Endothelial cells (ECs) lining arteries and veins have distinct molecular/functional signatures. The underlying regulatory mechanisms are incompletely understood. Here, we established a specific fingerprint of freshly isolated arterial and venous ECs from human umbilical cord comprising 64 arterial and 12 venous genes, representing distinct functions/pathways. Among the arterial genes were 8 transcription factors (TFs), including Notch target HEY2, the current "gold standard" determinant for arterial EC (aEC) specification. Culture abrogated differential gene expression in part due to gradual loss of canonical Notch activity and HEY2 expression. Notably, restoring HEY2 expression or Delta-like4-induced Notch signaling in cultured ECs only partially reinstated the aEC gene signature, whereas combined overexpression of the 8 TFs restored this fingerprint more robustly. Whereas some TFs stimulated few genes, others boosted a large proportion of arterial genes. Although there was some overlap and crossregulation, the TFs largely complemented each other in regulating the aEC gene profile. Finally, overexpression of the 8 TFs in human umbilical vein ECs conveyed an arterial-like behavior upon their implantation in a Matrigel plug in vivo. Thus, our study shows that Notch signaling determines only part of the aEC signature and identifies additional novel and complementary transcriptional players in the complex regulation of human arteriovenous EC identity.
Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer that is characterized by rapid and uncontrolled proliferation of immature B- or T-lymphoid precursors. Although ALL has been regarded as a genetic disease for many years, the crucial importance of epigenetic alterations in leukemogenesis has become increasingly evident. Epigenetic mechanisms, which include DNA methylation and histone modifications, are critical for gene regulation during many key biological processes. Here, we review the cell signaling pathways that are regulated by DNA methylation or histone modifications in ALL. Recent studies have highlighted the fundamental role of these modifications in ALL development, and suggested that future investigation into the specific genes and pathways that are altered by epigenetic mechanisms can contribute to the development of novel drug-based therapies for ALL.
Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies <20%, likely below the detection limit of traditional sequencing methods. Genomic array data showed that cases of advanced del(5q) myelodysplastic syndrome had a complex background of cytogenetic aberrations, often encompassing genes involved in myeloid disorders. Our study is the first to investigate the molecular pathogenesis of early and advanced del(5q) myelodysplastic syndromes using next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies, further illuminating the molecular landscape of del(5q) myelodysplastic syndromes.
Long non-coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. LncRNAs are as diverse as mRNAs and they normally share the same biosynthetic machinery based on RNA polymerase II, splicing and polyadenylation. However, lncRNAs have low coding potential. Compared to mRNAs, lncRNAs are preferentially nuclear, more tissue specific and expressed at lower levels. Most of the lncRNAs described to date modulate the expression of specific genes by guiding chromatin remodelling factors; inducing chromosomal loopings; affecting transcription, splicing, translation or mRNA stability; or serving as scaffolds for the organization of cellular structures. They can function in cis, cotranscriptionally, or in trans, acting as decoys, scaffolds or guides. These functions seem essential to allow cell differentiation and growth. In fact, many lncRNAs have been shown to exert oncogenic or tumor suppressor properties in several cancers including haematological malignancies. In this review, we summarize what is known about lncRNAs, the mechanisms for their regulation in cancer and their role in leukemogenesis, lymphomagenesis and hematopoiesis. Furthermore, we discuss the potential of lncRNAs in diagnosis, prognosis and therapy in cancer, with special attention to haematological malignancies.
Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-?B pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.
The ASXL1 gene encodes a chromatin-binding protein involved in epigenetic regulation in haematopoietic cells. Loss-of-function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral-based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34(+) cells differentiated along the myeloid lineage in vitro. ASXL1-deficient cells showed a significant decrease in the generation of CD11b(+) and CD15(+) cells, implicating impaired granulomonocytic differentiation. Furthermore, colony-forming assays showed a significant increase in the number of multipotent mixed lineage colony-forming unit (CFU-GEMM) colonies and a significant decrease in the numbers of granulocyte-macrophage CFU (CFU-GM) and granulocyte CFU (CFU-G) colonies in ASXL1-deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1-deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over-representation of PRC2 targets among the deregulated genes in ASXL1-deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.
The disruption of RUNX1 function is one of the main mechanisms of disease observed in hematopoietic malignancies and the description of novel genetic events that lead to a RUNX1 loss of function has been accelerated with the development of genomic technologies. Here we describe the molecular characterization of a new t(4;21)(q21;q22) in a de novo myelodysplastic syndrome that resulted in the deletion of the RUNX1 gene. We demonstrated by quantitative real-time RT-PCR an almost complete depletion of the expression of the RUNX1 gene in our t(4;21) case compared with CD34(+) cells that was independent of mutation or DNA methylation. More importantly, we explored and confirmed the possibility that this abrogation also prevented transactivation of RUNX1 target genes, perhaps confirming the genetic origin of the thrombocytopenia and the myelodysplastic features observed in our patient, and certainly mimicking what has been observed in the presence of the RUNX1/ETO fusion protein.
The role of epigenetic mechanisms in the regulation of microRNAs (miRNAs) with a tumour-suppressor function in human neoplasms has recently been established. Several miRNAs have been found to be inappropriately regulated by DNA methylation in patients with acute lymphoblastic leukaemia (ALL). We analysed the methylation status of the three members of the MIR9 family (MIR9-1, MIR9-2 and MIR9-3) in a uniformly treated cohort of 200 newly diagnosed ALLs. MIR9 was methylated in 54% of the patients and was associated with downregulation of MIR9 (P?0·01). Hypermethylation of MIR9 was an independent prognostic factor for disease-free survival, overall survival and event-free survival in a multivariate analysis (P?0·01). Epigenetic downregulation of MIR9 induced upregulation of its targets, FGFR1 and CDK6, while treatment of ALL cells with FGFR1 (PD-173074) and CDK6 (PD-0332991) inhibitors induced a decrease in cell proliferation and an increase in apoptosis of ALL cells. Our results indicate that the MIR9 family is involved in the pathogenesis and clinical behaviour of ALL and provide the basis for new therapeutic strategies in the treatment of ALL, targeting the epigenetic regulation of miRNAs and/or the FGFR1 or CDK6-RB pathway directly.
The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia.
Transcription factors are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of transcription factors can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of Ikaros family genes, which code for essential transcription factors in cell differentiation and exhibit genetic defects in hematologic neoplasias. Unexpectedly, our analysis revealed that Ikaros undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Dukes stages. Ikaros hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis, and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma.
Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.
Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.
LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored.
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n?=?48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p?=?0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p?=?0.006) and overall survival (OS) (p?=?0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.
Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.
In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2(-/-)?c(-/-) mice and of murine B220(+)IgM(+) B-cell lymphomas generated in E?-MYC and E?-MYC-BIM(+/-) transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL.
To measure a combination of novel molecular biomarkers in urine/blood samples of consecutive patients referring lower urinary tract symptoms (LUTS) not previously diagnosed, to improve prostate cancer diagnosis.
Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.
Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+)pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter-exon 1 was analyzed by methylation-specific PCR and sequencing in eleven AML-derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5-Aza-2-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non-methylated group. Disease-free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML.
Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required.
In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor-supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down-regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa-miR-9, hsa-miR-129 and hsa-miR-137) in 3 CRC cell lines. Expression of hsa-miR-9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa-miR-9-1, hsa-miR-129-2 and hsa-miR-137 CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa-miR-9-1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.
The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.
The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles.
Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.
Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.
Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1(+) cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established.
The specific ablation of Rb1 gene in stratified epithelia (Rb(F/F);K14cre) promotes proliferation and altered differentiation but is insufficient to produce spontaneous tumors. The pRb relative, p107, compensates some of the functions of pRb in these tissues; however, Rb(F/F);K14cre;p107(-/-) mice die postnatally. Here we show, using an inducible mouse model (Rb(F/F);K14creER(TM)), that p107 exerts specific tumor suppressor functions in the absence of pRb in stratified epithelia. The simultaneous absence of pRb and p107 produces impaired p53 transcriptional functions and reduction of Pten expression, allowing spontaneous squamous carcinoma development. These tumors display significant overlap with human squamous carcinomas, supporting that Rb(F/F);K14creER(TM);p107(-/-) mice might constitute a new model for these malignancies. Remarkably tumor development in vivo is partially alleviated by mTOR inhibition. These data demonstrate the existence of a previously unreported functional connection between pRb, Pten and p53 tumor suppressors, through p107, of a particular relevance in squamous tumor development.
MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290-295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.
Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.
Using high-resolution genomic microarray analysis, a distinct genomic profile was defined in 114 samples from patients with splenic marginal zone lymphoma (SMZL). Deletion or uniparental disomy of chromosome 7q were detected in 42 of 114 (37%) SMZLs but in only nine of 170 (5%) mature B-cell lymphomas (P < 0·00001). The presence of unmutated IGHV, genomic complexity, 17p13-TP53 deletion and 8q-MYC gain, but not 7q deletion, correlated with shorter overall survival of SMZL patients. Mapping studies narrowed down a commonly deleted region of 2·7 Mb in 7q32.1-q32.2 spanning a region between the SND1 and COPG2 genes. High-throughput sequencing analysis of the 7q32-deleted segment did not identify biallelic deletions/insertions or clear pathogenic gene mutations, but detected six nucleotide changes in IRF5 (n = 2), TMEM209 (n = 2), CALU (n = 1) and ZC3HC1 (n = 1) not found in healthy individuals. Comparative expression analysis found a fourfold down-regulation of IRF5 gene in lymphomas with 7q32 deletion versus non-deleted tumours (P = 0·032). Ectopic expression of IRF5 in marginal-zone lymphoma cells decreased proliferation and increased apoptosis in vitro, and impaired lymphoma development in vivo. These results show that cryptic deletions, insertions and/or point mutations inactivating genes within 7q32 are not common in SMZL, and suggest that IRF5 may be a haploinsufficient tumour suppressor in this lymphoma entity.
Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL(+) acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL-dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2-deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL-expressing acute lymphoblastic leukemia.
Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-?B activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.
Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.
miRNAs are small RNA molecules ( 22nt) that interact with their corresponding target mRNAs inhibiting the translation of the mRNA into proteins and cleaving the target mRNA. This second effect diminishes the overall expression of the target mRNA. Several miRNA-mRNA relationship databases have been deployed, most of them based on sequence complementarities. However, the number of false positives in these databases is large and they do not overlap completely. Recently, it has been proposed to combine expression measurement from both miRNA and mRNA and sequence based predictions to achieve more accurate relationships. In our work, we use LASSO regression with non-positive constraints to integrate both sources of information. LASSO enforces the sparseness of the solution and the non-positive constraints restrict the search of miRNA targets to those with down-regulation effects on the mRNA expression. We named this method TaLasso (miRNA-Target LASSO).We used TaLasso on two public datasets that have paired expression levels of human miRNAs and mRNAs. The top ranked interactions recovered by TaLasso are especially enriched (more than using any other algorithm) in experimentally validated targets. The functions of the genes with mRNA transcripts in the top-ranked interactions are meaningful. This is not the case using other algorithms.TaLasso is available as Matlab or R code. There is also a web-based tool for human miRNAs at http://talasso.cnb.csic.es/.
Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.
Fanconi anemia (FA) is an inherited genetic disorder associated with BM failure and cancer predisposition. In the present study, we sought to elucidate the role of microRNAs (miRNAs) in the hematopoietic defects observed in FA patients. Initial studies showed that 3 miRNAs, hsa-miR-133a, hsa-miR-135b, and hsa-miR-181c, were significantly down-regulated in lymphoblastoid cell lines and fresh peripheral blood cells from FA patients. In vitro studies with cells expressing the luciferase reporter fused to the TNF? 3-untranslated region confirmed in silico predictions suggesting an interaction between hsa-miR-181c and TNF? mRNA. These observations were consistent with the down-regulated expression of TNF? mediated by hsa-miR-181c in cells from healthy donors and cells from FA patients. Because of the relevance of TNF? in the hematopoietic defects of FA patients, in the present study, we transfected BM cells from FA patients with hsa-miR-181c to evaluate the impact of this miRNA on their clonogenic potential. hsa-miR-181c markedly increased the number and size of the myeloid and erythroid colonies generated by BM cells from FA patients. Our results offer new clues toward understanding the biologic basis of BM failure in FA patients and open new possibilities for the treatment of the hematologic dysfunction in FA patients based on miRNA regulation.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.