BackgroundAberrant 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 FindingsFunctional 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.ConclusionThis 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.

In a fraction of families fulfilling the Amsterdam criteria for hereditary non-polyposis colorectal cancer, colorectal cancers are microsatellite stable and DNA mismatch repair gene (MMR) mutations are not found. These families were designated as familial colorectal cancer type X (FCCTX). We aimed to characterise a group of FCCTX families defined by the Amsterdam criteria and MSS tumours at clinical and molecular level. Twenty-four tumours from 15 FCCTX families were analysed for loss of known tumour suppressor gene (TSG) loci (APC, TP53, SMAD4 and DCC), MGMT and MMR genes promoter methylation, and also APC and KRAS somatic mutations. FCCTX families presented specific clinical features: absence of endometrial tumours, high adenoma/carcinoma ratio (1.91) and prevalence of rectal cancers (13/27, 48%). New molecular features were found: the majority of FCCTX tumours (13/18; 72%) presented TSG loss. TSG loss positive tumours presented frequent APC and KRAS somatic mutations and MGMT methylation [10/13 (77%), 7/13 (54%) and 6/11 (54%), respectively]. In TSG loss negative tumours (5/18; 28%), the same molecular events were found in 2/5 (40%), 2/5 (40%) and 1/3 (33%) tumours, respectively. Transition mutations in KRAS were more frequent among MGMT methylated tumours than in unmethylated [5/8 (63%) vs. 1/10 (10%), P = 0.03]. Although sharing similar clinical features, at least two different molecular entities should exist among FCCTX families, one whose tumours present frequent TSG loss, APC and KRAS somatic mutations, and MGMT promoter methylation, and a second, lesser predominant, with no evidence of TSG loss and rarely presenting promoter methylation.

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.

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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-κB. 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.

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 DNM3Tβ 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.

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Background: The classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are characterized by increased proliferation of hematopoietic precursors in the bone marrow resulting in elevated numbers of terminally differentiated cells. Despite the recent advances in the understanding of the biology of MPN, there is still no curative treatment for MPN except for bone marrow transplantation. The discovery of JAK-STAT constitutive activation in the majority of MPN patients led to the development of clinical studies targeting MPN with Ruxolitinib, a JAK1/2-specific inhibitor. However, despite achieving significant reductions in splenomegaly and symptomatic improvement in MPN patients, JAK inhibition failed to induce complete remissions and eradicate the malignant clone. In alternative, histone deacetylase inhibitors have shown success in the treatment of several hematological malignancies but their efficacy in MPN is limited. Aims: Ruxolitinib and the HDAC inhibitor Vorinostat both fail to eradicate the neoplastic clone in MPN patients. Here, we tested the hypothesis that the bone marrow stroma confers resistance to these drugs by preventing their cytotoxic effects on neoplastic cells. Material and Methods: MPN patient derived cell lines (SET-2 and HEL) were cultured alone, with HS-5 bone marrow stroma or with HS-5 conditioned medium in the presence of clinically-relevant concentrations of Ruxolitinib and Vorinostat. At different time points, cellular viability was analyzed by staining with Annexin-V/PI and CD45-APC (to distinguish MPN cells from the HS-5 cells). Furthermore, cells were lysed and RNA was extracted for gene expression analysis. Activation of specific signaling pathways was determined by Western-blot using phospho-specific antibodies for effectors of the following signaling pathways: PI3K-Akt-mTOR; MEK-Erk; MAPK-JNK; NF-κB and JAK-STAT. Results: Treatment of SET-2 and HEL cells with both Vorinostat and Ruxolitinib promoted apoptosis and decreased proliferation. Importantly, apoptosis was significantly abrogated when MPN cells were cultured in the presence of a stromal layer of HS-5 cells or HS-5 conditioned medium. The stroma protective effect was maintained for up to 6 six days and was also dependent on the concentration of the drugs. These effects correlated with altered expression in MPN cells of genes associated with inflammatory processes, apoptosis and proliferation (CDKN1A, IER3, BIRC3, TNFRSF8, TNFRSF9, COX2, IL1B, XIAP), and with the activation of signaling pathways important for cellular homeostasis, such as PI3K-Akt-mTOR, MAPK-JNK, JAK-STAT and NF-κB as shown by increased phosphorylation of Akt (Ser473); GSK3α/β (Ser9/Ser21); S6 (Ser235/236); STAT3/5 (Tyr705/Tyr694) and IKBα (Ser32). Summary/Conclusions: Overal, we show that bone marrow stroma protects MPN cells from the cytotoxic effects of two clinically effective pharmacological agents of different classes: Vorinostat and Ruxolitinib. This protective effect is likely achieved, through the up-regulation of genes that inhibit apoptosis (BIRC3, TNFRSF8, TNFRSF9) and also through the activation of pro-survival signaling pathways such as PI3K-Akt-mTOR. We did not observe any effects of the bone marrow stroma on proliferation or differentiation, suggesting that the main effect of stromal cells is to prevent apoptosis of the neoplastic cells. Our results identify a possible cell non-autonomous mechanism by which Ruxolitinib fails to eradicate the neoplastic clone in MPN patients. Through the identification of the factors released by the bone marrow stroma, we hope to uncover why the bone marrow protects MPN cells and possibly define novel therapeutic strategies maximizing the use of Ruxolitinib and Vorinostat in MPN. Disclosures Almeida: Shire: Speakers Bureau; Amgen: Speakers Bureau; Novartis: Consultancy; Celgene: Consultancy; Bristol-Meyer Squibb: Speakers Bureau.