Astroblastoma is a rare glial neoplasm that occurs mostly in the cerebral hemisphere of children, adolescents and young adults. Although astroblastic perivascular pseudorosettes are unique histopathology of this neoplasm, diagnosis is usually challenging. Recently, it was discovered that the meningioma 1 gene (MN1)-altered pediatric central nervous system high-grade neuroepithelial tumors are actually astroblastomas. This case report presents a rare brainstem astroblastoma, with an unusual immunoprofile: negative for glial fibrillary acidic protein and oligodendrocyte transcription factor 2, but with a robust expression of pancytokeratin and epithelial membrane antigen. The diagnosis was confirmed based on the detection of MN1 rearrangement in a fluorescence in situ hybridization study, in addition to typical histopathology. Here we discuss the diagnostic pitfalls and unclear grading system along with a literature review.

In intermediate-risk cytogenetic acute myeloid leukemia (IRC-AML) patients, novel biomarkers to guide post-remission therapy are needed. We analyzed with high-density arrays 40 IRC-AML patients who received a non-allogeneic hematopoietic stem-cell transplantation-based post-remission therapy, and identified a signature that correlated with early relapse. Subsequently, we analyzed selected 187 genes in 49 additional IRC-AML patients by RT-PCR. BAALC, MN1, SPARC and HOPX overexpression correlated to refractoriness. BAALC or ALDH2 overexpression correlated to shorter overall survival (OS) (5-year OS: 33 ± 8.6% vs. 73.7 ± 10.1%, p = .006; 32 ± 9.3% vs. 66.4 ± 9.7%, p = .016), whereas GPR44 or TP53INP1 overexpression correlated to longer survival (5-year OS: 66.7 ± 10.3% vs. 35.4 ± 9.1%, p = .04; 58.3 ± 8.2% vs. 23.1 ± 11.7%, p = .029). A risk-score combining these four genes expression distinguished low-risk and high-risk patients (5-year OS: 79 ± 9% vs. 30 ± 8%, respectively; p = .001) in our cohort and in an independent set of patients from a public repository. Our 4-gene signature may add prognostic information and guide post-remission treatment in IRC-AML patients.

Astroblastomas are rare brain tumours which predominate in children and young adults, and have a controversial claim as a distinct entity, with no established WHO grade. Reports suggest a better outcome than high grade gliomas, though they frequently recur. Recently, they have been described to overlap with a newly-discovered group of tumours described as'high grade neuroepithelial tumour with MN1 alteration' (CNS HGNET-MN1), defined by global methylation patterns and strongly associated with gene fusions targeting MN1. We have studied a unique case of astroblastoma arising in a 6 year-old girl, with multiple recurrences over a period of 10 years, with the pathognomonic MN1:BEND2 fusion. Exome sequencing allowed for a phylogenetic reconstruction of tumour evolution, which when integrated with clinical, pathological and radiological data provide for a detailed understanding of disease progression, with initial treatment driving tumour dissemination along four distinct trajectories. Infiltration of distant sites was associated with a later genome doubling, whilst there was evidence of convergent evolution of different lesions acquiring distinct alterations targeting NF-κB. These data represent an unusual opportunity to understand the evolutionary history of a highly recurrent childhood brain tumour, and provide novel therapeutic targets for astroblastoma/CNS HGNET-MN1.

t(12;22)(p13;q12) is a rare but recurrent chromosomal abnormality involving the ETS transcription factor ETV6 and meningioma 1 (MN1) genes. In this study, we analyzed the clinical, cytogenetic, and molecular features of five new patients with the t(12;22)/MN1-EVT6 who presented with acute myeloid leukemia or chronic myelomonocytic leukemia. We subsequently reviewed the literature and identified seven additional cases reported with t(12;22)/MN1-EVT6. Our data suggest that neoplasms carrying the t(12;22)/MN1-ETV6, although rare, can commonly present as myeloid neoplasms at the initial diagnosis, including acute myeloid leukemia (n = 8), myelodysplastic syndrome (n = 2), and myelodysplastic/myeloproliferative neoplasms (n = 2). There were five men and seven women with a median age of 43 years (range, 15-63 years) at initial diagnosis. Cytogenetics revealed t(12;22) as the sole abnormality in five patients, with the remaining seven patients harboring additional chromosomal aberrations. Of the five patients who received known therapy regimens, all of them had poor response to the idarubicin/mitoxantrone + cytarabine regimen. Of the seven patients with follow-up information, six patients died with a median overall survival time of only 5 months (range, 1-12 months) after the emergence of t(12;22). In summary, patients with t(12;22) are frequently associated with myeloid neoplasms, poor response to chemotherapy, and inferior outcome.

Astroblastoma is a rare and controversial glioma with variable clinical behavior. The diagnosis currently rests on histologic findings of a circumscribed glioma with astroblastomatous pseudorosettes and vascular hyalinization. Immunohistochemical studies have suggested different oncogenic drivers, such as BRAF p.V600E, but very few cases have been studied using genome-wide methodologies. Recent genomic profiling identified a subset of CNS embryonal tumors with astroblastoma-like morphology that harbored MN1 gene fusions, termed "CNS high-grade neuroepithelial tumors with MN1 alteration" (CNS-HGNET-MN1). To further characterize the genetic alterations that drive astroblastomas, we performed targeted next-generation sequencing (NGS) of 500 cancer-associated genes in a series of eight cases. We correlated these findings with break-apart fluorescence in situ hybridization (FISH) analysis of the MN1 locus and genome-wide DNA methylation profiling. Four cases showed MN1 alteration by FISH, including two pediatric cases that lacked other pathogenic alterations, and two adult cases that harbored other cancer-associated gene mutations or copy number alterations (eg, CDKN2A/B homozygous deletion, TP53, ATM and TERT promoter mutations). Three of these cases grouped with the CNS-HGNET-MN1 entity by methylation profiling. Two of four MN1 intact cases by FISH showed genetic features of either anaplastic pleomorphic xanthoastrocytoma (BRAF p.V600E mutation, CDKN2A/B homozygous deletion and TERT promoter mutation) or IDH-wildtype glioblastoma (trisomy 7, monosomy 10, CDK4 amplification and TP53, NRAS and TERT promoter mutations) and these cases had an aggressive clinical course. Two clinically indolent cases remained unclassifiable despite multimodal molecular analysis. We conclude that astroblastoma histology is not specific for any entity including CNS-HGNET-MN1, and that additional genetic characterization should be considered for astroblastomas, as a number of these tumors likely contain a methylation profile or genetic alterations that suggest classification as other tumor entities. Our heterogeneous molecular findings help to explain the clinical unpredictability of astroblastoma.

In acute myeloid leukemia (AML), specific genomic aberrations induce aberrant methylation, thus directly influencing the transcriptional programing of leukemic cells. Therefore, therapies targeting epigenetic processes are advocated as a promising therapeutic tool for AML treatment. However, to develop new therapies, a comprehensive understanding of the mechanism(s) driving the epigenetic changes as a result of acquired genetic abnormalities is necessary. This understanding is still lacking. In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. Six differentially methylated genomic regions within two genes, discriminating inv(16)(p13;q22) from non-inv(16) pediatric AML samples, were identified. All six regions had a hypomethylated phenotype in inv(16) AML samples, and this was most prominent at the regions encompassing the meningioma (disrupted in balanced translocation) 1 (MN1) oncogene. MN1 expression primarily correlated with the methylation level of the 3' end of the MN1 exon-1 locus. Decitabine treatment of different cell lines showed that induced loss of methylation at the MN1 locus can result in an increase of MN1 expression, indicating that MN1 expression is coregulated by DNA methylation. To investigate this methylation-associated mechanism, we determined the expression of DNA methyltransferases in inv(16) AML. We found that DNMT3B expression was significantly lower in inv(16) samples. Furthermore, DNMT3B expression correlated negatively with MN1 expression in pediatric AML samples. Importantly, depletion of DNMT3B impaired remethylation efficiency of the MN1 exon-1 locus in AML cells after decitabine exposure. These findings identify DNMT3B as an important coregulator of MN1 methylation. Taken together, this study shows that the methylation level of the MN1 exon-1 locus regulates MN1 expression levels in inv(16) pediatric AML. This methylation level is dependent on DNMT3B, thus suggesting a role for DNMT3B in leukemogenesis in inv(16) AML, through MN1 methylation regulation.

BACKGROUND: Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a "physiological" dose of mutant ASXL1 remain unexplored.
METHODS: We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system.
RESULTS: Heterozygotes (Asxl1
CONCLUSIONS: We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies.

BACKGROUND: Molecular heterogeneity accounts for the variable and often poor prognosis in acute myeloid leukemia (AML). The current risk stratification strategy in clinical practice is limited to karyotyping and limited molecular studies screening for genetic mutations such as FLT-3 and NPM1. There is opportunity to identify further molecular prognostic markers, which may also lay the groundwork for the development of novel targeted therapies. Complex molecular technologies require transition into widely available laboratory platforms, for better integration into routine clinical practice.
METHOD: In a defined subset (MYC/BCL2 or MYC/BCL2) of AML patients (n=20), we examined expression signature of several genes (n=12) of established prognostic value in AML. RNA expression and MYC/BCL2 protein pattern was correlated with 3 cytogenetic risk groups and overall survival.
RESULTS: K-means++ unsupervised clustering defined 2 distinct groups with high and low transcript levels of BAALC/MN1/MLLT11/EVI1/SOCS2 genes (>2.5-fold difference; P<0.001). This mRNA signature trended with higher prevalence of MYC/BCL2 coexpression (P<0.057) and poor overall survival (P<0.036), but did not correlate with conventional cytogenetic risk groups (P<0.084).
CONCLUSIONS: This pilot study provides useful data, which may help further refine the prognostic scheme of AML patients outside conventional cytogenetic risk groups. It also presents some biological rationale for future studies to explore the use of novel agents targeting MYC and/or BCL2 genes in combination with conventional chemotherapy protocols for AML.

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.

OBJECTIVE: Multiple genetic alterations with prognostic significance have been discovered in acute myeloid leukemia (AML). We studied the expression level of two genes, Meningioma1 (MN1) and Phosphatase and Tensin homolog (PTEN) to determine their expression in AML patients and their role as prognostic markers.
METHODS: The study included 50 cytogenetic normal de novo AML cases and 10 controls, Their level was detected by Real time Reverse Transcription-Polymerase Chain Reaction.
RESULT: Relative mRNA expression of MN1 was significantly higher (p value < 0.001) and PTEN expression was significantly lower (p value = 0.002). No correlation was found between neither MN1 nor PTEN mRNA expression and overall survival (p value = 0.212 and 0.310) respectively.
CONCLUSION: Although our study suggests a role for MN1 gene and PTEN genes in AML, we could not recommend their use as routine diagnostic and prognostic markers for AML in Egyptian population.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.

OBJECTIVE: To identify a gene-based signature as a novel prognostic model in lower grade gliomas.
RESULTS: A gene signature developed from HOXA7, SLC2A4RG and MN1 could segregate patients into low and high risk score groups with different overall survival (OS), and was validated in TCGA RNA-seq and GSE16011 mRNA array datasets. Receiver operating characteristic (ROC) was performed to show that the three-gene signature was more sensitive and specific than histology, grade, age, IDH1 mutation and 1p/19q co-deletion. Gene Set Enrichment Analysis (GSEA) and GO analysis showed high-risk samples were associated with tumor associated macrophages (TAMs) and highly invasive phenotypes. Moreover, HOXA7-siRNA inhibited migration and invasion in vitro, and downregulated MMP9 at the protein level in U251 glioma cells.
METHODS: A cohort of 164 glioma specimens from the Chinese Glioma Genome Atlas (CGGA) array database were assessed as the training group. TCGA RNA-seq and GSE16011 mRNA array datasets were used for validation. Regression analyses and linear risk score assessment were performed for the identification of the three-gene signature comprising HOXA7, SLC2A4RG and MN1.
CO NCLUSIONS: We established a three-gene signature for lower grade gliomas, which could independently predict overall survival (OS) of lower grade glioma patients with higher sensitivity and specificity compared with other clinical characteristics. These findings indicate that the three-gene signature is a new prognostic model that could provide improved OS prediction and accurate therapies for lower grade glioma patients.

Umbilical cord blood is a valuable source of hematopoietic stem cells. While cytokine stimulation can induce ex vivo hematopoietic cell proliferation, attempts have been made to use epigenetic-modifying agents to facilitate stem cell expansion through the modulation of cellular epigenetic status. However, the potential global effect of these modifying agents on epigenome raises concerns about the functional normality of the expanded cells. We studied the ex vivo expansion of cord blood hematopoietic stem and progenitor cells (HSPCs) by histone deacetylase (HDAC) inhibitors, trichostatin A and valproic acid. Treatment with HDAC inhibitors resulted in mild expansion of the total hematopoietic cell number when compared with cytokine stimulated sample. Nevertheless, we observed 20-30-fold expansion of the CD34

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Meningiomas are frequent central nervous system tumors. Although most meningiomas are benign (WHO grade I) and curable by surgery, WHO grade II and III tumors remain therapeutically challenging due to frequent recurrence. Interestingly, relapse also occurs in some WHO grade I meningiomas. Hence, we investigated the transcriptional features defining aggressive (recurrent, malignantly progressing or WHO grade III) meningiomas in 144 cases. Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M) in addition to their WHO grade. Unsupervised transcriptomic analysis in 62 meningiomas revealed transcriptional profiles lining up according to WHO grade and clinical subgroup. Notably aggressive subgroups (R+M tumors and WHO grade III) shared a large set of differentially expressed genes (n=332; p<0.01, FC>1.25). In an independent multicenter validation set (n=82), differential expression of 10 genes between WHO grades was confirmed. Additionally, among WHO grade I tumors differential expression between NR and aggressive R+M tumors was affirmed for PTTG1, AURKB, ECT2, UBE2C and PRC1, while MN1 and LEPR discriminated between NR and R+M WHO grade II tumors. Univariate survival analysis revealed a significant association with progression-free survival for PTTG1, LEPR, MN1, ECT2, PRC1, COX10, UBE2C expression, while multivariate analysis identified a prediction for PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. Finally, stainings of PTTG1 and LEPR confirmed malignancy-associated protein expression changes. In conclusion, based on the so far largest study sample of WHO grade III and recurrent meningiomas we report a comprehensive transcriptional landscape and two prognostic markers.

In double-stranded miRNA/miRNA* duplexes, one of the strands represents an active miRNA, whereas another, known as a passenger strand (miRNA*), is typically degraded. MiR-9* is not detectable in normal myeloid cells. Here we show that miR-9* is expressed in 59% of acute myeloid leukemia (AML) cases and we investigate its clinical impact in 567 adults with de novo AML (age⩽60 years). AML cases with detectable miR-9* included a lower percentage of cases with favorable risk (P<0.001) as compared with those with no detectable miR-9*. High levels of miR-9* expression independently predicted for higher complete remission (odds ratio=1.28, P=0.013) and better event-free survival (EFS) (hazard ratio (HR)=0.86, P=0.001), relapse-free survival (RFS) (HR=0.84, P=0.008) and overall survival (OS) (HR=0.86, P=0.002). Among the subgroup of adverse risk patients, high miR-9* expressers had strikingly longer median survival than low miR-9* expressers (EFS: 16 vs 5 months, P=0.020; RFS: 12 vs 4, P=0.060; OS: 23 vs 8, P=0.021). Comparative transcriptome analysis suggests that miR-9* regulates genes involved in leukemogenesis, for example, MN1 and MLLT3. This is the first report showing that an miRNA* has prognostic value in AML.

Immune checkpoint molecules are highly relevant as potential prognostic markers and therapeutic targets in malignant diseases. HVEM belongs to the TNF receptor family and provides stimulatory as well as inhibitory signals depending on the ligand. Abnormal HVEM expression has been described in various malignancies, but the role in AML is unknown. Here we report extensive data on HVEM surface protein expression analyzed by flow cytometry on bone marrow leukemic cells of 169 AML patients at diagnosis. An independent cohort of 512 AML patients was analyzed for HVEM mRNA expression in bone marrow samples by Affymetrix microarrays. Consistently for both cohorts and methods, we show that HVEM was differentially expressed and that expression levels were associated with defined genetic markers. HVEM expression was lower in cases with FLT3-ITD (p = 0.001, p < 0.001), with mutations in NPM1 (p = 0.001, p < 0.001) or with the combination of NPM1 mutation and FLT3 wild type (p = 0.049, p = 0.050), while a biallelic mutation in CEBPA correlated positively with higher HVEM expression (p = 0.015, p < 0.001). In a differential gene expression analysis, we found 13 genes including HOXA9, MEIS1 and MN1 that were closely associated with HVEM expression. Besides, four gene sets closely linked to immunity were enriched in HVEM (high) samples. Finally, high expression of HVEM was associated with a trend toward longer relapse-free survival. The results of this study provide new information on the potential significance of HVEM in AML.

The MN1 (Meningioma 1) gene is overexpressed in certain subtypes of acute myeloid leukemia (AML) and high levels of MN1 expression in mouse bone marrow cells results in myeloid leukemia. We showed that compared with control bone marrow (BM) MN1 expression was increased (2-fold or more) in 29 out of 73 (40%) pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient BM. Additional analysis of MN1 expression in sub-groups within our cohort carrying different chromosome translocations showed that carriers of the good prognostic marker t(12;21)(TEL-AML1) (n=27) expressed significantly more MN1 than both healthy controls (n=9) (P=0.02) and the group carrying the t(9;22)(BCR-ABL) (n=9) (P=0.001). In addition, AML1 expression was also upregulated in 31 out of 45 (68%) B-ALL patient BM compared with control and there was a significant correlation between MN1 and AML1 expression (r=0.3552, P=0.0167). Retroviral MN1 overexpression increased the colony forming activity of mouse Pro-B/Pre-B cells in vitro. Our results suggest that deregulated MN1 expression contributes to the pathogenesis of pediatric B-ALL. Further investigation into the clinical and biological significance of elevated MN1 expression in TEL-AML1(positive) leukemia might provide insight into additional molecular mechanisms contributing to B-ALL and may lead to improved treatment options for patients.

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

We report on seven novel patients with a submicroscopic 22q12 deletion. The common phenotype constitutes a contiguous gene deletion syndrome on chromosome 22q12.1q12.2, featuring NF2-related schwannoma of the vestibular nerve, corpus callosum agenesis and palatal defects. Combining our results with the literature, eight patients are recorded with palatal defects in association with haploinsufficiency of 22q12.1, including the MN1 gene. These observations, together with the mouse expression data and the finding of craniofacial malformations including cleft palate in a Mn1-knockout mouse model, suggest that this gene is a candidate gene for cleft palate in humans.

BACKGROUND: Meningiomas are tumors originating from the membranous layers surrounding the central nervous system, and are generally regarded as "benign" tumors of the brain. Malignant meningiomas are rare and are typically associated with a higher risk of local tumor recurrence and a poorer prognosis (median survival time <2 years). Previous genome-wide association studies and exome sequencing studies have identified genes that play a role in susceptibility to meningiomas, but these studies did not focus specifically on malignant tumors.
METHODS: We performed exome sequencing on five malignant meningiomas on the Illumina HiSeq2000 platform using Agilent SureSelect Human All Exon kits. We used wANNOVAR web server to annotate and prioritize variants, identified candidate genes with recurrent mutations, and validated selected mutations by Sanger sequencing. We next designed custom NimbleGen targeted region arrays on five candidate genes, and sequenced four additional malignant meningiomas.
RESULTS: From exome sequencing data, we identified several frequently mutated genes including NF2, MN1, ARID1B, SEMA4D, and MUC2, with private mutations in tumors. We sequenced these genes in four additional samples and identified potential driver mutations in NF2 (neurofibromatosis type 2) and MN1 (meningioma 1).
CONCLUSIONS: We confirmed that mutations in NF2 may play a role in progression of meningiomas, and nominated MN1 as a candidate gene for malignant transformation of meningiomas. Our sample size is limited by the extreme rarity of malignant meningiomas, but our study represents one of the first sequencing studies focusing on the malignant subtype.

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. Meningioma 1 (MN1) is overexpressed in AML patients and confers resistance to all-trans retinoic acid-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like interferon-response factor (Irf) 8 and Ccl9. As MN1 is a cofactor of MEIS1 and retinoic acid receptor alpha (RARA), we compared chromatin occupancy between these genes. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of murine and human leukemias in vivo. Our data show that MN1 prevents activation of the immune response pathway, and suggest restoration of IRF8 signaling as therapeutic target in AML.

BACKGROUND: Multiple genetic alterations that affect the process of acute myeloid leukemia (AML) have been discovered, and more evidence also indicates that aberrant splicing plays an important role in cancer.
MATERIALS AND METHODS: We present a RNA-Seq profiling of an AML patient with complete remission after treatment, to analyze the aberrant splicing of genes during treatment. We sequenced 3.97 and 3.32 Gbp clean data of the AML and remission sample, respectively. Firstly, by analyzing biomarkers associated with AML, to assist normal clinical tests, we confirmed that the patient was anormal karyo type, with NPM1 and IDH2 mutations and deregulation patterns of related genes, such as BAALC, ERG, MN1 and HOX family. Then, we performed alternative splicing detection of the AML and remission sample.
RESULTS: We detected 91 differentially splicing events in 68 differentially splicing genes (DSGs) by mixture of isoforms (MISO). Considering Psi values (Ψ) and confidence intervals, 25 differentially expressed isoforms were identified as more confident isoforms, which were associated with RNA processing, cellular macromolecule catabolic process and DNA binding according to GO enrichment analysis. An exon2-skipping event in oncogene FOS (FBJ murine osteosarcoma viral oncogene homolog) were detected and validated in this study. FOS has a critical function in regulating cell proliferation, differentiation and transformation. The exon2-skipping isoform of FOS was increased significantly after treatment.
CONCLUSIONS: All the data and information of RNA-Seq provides highly accurate and comprehensive supplements to conventional clinical tests of AML. Moreover, the splicing aberrations would be another source for biomarker and even therapeutic target discovery. More information of splicing may also assist the better understanding of leukemogenesis.

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

OBJECTIVE: Mutations in the gene encoding isocitrate dehydrogenease 1 (IDH1) occur in various hematopoietic tumors including acute myeloid leukemia (AML), myeloproliferative neoplasms and myelodysplastic syndromes. IDH1 mutations are significant in both diagnosis and prognosis of these conditions. In the present study we determined the prevalence and clinical significance of IDH1 mutations in 349 samples from newly diagnosed AML patients.
RESULTS: Of the 349 AML patient specimens analyzed, 35 (10.03%) were found to have IDH1 mutations including 4 IDH1 R132 mutations and 31 non-R132 mutations. IDH1 non-R132 mutations were largely concentrated within AML-M1 (35.72%, p<0.01). We identified five IDH1 mutations that were novel to AML: (1) c.299 G>A, p.R100Q; (2) c.311G>T, p.G104V; (3) c.322T>C, p.F108L; (4) c.356G>A, p.R119Q; and (5) c.388A>G, p.I130V. In addition, we identified three IDH1 mutations that were previously described in AML. The frequency of IDH1 mutations in AML patients with normal karyotype was 9.9%. IDH1 non-R132 mutations were concurrent with mutations in FLT3-ITD (p<0.01), CEBPA (p<0.01), and NRAS (p<0.01), as well as the overexpression of MN1 (p<0.01) and WT1(p<0.01). The overall survival (OS) in the patients with IDH1 non-R132 mutations compared to patients without IDH1 mutations don't reach statistically significance (median 521 days vs median: not reached; n.s.).
CONCLUSION: IDH1 non-R132 mutations occurred frequently in newly diagnosed adult Chinese AML patients, and these mutations were associated with genetic alterations. The OS was not influenced by IDH1 non-R132 mutations in the present study.

Emerging data demonstrate important roles for the TYRO3/AXL/MERTK receptor tyrosine kinase (TAM RTK) family in diverse cancers. We investigated the prognostic relevance of GAS6 expression, encoding the common TAM RTK ligand, in 270 adults (n=71 aged<60 years; n=199 aged ⩾60 years) with de novo cytogenetically normal acute myeloid leukemia (CN-AML). Patients expressing GAS6 (GAS6+), especially those aged ⩾60 years, more often failed to achieve a complete remission (CR). In all patients, GAS6+ patients had shorter disease-free (DFS) and overall (OS) survival than patients without GAS6 expression (GAS6-). After adjusting for other prognostic markers, GAS6+ predicted CR failure (P=0.02), shorter DFS (P=0.004) and OS (P=0.04). To gain further biological insights, we derived a GAS6-associated gene-expression signature (P<0.001) that in GAS6+ patients included overexpressed BAALC and MN1, known to confer adverse prognosis in CN-AML, and overexpressed CXCL12, encoding stromal cell-derived factor, and its receptor genes, chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. This study reports for the first time that GAS6 expression is an adverse prognostic marker in CN-AML. Although GAS6 decoy receptors are not yet available in the clinic for GAS6+ CN-AML therapy, potential alternative therapies targeting GAS6+-associated pathways, for example, CXCR4 antagonists, may be considered for GAS6+ patients to sensitize them to chemotherapy.