2 µM) for 24 and 48 h. B upper panel, immunoblot showing SP1 protein levels in cells transfected with control (Ctr) and SP1 siRNAs in 1 out of 5 assays at 48 h after transfection. B lower panel, SP1 mRNA levels in cells transfected with control (Ctr) and SP1 siRNAs. Graph shows average percentage of SP1 mRNA ± S.D. from 5 independent assays at 24 h after transfection. C, Graph shows average mRNA fold change of the indicated genes over vehicle treated cells transfected with the same siRNA 48 h after transfection ± SEM from 3 independent experiments. D, K562 and HL60 cells were treated with vorinostat (5 µM) or vehicle (Control) for 7 h and SP1 binding to the proximal promoter regions of the indicated genes determined by ChIP assays and qPCR. Results are expressed as fold change over control IgG and represent average values of at least three independent experiments ± SEM. Data were analyzed using paired Student’s t test. *p<0.05. **p<0.05.</p

Effect of vorinostat on genes involved in regulation of cell cycle control, proliferation, apoptosis, and differentiation in CD33 myeloid cells from MDS patients.

<p>BM-CD33 cells from MDS patients were treated with 5 µM vorinostat or vehicle (Control) for 8 h and gene expression quantified by qPCR. A, Effect of vorinostat on the expression of genes responsive to vorinostat in K562, HL60 and THP1; 1- genes with altered expression in haematologic malignancies; 2- genes with altered expression in haematologic malignancies that respond to epigenetic therapy; and 3- transcription factors. B, Effect of vorinostat on the expression of genes that control: 4- cell cycle arrest/check point/DNA repair and 5- cell cycle transition. C, Effect of vorinostat on the expression of: 6- pro-apoptotic and 7-anti-apoptotic genes. In all panels, each dot represents data from one patient. *p<0.05.</p