Long non-coding RNAs and MYC association in hematological malignancies.

Long non-coding RNAs (lncRNAs) have an established role in cell biology. Among their functions is the regulation of hematopoiesis. They characterize the different stages of hematopoiesis in a more lineage-restricted expression pattern than coding mRNAs. They affect hematopoietic stem cell renewal, proliferation, and differentiation of committed progenitors by interacting with master regulators transcription factors. Among these transcription factors, MYC has a prominent role. Similar to MYC's transcriptional activation/amplification of protein coding genes, MYC also regulates lncRNAs' expression profile, while it is also regulated by lncRNAs. Both myeloid and lymphoid malignancies are prone to the association of MYC with lncRNAs. Such interaction inhibits apoptosis, enhances cell proliferation, deregulates metabolism, and promotes genomic instability and resistance to treatment. In this review, we discuss the recent findings that encompass the crosstalk between lncRNAs and describe the pathways that very probably have a pathogenetic role in both acute and chronic hematologic malignancies.

Enhancer DNA methylation in acute myeloid leukemia and myelodysplastic syndromes.

DNA methylation (CpG methylation) exerts an important role in normal differentiation and proliferation of hematopoietic stem cells and their differentiated progeny, while it has also the ability to regulate myeloid versus lymphoid fate. Mutations of the epigenetic machinery are observed in hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) resulting in hyper- or hypo-methylation affecting several different pathways. Enhancers are cis-regulatory elements which promote transcription activation and are characterized by histone marks including H3K27ac and H3K4me1/2. These gene subunits are target gene expression 'fine-tuners', are differentially used during the hematopoietic differentiation, and, in contrast to promoters, are not shared by the different hematopoietic cell types. Although the interaction between gene promoters and DNA methylation has extensively been studied, much less is known about the interplay between enhancers and DNA methylation. In hematopoiesis, DNA methylation at enhancers has the potential to discriminate between fetal and adult erythropoiesis, and also is a regulatory mechanism in granulopoiesis through repression of neutrophil-specific enhancers in progenitor cells during maturation. The interplay between DNA methylation at enhancers is disrupted in AML and MDS and mainly hyper-methylation at enhancers raising early during myeloid lineage commitment is acquired during malignant transformation. Interactions between mutated epigenetic drivers and other oncogenic mutations also affect enhancers' activity with final result, myeloid differentiation block. In this review, we have assembled recent data regarding DNA methylation and enhancers' activity in normal and mainly myeloid malignancies.

On the potential role of DNMT1 in acute myeloid leukemia and myelodysplastic syndromes: not another mutated epigenetic driver.

DNA methylation is the most common epigenetic modification in the mammalian genome. DNA methylation is governed by the DNA methyltransferases mainly DNMT1, DNMT3A, and DNMT3B. DNMT1 methylates hemimethylated DNA ensuring accurate DNA methylation maintenance. DNMT1 is involved in the proper differentiation of hematopoietic stem cells (HSCs) through the interaction with effector molecules. DNMT1 is deregulated in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) as early as the leukemic stem cell stage. Through the interaction with fundamental transcription factors, non-coding RNAs, fusion oncogenes and by modulating core members of signaling pathways, it can affect leukemic cells biology. DNMT1 action might be also catalytic-independent highlighting a methylation-independent mode of action. In this review, we have gathered some current facts of DNMT1 role in AML and MDS and we also propose some perspectives for future studies.

Polycomb group proteins and MYC: the cancer connection.

Polycomb group proteins (PcGs) are transcriptional repressors involved in physiological processes whereas PcG deregulation might result in oncogenesis. MYC oncogene is able to regulate gene transcription, proliferation, apoptosis, and malignant transformation. MYC deregulation might result in tumorigenesis with tumor maintenance properties in both solid and blood cancers. Although the interaction of PcG and MYC in cancer was described years ago, new findings are reported every day to explain the exact mechanisms and results of such interactions. In this review, we summarize recent data on the PcG and MYC interactions in cancer, and the putative involvement of microRNAs in the equation.

DLK1-DIO3 imprinted cluster in induced pluripotency: landscape in the mist.

DLK1-DIO3 represents an imprinted cluster which genes are involved in physiological cell biology as early as the stem cell level and in the pathogenesis of several diseases. Transcription factor-mediated induced pluripotent cells (iPSCs) are considered an unlimited source of patient-specific hematopoietic stem cells for clinical application in patient-tailored regenerative medicine. However, to date there is no marker established able to distinguish embryonic stem cell-equivalent iPSCs or safe human iPSCs. Recent findings suggest that the DLK1-DIO3 locus possesses the potential to represent such a marker but there are also contradictory data. This review aims to report the current data on the topic describing both sides of the coin.

The microRNAs within the DLK1-DIO3 genomic region: involvement in disease pathogenesis.

The mammalian genome is transcribed in a developmentally regulated manner, generating RNA strands ranging from long to short non-coding RNA (ncRNAs). NcRNAs generated by intergenic sequences and protein-coding loci, represent up to 98 % of the human transcriptome. Non-coding transcripts comprise short ncRNAs such as microRNAs, piwi-interacting RNAs, small nucleolar RNAs and long intergenic RNAs, most of which exercise a strictly controlled negative regulation of expression of protein-coding genes. In humans, the DLK1-DIO3 genomic region, located on human chromosome 14 (14q32) contains the paternally expressed imprinted genes DLK1, RTL1, and DIO3 and the maternally expressed imprinted genes MEG3 (Gtl2), MEG8 (RIAN), and antisense RTL1 (asRTL1). This region hosts, in addition to two long intergenic RNAs, the MEG3 and MEG8, one of the largest microRNA clusters in the genome, with 53 miRNAs in the forward strand and one (mir-1247) in the reverse strand. Many of these miRNAs are differentially expressed in several pathologic processes and various cancers. A better understanding of the pathophysiologic importance of the DLK1-DIO3 domain-containing microRNA cluster may contribute to innovative therapeutic strategies in a range of diseases. Here we present an in-depth review of this vital genomic region, and examine the role the microRNAs of this region may play in controlling tissue homeostasis and in the pathogenesis of some human diseases, mostly cancer, when aberrantly expressed. The potential clinical implications of this data are also discussed.

Non-coding RNAs and EZH2 interactions in cancer: long and short tales from the transcriptome.

A large amount of data indicates that non-coding RNAs represent more than the "dark matter" of the genome. Both microRNAs and long non-coding RNAs are involved in several fundamental biologic processes, and their deregulation may lead in oncogenesis. Interacting with the Polycomb-repressive complex 2 subunit EZH2, they could affect the expression of protein-coding genes and form feedback networks and autoregulatory loops. They can also form networks with upstream and downstream important factors, in which EZH2 represent the stabilizing factor of the pathway. As such non-coding RNAs affect the epigenetic modifications leading to malignant transformation.

MicroRNAs mark in the MLL-rearranged leukemia.

MicroRNAs are short noncoding RNAs, known regulators of several signaling pathways cell differentiation and proliferation, development, and apoptosis, which are deregulated in acute leukemia. Mixed lineage leukemia (MLL) gene encodes a protein with histone methyltransferase activity, which is essential for the fine tuning of hematopoietic stem cell development and differentiation through the regulation of HOXA and MEIS1. MLL gene rearrangements characterize both acute myeloid and acute lymphoblastic leukemia associated with poor outcomes. MicroRNAs and MLL rearrangements are in tight association regulating each other expression, affecting cell cycle regulators, and composing complex networks with factors involved in leukemogenesis such as MYC and FLT3. MLL fusion genes are also capable of recruiting DNA methyltransferases at microRNAs promoters controlling their expression through epigenetic changes. Direct drug targeting of MLL has been difficult to achieve, and in this context, microRNA expression modulation represents an attractive approach.

DLK1-MEG3 imprinted domain microRNAs in cancer biology.

A rapidly growing body of evidence highlights the involvement of DLK1-MEG3 imprinted domain in cell biology and cancer pathogenesis. The imprinted domain contains protein-coding genes, long non-coding RNAs, and various small non-coding RNAs. The imprinted microRNAs located here interact with important transcription factors, modulate fundamental signaling cascades, form molecular signatures with diagnostic and prognostic potential, and could differentiate chemoresistant from chemosensitive disease. Moreover, as they can be detected in patients' serum, are easy to obtain, and can be used as adjuvant diagnostic biomarkers with the potential of monitoring disease progression and response to treatment.

Deregulated microRNAs in multiple myeloma.

MicroRNAs are short noncoding RNAS involved in gene expression regulation under physiological and pathological situations. They bind to mRNA of target genes and are potential regulators of gene expression at a post-transcription level through the RNA interference pathway. They are estimated to represent 1% to 2% of the known eukaryotic genome, and it has been demonstrated that they are involved in the pathogenesis of neurodegenerative diseases, cancer, metabolism disorders, and heart disease. MicroRNAs are known to act as tumor suppressors or oncogenes in cancer biology. The authors describe the current knowledge on microRNA involvement in regulatory pathways that characterize multiple myeloma pathogenesis gained from in vitro and in vivo studies. These small molecules interact with important factors such as p53, SOCS1, IGF-1, IGF-1R, vascular endothelial growth factor, NF-κB, and others. As such, microRNAs represent an attractive therapeutic target in the context of multiple myeloma interfering with the myeloma regulatory networks. Further studies are needed to better understand their role in myelomagenesis and their therapeutic potential.

Epialleles and epiallelic heterogeneity in hematological malignancies.

DNA methylation has a well-established role in the pathogenesis, prognosis, and response to treatment in all the spectra of hematological malignancies. However, most of the data reported involve average DNA methylation observed in a sample. The emergence of bisulfite sequencing methods such as enhanced reduced representation that permit analyze adjacent CpGs led to exciting findings. Among these are the epialleles shift and the resulting epigenetic heterogeneity observed in leukemias and lymphomas. Epialleles seem to have an influential role as the cause of mutations that characterize leukemias, may stratify groups with different prognosis and response to treatment, and may be redistributed in the genome at different time points of the disease promoting activation of alternate transcriptional networks. Epiallelic shift may be responsible for the intratumor heterogeneity observed within the cells of the same tumor which increases with disease aggressiveness. It may also responsible for the interpatient heterogeneity explaining why blood cancers exhibit different behavior among different patients. Understanding better epiallelic conformation and the consequent chromatin conformational changes and the pathways that may be affected will permit deeper understanding of hematological malignancies pathogenesis and treatment.

Luspatercept: A New Tool for the Treatment of Anemia Related to ß-Thalassemia, Myelodysplastic Syndromes and Primary Myelofibrosis.

Anemia is a common feature of both benign and malignant hematologic diseases. Beta-thalassemia (ß-thalassemia) syndromes are a group of hereditary disorders characterized by ineffective erythropoiesis, due to a genetic deficiency in the synthesis of the beta chains of hemoglobin, often accompanied by severe anemia and the need for red blood cell (RBC) transfusions. Myelodysplastic syndromes (MDS) are characterized by cytopenia(s) and ineffective hematopoiesis, despite a hypercellular bone marrow. Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm characterized by reactive fibrosis of the bone marrow, accompanied by extramedullary hematopoiesis. Luspatercept, previously known as ACE-536, is a fusion protein that combines a modified activin receptor IIB (ActRIIB), a member of the transforming growth factor-ß (TGF-ß) superfamily, with the Fc domain of human immunoglobulin G (IgG1). It has shown efficacy in the treatment of anemia due to beta ß-thalassemia, MDS and PMF and recently gained approval by the Federal Drug Agency (FDA) and the European Medicines Agency (EMA) for transfusion-dependent (TD) patients with ß-thalassemia and very low to intermediate-risk patients with MDS with ringed sideroblasts who have failed to respond to, or are ineligible for, an erythropoiesis-stimulating agent. In this review, we describe the key pathways involved in normal hematopoiesis and the possible mechanism of action of luspatercept, present its development and data from the most recent clinical trials in ß-thalassemia, MDS and PMF, and discuss its potential use in the treatment of these hematological disorders.

MEG3 imprinted gene contribution in tumorigenesis.

Maternally expressed gene 3 (MEG3) is a maternally expressed imprinted gene representing a large noncoding RNA in which microRNAs (miRNAs) and small nucleolar RNAs are also hosted. It is capable of interacting with cyclic AMP, p53, murine double minute 2 (MDM2) and growth differentiation factor 15 (GDF15) playing a role in cell proliferation control. MEG3 expression is under epigenetic control, and aberrant CpG methylation has been observed in several types of cancer. Moreover, gene copy number loss has been reported as additional mechanism associated with tumorigenesis. MEG3 deletion seems to upregulate the paternally expressed genes and on the other hand downregulate the expression of downstream maternally expressed genes and tumor suppressor miRNAs, although there are conflicting data on the topic. MEG3 could represent a tumor suppressor gene located in chromosome 14q32 and its association with tumorigenesis is growing every day.

Polo-like kinase 2 (SNK/PLK2) is a novel epigenetically regulated gene in acute myeloid leukemia and myelodysplastic syndromes: genetic and epigenetic interactions.

Polo-like kinase 2 (SNK/PLK2), a transcriptional target for wild-type p53 and is hypermethylated in a high percentage of multiple myeloma and B cell lymphomas patients. Given these data, we sought to study the methylation status of the specific gene in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and to correlate it with clinical and genetic features. Using methylation-specific PCR MSP, we analyzed the methylation profile of 45 cases of AML and 43 cases of MDS. We also studied the distribution of MTHFR A1298C and MTHFR C677T polymorphisms and FLT3 mutations in AML patients and correlated the results with hypermethylation in the SNK/PLK2 CpG island. The SNK/PLK2 CpG island was hypermethylated in 68.9% and 88.4% of AML and MDS cases, respectively. Cases with hypermethylation had a trend towards more favorable overall survival (OS). There was no association between different MTHFR genotypes and susceptibility to develop AML. SNK/PLK2 hypermethylation combined with the MTHFR AA1298 genotype was associated with a tendency for a better OS. Similarly, patients with SNK/PLK2 hypermethylation combined with the MTHFR CT677 polymorphism had a better OS (HR = 0.34; p = 0.017). SNK/PLK2 methylation associated with unmutated FLT3 cases had a trend for better OS compared to patients with mutated FLT3 gene. SNK/PLK2 is a novel epigenetically regulated gene in AML and MDS, and methylation occurs at high frequency in both diseases. As such, SNK/PLK2 could represent a potential pathogenetic factor, although additional studies are necessary to verify its exact role in disease pathogenesis.

Enhancers and MYC interplay in hematopoiesis.

Transcription requires the fine interplay between enhancers and transcription factors. Enhancers are able to activate transcription of genes involved in normal cell biology, whereas aberrant enhancer activity leads to oncogenesis. MYC is a well-established proto-oncogene involved in half of human cancers amplifying the output of its targets. The crosstalk between MYC and enhancers is known for many years since the discovery of IgH enhancer juxtaposition with MYC in high-grade lymphomas. Here, we focus mainly in the enhancers surrounding MYC in the 8q24 locus. That region comprises several enhancers that associate with other transcription factors, transmembrane receptors, and fusion genes composing complex regulatory networks aberrantly expressed in almost all types of hematological malignancies. Understanding the nature of these interactions in normal blood cells and in leukemias/lymphomas will expand MYC targeting options in the armamentarium against hematological cancers.

Promoter hypermethylation of the MEG3 (DLK1/MEG3) imprinted gene in multiple myeloma.

BACKGROUND: Methylation represents the most studied epigenetic modification and results in the silencing of genes involved in various processes such as differentiation and cell-cycle regulation. MEG3 represents an imprinted gene maternally expressed in humans that encodes a nontranslated product. In this survey, we studied the methylation status of the specific gene in multiple myeloma (MM). PATIENTS AND METHODS: Twenty-one patients with MM (17 with immunoglobulin [Ig] G, 3 with IgA, and 1 with IgM) were evaluated using methylation-specific polymerase chain reaction (after DNA bisulphite modification). RESULTS: Promoter hypermethylation was observed in 12 (57.14%) bone marrow samples and in 9 of 14 (64.28%) available peripheral blood samples. A correlation with disease stage was also observed and also with the disease subtype (IgG, 64.7%; IgA, 0; IgM, 100%). CONCLUSION: We conclude that promoter hypermethylation of the differentially methylated region of the MEG3 imprinted gene is observed in patients with MM.

The crosstalk between long non-coding RNAs and PI3K in cancer.

Long non-coding RNAs (lncRNAs) are able to positively or negatively regulate other genes expression in cis or in trans. Their effect can be achieved through RNA-protein, RNA-DNA, or RNA-RNA interactions. They can recruit transcription factors and act as scaffolds or guides for chromatin-modifying enzymes. PI3K kinases transform external stimuli to intracellular signals regulating cell growth, differentiation, proliferation, survival, intracellular trafficking, cytoskeletal changes, cell migration and motility, and metabolism. PI3K is activated in cancer and affects several aspects of oncogenesis. LncRNAs and PI3K have been shown to be interconnected in several different cancer subtypes enhancing aberrant cell proliferation, epithelial-to-mesenchymal transition, migration and invasion, and also cancer cell metabolism. In this review, we have assembled recent data describing the interaction between lncRNAs and PI3K and the results of such interaction.

Delta-like homologue 1 and its role in the bone marrow niche and hematologic malignancies.

Delta-like homologue 1 (DLK1) is an imprinted gene, that acts as a Notch pathway antagonist. It is deregulated in solid and blood cancers, conferring malignant cells a cancer stem cell-like phenotype. DLK1 is important for normal hematopoiesis and for bone marrow homeostasis, because it directly affects the differentiation of mesenchymal stem cells into the nonhematopoietic components. It is possible that the resulting abnormal biology of mesenchymal stem cells promotes leukemic blasts survival providing also a sanctuary against chemotherapeutic agents. In this review, the effects of DLK1 on mesenchymal stem cells and the bone marrow microenvironment with perspectives are discussed.