CircRNAs Dysregulated in Juvenile Myelomonocytic Leukemia: CircMCTP1 Stands Out

Juvenile myelomonocytic leukemia (JMML), a rare myelodysplastic/myeloproliferative neoplasm of early childhood, is characterized by clonal growth of RAS signaling addicted stem cells. JMML subtypes are defined by specific RAS pathway mutations and display distinct gene, microRNA (miRNA) and long non-coding RNA expression profiles. Here we zoom in on circular RNAs (circRNAs), molecules that, when abnormally expressed, may participate in malignant deviation of cellular processes. CirComPara software was used to annotate and quantify circRNAs in RNA-seq data of a “discovery cohort” comprising 19 JMML patients and 3 healthy donors (HD). In an independent set of 12 JMML patients and 6 HD, expression of 27 circRNAs was analyzed by qRT-PCR. CircRNA-miRNA-gene networks were reconstructed using circRNA function prediction and gene expression data. We identified 119 circRNAs dysregulated in JMML and 59 genes showing an imbalance of the circular and linear products. Our data indicated also circRNA expression differences among molecular subgroups of JMML. Validation of a set of deregulated circRNAs in an independent cohort of JMML patients confirmed the down-regulation of circOXNAD1 and circATM, and a marked up-regulation of circLYN, circAFF2, and circMCTP1. A new finding in JMML links up-regulated circMCTP1 with known tumor suppressor miRNAs. This and other predicted interactions with miRNAs connect dysregulated circRNAs to regulatory networks. In conclusion, this study provides insight into the circRNAome of JMML and paves the path to elucidate new molecular disease mechanisms putting forward circMCTP1 up-regulation as a robust example.

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Ovary-derived circular RNAs profile analysis during the onset of puberty in gilts

BMC Genomics ◽

10.1186/s12864-021-07786-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Xiangchun Pan ◽

Wentao Gong ◽

Yingting He ◽

Nian Li ◽

Hao Zhang ◽

...

Keyword(s):

Alternative Splicing ◽

Signaling Pathway ◽

Gene Networks ◽

Profile Analysis ◽

Mirna Gene ◽

Mapk Signaling ◽

Circular Rnas ◽

Ras Signaling ◽

Female Reproduction ◽

Non Coding Rna

Abstract Background In mammals, the ovary is the essential system of female reproduction for the onset of puberty, and the abnormal puberty has negative outcomes on health. CircRNA is a non-coding RNA produced by non-canonical alternative splicing (AS). Several studies have reported that circRNA is involved in the gene regulation and plays an important role in some human diseases. However, the contribution of circRNA has received little known within the onset of puberty in ovary. Results Here, the profiles of ovarian circRNAs across pre-, in- and post-pubertal stages were established by RNA-sEq. In total, 972 circRNAs were identified, including 631 stage-specific circRNAs and 8 tissue-specific circRNAs. The biological functions of parental genes of circRNAs were enriched in steroid biosynthesis, autophagy-animal, MAPK signaling pathway, progesterone-mediated oocyte maturation and ras signaling pathway. Moreover, 5 circRNAs derived from 4 puberty-related genes (ESR1, JAK2, NF1 and ARNT) were found in this study. The A3SS events were the most alternative splicing, but IR events were likely to be arose in post-pubertal ovaries. Besides, the circRNA-miRNA-gene networks were explored for 10 differentially expressed circRNAs. Furthermore, the head-to-tail exon as well as the expressions of 10 circRNAs were validated by the divergent RT-qPCR and sanger sequencing. Conclusions In summary, the profiles of ovarian circRNAs were provided during pubertal transition in gilts, and these results provided useful information for the investigation on the onset of puberty at the ovarian-circRNAs-level in mammals.

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CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.2604.2604 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2604-2604

Author(s):

Y. Lucy Liu ◽

Priyangi A Malaviarachchi ◽

Shelly Y. Lensing ◽

Robert P. Castleberry ◽

Peter Dean Emanuel

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Camp Response Element ◽

Response Element ◽

Creb Protein ◽

Gm Csf ◽

Myelomonocytic Leukemia ◽

Normal Controls

Abstract Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.

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Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Blood ◽

10.1182/blood.v122.21.45.45 ◽

2013 ◽

Vol 122 (21) ◽

pp. 45-45 ◽

Cited By ~ 1

Author(s):

Hui Huang ◽

Daniel E. Bauer ◽

Mignon L. Loh ◽

Govind Bhagat ◽

Alan B. Cantor ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Phosphorylation ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Brdu Incorporation ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

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Bedside to bench in juvenile myelomonocytic leukemia: insights into leukemogenesis from a rare pediatric leukemia

Blood ◽

10.1182/blood-2014-03-300319 ◽

2014 ◽

Vol 124 (16) ◽

pp. 2487-2497 ◽

Cited By ~ 55

Author(s):

Tiffany Y. Chang ◽

Christopher C. Dvorak ◽

Mignon L. Loh

Keyword(s):

Myeloproliferative Neoplasm ◽

World Health ◽

Driver Mutations ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Cell Transplant ◽

Pediatric Leukemia ◽

Hematopoietic Stem ◽

Molecular Features ◽

Myelomonocytic Leukemia

AbstractJuvenile myelomonocytic leukemia (JMML) is a typically aggressive myeloid neoplasm of childhood that is clinically characterized by overproduction of monocytic cells that can infiltrate organs, including the spleen, liver, gastrointestinal tract, and lung. JMML is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) by the World Health Organization and also shares some clinical and molecular features with chronic myelomonocytic leukemia, a similar disease in adults. Although the current standard of care for patients with JMML relies on allogeneic hematopoietic stem cell transplant, relapse is the most frequent cause of treatment failure. Tremendous progress has been made in defining the genomic landscape of JMML. Insights from cancer predisposition syndromes have led to the discovery of nearly 90% of driver mutations in JMML, all of which thus far converge on the Ras signaling pathway. This has improved our ability to accurately diagnose patients, develop molecular markers to measure disease burden, and choose therapeutic agents to test in clinical trials. This review emphasizes recent advances in the field, including mapping of the genomic and epigenome landscape, insights from new and existing disease models, targeted therapeutics, and future directions.

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A simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia

Blood Advances ◽

10.1182/bloodadvances.2021005080 ◽

2021 ◽

Author(s):

Hironobu Kitazawa ◽

Yusuke Okuno ◽

Hideki Muramatsu ◽

Kosuke Aoki ◽

Norihiro Murakami ◽

...

Keyword(s):

Myeloproliferative Neoplasm ◽

Enzyme Analysis ◽

Support Vector ◽

Juvenile Myelomonocytic Leukemia ◽

Restriction Enzyme Analysis ◽

Clinical Test ◽

Consensus Clustering ◽

International Consensus ◽

Consensus Definition ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following three groups: high methylation (HM), intermediate methylation (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML have been analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n=99) using the DREAM data has identified HM and LM subgroups (HM_DREAM, n=35; LM_DREAM; n=64). Of the 98 cases that could be compared with the international consensus classification, 90 cases of HM (n=30) and LM (n=60) had 100% concordance with the DREAM clustering results. For the remaining eight cases classified as the IM group, four cases were classified into the HM_DREAM group and four cases into the LM_DREAM group. A machine-learning classifier has been successfully constructed using a Support Vector Machine (SVM), which divided the validation cohort (n=38) into HM (HM_SVM; n=18) and LM (LM_SVM; n=20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, a robust methylation test has been developed using the DREAM analysis for patients with JMML. This simple and straightforward test can be easily incorporated in diagnosis to generate a methylation classification for patients so that they can receive risk-adapted treatment in the context of future clinical trials.

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Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment

Leukemia ◽

10.1038/s41375-019-0662-y ◽

2019 ◽

Vol 34 (6) ◽

pp. 1658-1668

Author(s):

Aurélie Caye ◽

Kevin Rouault-Pierre ◽

Marion Strullu ◽

Elodie Lainey ◽

Ander Abarrategi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Integrated Analysis ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Activating Mutations ◽

Colony Forming Assay ◽

Myelomonocytic Leukemia

AbstractJuvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

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Rapamycin -- a Potential Mechanistically Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2378.2378 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2378-2378

Author(s):

Y. Lucy Liu ◽

Robert P. Castleberry ◽

Peter Dean Emanuel

Keyword(s):

Philadelphia Chromosome ◽

Disease Free Survival ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Sensitivity Testing ◽

Blood Concentrations ◽

Patient Sample ◽

Gm Csf ◽

Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD) of infancy and early childhood. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. The pathogenesis of JMML has been clearly and definitively linked to dysregulated signal transduction through the RAS signaling pathway. A series of studies conducted over the last decade have shown that mutations or other abnormalities in RAS, NF1, and PTPN11, are potentially responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult. There is no effective therapy for JMML. Only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28–55%), with 5-year disease-free survival rates of 25-40%. Rapamycin is a macrolide antibiotic with established clinical applications in organ transplantation. Recent studies have proved that the Mammalian Target of Rapamycin (mTOR) plays an important role in cytokine receptor signaling and induction of apoptosis. Numerous studies have suggested that mTOR functions as a nutritional checkpoint and is connected to energy sensing through AMP-dependent kinase (AMPK) which senses the AMP: ATP ratio in cells. Its function is regulated by the RAS/PI3-kinase pathway. In searching for novel mechanistically-targeted reagents to treat JMML, we conducted an in vitro pilot study with JMML cells. The CFU-GM formation assay was used to test the therapeutic sensitivity of rapamycin to JMML cells. Mononuclear cells (MNCs) from peripheral blood of 9 JMML patients were collected and plated on 0.3% agar medium with rapamycin at a concentration of 1-8nM(0.91-7.28μg/L) and carrier (DMSO). Greater than 50% inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion, with the exception of one patient sample which had colonies resistant to rapamycin. The effective concentrations in our cultures are equivalent to the safe and tolerable whole blood concentrations achieved in organ transplant patients in clinical settings (5-30μg/L). Our data suggests that rapamycin may be considered as a potentially safe and effective reagent to treat JMML, but that in vitro sensitivity testing might be recommended since one patient sample demonstrated complete resistance to rapamycin in vitro. Further studies are ongoing to explore the mechanism of rapamycin in inhibiting hypersensitivity of JMML cells to GM-CSF.

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Analyzing the miRNA-Gene Networks to Mine the Important miRNAs under Skin of Human and Mouse

BioMed Research International ◽

10.1155/2016/5469371 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Jianghong Wu ◽

Husile Gong ◽

Yongsheng Bai ◽

Wenguang Zhang

Keyword(s):

Gene Networks ◽

Regulatory Networks ◽

Mirna Gene ◽

Genetic Regulatory Networks ◽

Host Immunity ◽

Genetic Networks ◽

Target Sites ◽

Enormous Number ◽

Human And Mouse

Genetic networks provide new mechanistic insights into the diversity of species morphology. In this study, we have integrated the MGI, GEO, and miRNA database to analyze the genetic regulatory networks under morphology difference of integument of humans and mice. We found that the gene expression network in the skin is highly divergent between human and mouse. The GO term of secretion was highly enriched, and this category was specific in human compared to mouse. These secretion genes might be involved in eccrine system evolution in human. In addition, total 62,637 miRNA binding target sites were predicted in human integument genes (IGs), while 26,280 miRNA binding target sites were predicted in mouse IGs. The interactions between miRNAs and IGs in human are more complex than those in mouse. Furthermore,hsa-miR-548,mmu-miR-466, andmmu-miR-467have an enormous number of targets on IGs, which both have the role of inhibition of host immunity response. The pattern of distribution on the chromosome of these three miRNAs families is very different. The interaction of miRNA/IGs has added the new dimension in traditional gene regulation networks of skin. Our results are generating new insights into the gene networks basis of skin difference between human and mouse.

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Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia

Current Drug Targets ◽

10.2174/138945007780830773 ◽

2007 ◽

Vol 8 (6) ◽

pp. 715-725 ◽

Cited By ~ 30

Author(s):

Christian Flotho ◽

Christian Kratz ◽

Charlotte Niemeyer

Keyword(s):

Signaling Pathways ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Myelomonocytic Leukemia

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miRGTF-net: Integrative miRNA-gene-TF network analysis reveals key drivers of breast cancer recurrence

PLoS ONE ◽

10.1371/journal.pone.0249424 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249424

Author(s):

Stepan Nersisyan ◽

Alexei Galatenko ◽

Vladimir Galatenko ◽

Maxim Shkurnikov ◽

Alexander Tonevitsky

Keyword(s):

Breast Cancer ◽

Regulatory Networks ◽

Estrogen Receptor Alpha ◽

Expression Profiles ◽

Topological Analysis ◽

Mirna Gene ◽

Cancer Recurrence ◽

The Cancer Genome Atlas ◽

Sequencing Data ◽

Source Codes

Analysis of regulatory networks is a powerful framework for identification and quantification of intracellular interactions. We introduce miRGTF-net, a novel tool for construction of miRNA-gene-TF networks. We consider multiple transcriptional and post-transcriptional interaction types, including regulation of gene and miRNA expression by transcription factors, gene silencing by miRNAs, and co-expression of host genes with their intronic miRNAs. The underlying algorithm uses information on experimentally validated interactions as well as integrative miRNA/mRNA expression profiles in a given set of samples. The latter ensures simultaneous tissue-specificity and biological validity of interactions. We applied miRGTF-net to paired miRNA/mRNA-sequencing data of breast cancer samples from The Cancer Genome Atlas (TCGA). Together with topological analysis of the constructed network we showed that considered players can form reliable prognostic gene signatures for ER-positive breast cancer. A number of signatures demonstrated remarkably high accuracy on transcriptomic data obtained by both microarrays and RNA sequencing from several independent patient cohorts. Furthermore, an essential part of prognostic genes were identified as direct targets of transcription factor E2F1. The putative interplay between estrogen receptor alpha and E2F1 was suggested as a potential recurrence factor in patients treated with tamoxifen. Source codes of miRGTF-net are available at GitHub (https://github.com/s-a-nersisyan/miRGTF-net).

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