Meningioma 1 Research Articles

PF208 MN1 IS INDISPENSABLE FOR MLL‐REARRANGED ACUTE MYELOID LEUKEMIA

Background:Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL‐r) are one of the most frequent chromosomal aberrations in AML. In addition, Meningioma 1 (MN1) expression serves as a prognostically important factor in AML patients being associated with poor prognosis. Overexpression of MN1 induces AML in mice and causes resistance to cytarabine and all‐trans‐retinoic acid (ATRA) induced cytotoxic/differentiation effects.Aims:To evaluate the function of MN1, a cofactor of HOXA9 and MEIS1, in human and murine AML cells by CRISPR‐Cas9 mediated deletion of MN1.Methods:MN1 was deleted in murine cells transformed by MLL‐AF9/HOXA9/HOXA9MEIS1/E2A‐HLF and 10 human leukemia cell lines (THP‐1, MV4–11, NB4, OCI‐AML2, OCI‐AML3, U937, K562, Kasumi‐1, HL‐60 and HEL). Murine and human leukemic models with engineered deletion of MN1 were studied as single cell clones and were characterized in vitro and in vivo.Results:Out of 96 to 288 CRISPR‐Cas9‐MN1‐transduced single sorted clones per cell line, 30 percent of them proliferated and only 0 to 8.9% of them were found to have a MN1‐deletion. Interestingly, only 1 MN1‐deleted clone was obtained from four different murine cell lines with HOX‐dependent oncogene expression suggesting that MN1 loss impairs clonogenicity of MLL‐r and HOXA9‐transformed cells. Loss of MN1 reduced in vitro proliferation and impaired colony‐forming potential up to 22‐fold in MLL‐r murine and human leukemia cells. MN1 deletion also suppressed cell cycle, promoted apoptosis and induced differentiation in MLL positive leukemia cells. MN1 was critically required for in vivo leukemogenicity of MLL‐AF9 murine leukemic cells. Mice transplanted with MLL‐AF9/Mn1null cells had <1% engraftment in all ten mice, whereas mice transplanted with MLL‐AF9/Mn1 wt cells showed >90% engraftment at 4 weeks with high WBC counts. Loss of Mn1 significantly prolonged survival of mice transplanted with MLL‐AF9/Mn1null cells as compared to mice transplanted with MLL‐AF9/Mn1 wt cells (median survival of 35 days). Additionally, we transplanted MN1 wildtype or MN1null cells of THP‐1 and MV‐4‐11 subcutaneously in NOD‐SCID mice and monitored tumor formation. Deletion of MN1 led to significantly reduced tumor volumes in both THP‐1 and MV‐4‐11 cells (MN1null) as compared to their MN1 wild type control. ChIP sequencing and gene expression analysis suggested that MN1 is required in MLL‐r leukemias to maintain expression of the distal Hoxa cluster and Meis1 and as a cofactor of the Hoxa9/Meis1 transcriptional complex and their target genes essential for leukemogenic activity. Also, we treated CD34+ cells from healthy donors and MLL‐AF9 positive AML cells with control/anti‐MN1 siRNA. MN1 expression was found to be critical for proliferation of MLL‐transformed leukemic cells while it had no effect on CD34+ cells from healthy donors, proposing MN1 as a therapeutic target in MLL‐transformed AML.Summary/Conclusion:Our study offers insight to the function of endogenous MN1 expression in leukemogenesis of MLL‐r AML. Our data shows that MN1 is essential for MLL fusion leukemias via regulation of Hoxa cluster and Meis genes and serves as a therapeutic target in MLL‐r AML.

High expression of meningioma 1 is correlated with reduced survival rates in colorectal cancer patients

The identification of prognostic markers for colorectal cancer (CRC) has important clinical implications. However, the association between meningioma 1 (MN1) expression and clinical outcomes of CRC has not been fully investigated. The aim of this study was to investigate the expression of MN1 in the clinical context of CRC. We first used immunohistochemistry (IHC) staining to examine and compare MN1 expression between multiple human cancer tissues and normal tissues. Initial screening revealed that the expression of MN1 proteins was significantly higher in tumor tissues of the breast, colon, and liver than in normal tissues. In further testing conducted on 59 paired CRC samples, we observed that the expression of MN1 in CRC tissue samples was significantly higher than in adjacent normal tissues. Moreover, high MN1 expression was not significantly associated with clinicopathological characteristics. Kaplan-Meier survival analysis revealed that high expression of MN1 mRNA or MN1 protein was significantly associated with poor CRC prognosis. Furthermore, univariate Cox analysis revealed that a high MN1 score was significantly associated with prognostic factors. Multivariate Cox analysis further indicated that gender, histologic grade, tumor-node-metastasis (TNM) stage, and a high MN1 score were independent factors of overall CRC survival rates. Finally, MN1 and PCNA protein levels were positively correlated, which suggests that MN1 may be involved in the cell proliferation process during CRC formation. Our results, which confirm those of other studies, indicate that (1) high levels of MN1 expression contribute to poor CRC prognosis and (2) MN1 can serve as a novel potential biomarker in predicting the prognosis of CRC patients.

Prognostic Impact of Blood MN1 Copy Numbers Before Allogeneic Stem Cell Transplantation in Patients With Acute Myeloid Leukemia.

High expression of the leukemia-associated gene meningioma-1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation- and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.

Menin Is a Therapeutic Target in MN1 High Leukemia

Meningioma-1 (MN1) has been found overexpressed in acute myeloid leukemia (AML). High MN1 expression levels are associated with poor prognosis and limited therapeutic options. Overexpression of MN1 in murine bone marrow progenitor cells causes leukemia as a single hit. Interestingly, MN1 induces a gene expression program that is reminiscent of KMT2a-rearranged leukemia, with high expression of the KMT2a (MLL-1) target genes HOXA7-13 and MEIS1. However, MN1 oncogenic functions remain unclear and no targetable therapies are available for MN1 high leukemia. We have previously shown, using a conditional knock-out mouse, that deletion of the histone methyltransferase Kmt2a increased latency and decreased penetrance of MN1 driven leukemia. We sought to determine what specific function of KMT2A is relevant to MN1 leukemogenesis in order to identify new therapeutic targets.KMT2a is a H3K4 histone methyltransferase that can perform mono, di and tri-methylation through its C-terminal catalytic site, the conserved SET domain. We first asked whether the enzymatic activity of Kmt2a was required in MN1 driven leukemia. Mice with a constitutive germline deletion of the SET domain of Kmt2a (∆SET) present some reduction of Hox gene expression during development but survive into adulthood with normal hematopoiesis. We established MN1 leukemia on a ∆SET background and found no difference in disease latency. Similarly, deletion of the SET domain did not influence colony forming potential in vitro.Menin, the product of the MEN1 suppressor gene, interacts with the N-terminal of KTM2a. The KMT2a-Menin interaction has been shown to be critical for leukemic transformation by KMT2a fusion proteins. Interestingly, this is a unique feature of leukemic cells since Menin is dispensable for steady state hematopoiesis. Since Kmt2a is required for MN1 leukemogenesis, we next investigated whether Menin was also necessary. We established MN1 leukemia on a conditional Menin knock-out background. In primary transplants, upon Cre-mediated excision of Menin, we found a slight decrease in colony forming potential as well as a modest but significant increase in survival of injected recipients. The effect of Menin deletion on MN1 leukemogenesis became much more dramatic in secondary transplants. In vitro, replating efficiency was severely impaired with increased apoptosis and cell cycle arrest. In vivo, we noted a significant increase in disease latency. Finally, we asked if Menin was a potentially relevant therapeutic target in MN1 high leukemia. In vitro, pharmacologic inhibition of the KMT2a-Menin interaction resulted in reduced growth and increased apoptosis of MN1 murine leukemia cells, at micromolar concentrations. We have identified a human leukemia cell line with high MN1 and high HOXA9 expression, Mutz3. To ensure that our findings translate to human AML, we will address the effect of Menin inhibition in Mutz3 cells as well as in patient samples with confirmed MN1 high leukemia.In summary, our data suggest that the role of Kmt2a in MN1 driven leukemia is mediated by the KMT2a-Menin interaction, identifying Menin as a druggable target in MN1 high leukemia. DisclosuresLibbrecht:Institut Servier: Research Funding. Bernt:GSK: Other: husband works at GSK.

Meis2 as a critical player in MN1-induced leukemia

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

MN1 and PTEN gene expression in acute myeloid leukemia.

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. 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. 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. 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.

The Meningioma 1 (MN1) Gene is an Independent Poor Prognostic Factor in Adult Egyptian Acute Myeloid Leukemia Patients

Aim:To determine the prognostic importance of meningioma 1 (MN1) gene expression levels in the context of other predictive markers for acute myeloid leukemia (AML) cases.Methods:MN1 expression was measured in 85 newly diagnosed adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction.Results:At diagnosis 67.4% of cases had elevated MN1 expression, this being associated with a worse prognosis, higher incidence of lymphadenopathy and CD34 transcript expression (p=0.02 and <0.001, respectively). No other molecular or clinical characteristics were significantly associated with MN1expression. Patients with high MN1 expression had lower complete response rate at day 15 compared to patients with low MN1 expression (p=0.09) and a significantly higher relapse rate (21.1% versus 7.7%, respectively, p=0.04). Patients with high MN1 expression had shorter TTP compared to those with low expression, p= 0.07.Conclusion:MN1 expression may predict outcome in AML patients. The MN1 gene and micro RNA expression suggest a biological feature that could be used as therapeutic targets.

The Role of Gata2 in Murine Models of Acute Myeloid Leukemia

The importance of stem cell and self-renewal programs in Acute Myeloid Leukemia (AML) is generally accepted, but the molecular details are incompletely understood. The master transcriptional regulator GATA2 is highly expressed in hematopoietic stem cells (HSCs) and has critically important roles in the hematopoietic system. Gata2 is required for murine HSC development and maintenance, and heterozygous loss of Gata2 compromises murine HSC- and progenitor cell-function. High levels of GATA2-expression have been correlated with adverse prognosis in human AML. GATA2 is also overexpressed in human chronic myeloid leukemia. These data suggest an important role for GATA2 in normal stem cells and in leukemia. However, genetic lesions resulting in compromised GATA2 function can lead to MDS and in some cases AML. In a murine AML model driven by Flt3-ITD and inactivation of Tet2, Gata2 is strongly downregulated. Furthermore, mouse models of leukemia suggest that high-level forced expression of Gata2 can have a tumor suppressor role. To clarify the role of Gata2in AML we used homozygous genetic inactivation in established murine models of leukemia, using a a conditional allele.We initially tested the role of Gata2 in a murine leukemia mediated by forced expression of Meningioma1 (MN1). This model has a HoxA9/Meis1 transcriptional program. We recently found that MN1-driven leukemia depends on the histone methyltransferase Dot1l (J Clin Invest. 2016 Feb 29. pii: 80825). Lineage marker negative (Lin-), Sca1+, Kit-positive (LSK) bone marrow cells from mice with a floxed exon 5 in the Gata2 gene, and a ROSA26-YFP Cre-reporter allele were transduced with an MSCV-based ecotropic retroviral vector expressing MN1 and linked via an internal ribosomal entry site (IRES) the selectable marker GFP. Floxed Gata2-sequences were excised using transduction with a self-excising Cre-expressing vector (HR-Cre). Cells were sorted and plated in methylcellulose. The GFP/YFP double positive Gata2ko cells showed a replating defect compared to GFP single positive Gata2-floxed cells, both with regard to colony number and colony size. Next, we tested the role of Gata2 in disease maintenance in vivo. We established MN1 Gata2ff leukemias in primary recipients. Primary leukemias were transduced with Cre-expressing vector and Gata2ko and Gata2ff MN1 cells were transplanted. While all mice in the Gata2ff group developed leukemia with a median survival of 35 days, the mice in the Gata2ko cohort developed leukemia with incomplete penetrance with a latency of 249 days (p=0.0005). These data suggest an important role for Gata2in MN1 leukemia in vitro and in vivo. Genetic inactivation of Gata2 resulted in increased protein levels of p53 in vitro as detected by Western blot. Furthermore, MN1-transduced cells showed accentuated p53 stabilization and apoptosis in response to the Mdm2-antagonist and p53 stabilizer Nutlin3.We next tested the role of Gata2 in leukemia driven by the oncogenic fusion MLL-AF9. In contrast to the MN1 model, recipients of Gata2ko leukemias only showed a trend towards prolonged latency in secondary recipients (median survival Gata2ff=48 days vs. Gata2ko=62 days, p=0.09). In this model, we also did not observe a substantial effect of Gata2-inactivation on p53 activation. We are currently characterizing the underlying molecular mechanisms.Our data document an important role for Gata2 in AML mediated by MN1 and to a lesser degree, MLL-AF9. The role of Gata2 in leukemia is complex and depends on expression levels and cellular context. A more detailed understanding of leukemic self-renewal, including the role of Gata2, will inform the development of more efficacious and less toxic therapies for this difficult-to-treat malignancy. DisclosuresBernt:Epizyme: Patents & Royalties: patent filed. Neff:Epizyme: Patents & Royalties: patent filed; Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel.

Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection.

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.

MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia.

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.

Meningeoma-1 Cooperates with MLL and DOT1L to Induce Leukemia

Meningioma-1 (MN1) is frequently overexpressed in AML, and associated with a poor prognosis. In addition, MN1-TEL fusions are found in AML, underscoring the importance and possible driver function of MN1 in AML. Forced expression of MN1 in murine hematopoietic progenitors induces a highly aggressive leukemia as a single hit. The mechanism by which MN1 induces AML is unclear. MN1 is a transcriptional co-activator with almost no sequence or structural similarity to any other protein, and no targeted approaches to MN1-high AML are currently available.We sought to understand the mechanism by which MN1 induces AML with the goal to identify targetable downstream effectors. We found that the gene expression program induced by forced expression of MN1 in hematopoietic progenitors substantially overlaps with a set of genes that is downregulated in response to loss of the histone methyltransferase Dot1l in normal hematopoietic progenitors. This led us to hypothesize that the MN1-induced leukemogenic gene expression program might be dependent on Dot1l. We established MN1 leukemias on a Dot1l conditional knockout background and found that of Dot1l indeed induced cell cycle arrest, differentiation and apoptosis, and prolonged the survival of transplanted mice in vivo. This was associated with the downregulation of the MN1-induced gene expression program. We next sought to investigate a possible mechanism for this observation. MN1 has been reported to be recruited to its target genes based on ChIP-seq, but it does not itself possess sequence specific DNA binding capacity. The mediator of this recruitment is thus unclear. Since Dot1l has been shown to be required for the high level expression of MLL-fusion target genes in MLL-rearranged leukemias, we asked whether MN1 might cooperate with wild type MLL, explaining the dependence on Dot1l. In order to test whether wild-type MLL is required for MN1 leukemias, we established AML on a MLL-conditional background. Results phenocopied the loss of Dot1l, suggesting that MN1 cooperates with both MLL and Dot1l to induce leukemia. We are currently investigating whether MN1 binds MLL and/or DOT1L directly to exert this function using biochemical and genomic approaches. Finally, we asked whether our findings had relevance for human AML. MN1 overexpression is found over a wide range of different molecular subgroups but is relatively under-represented in MLL-rearranged AML, suggesting redundant pathways. A subgroup that frequently displays very high levels of MN1 expression are AML with a complex karyotype with loss of 5q or 7 sequences, and high expression of HOXA cluster genes. We analyzed the response of a human cell line and two patient samples with high MN1/high HOXA9 expression to inhibition of DOT1L, and found induction of differentiation and apoptosis similar to our mouse model.In summary, our data suggest that MN1 cooperates with wild type MLL to induce a leukemogenic gene expression program that results in AML, and that this program may be targetable by inhibiting DOT1L. DisclosuresPollyea:GlycoMimetics: Other: Member of data safety monitoring board; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy. Ernst:Amgen: Other: 528 shares of stock. Neff:Epizyme: Patents & Royalties: US Patent 62/026583 Dot1l Inhibition in Patients with MN1 High AML filed 7/2014 Coinventor: Tobias Neff.

High MN1 expression increases the in vitro clonogenic activity of primary mouse B-cells

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–AML1positive leukemia might provide insight into additional molecular mechanisms contributing to B-ALL and may lead to improved treatment options for patients.

Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling.

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.

Meningioma 1 (MN1) expression: Refined risk stratification in acute myeloid leukemia with normal cytogenetics (CN-AML)

Prognostic stratification of cytogenetic normal acute myeloid leukemia (CN-AML) is an area of active research. The aim of this study was to determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in CN-AML. One hundred patients with CN-AML were diagnosed; MN1 expressions were analyzed using quantitative real-time polymerase chain reaction. High expressions were detected in 48 (48%) patients (expression range: 2.35–31.99, mean: 13.9 ± 8.49) in comparison with 52 (52%) patients with low expression (expression range: 0.02–2.3, mean: 0.68 ± 0.77). The course of the disease in patients with high MN1 expression was unfavorable. Patients with high MN1 expression was associated with significant low complete remission rate (62.5 vs. 8.4%, high vs. low MN1, P = 0.001) and high mortality rate (75% vs. 46.1, P = 0.03). AML patients with high MN1 expression tended to be refractory (37.5 vs. 19.2%, P = 0.00) and relapse risk (54.1 vs. 23%, P = 0.02). Multivariable analysis confirmed high MN1 expression as an independent risk factor for disease-free survival and overall survival. In conclusion, MN1 overexpression independently predicts bad clinical outcome in CN-AML patients.

MN1 regulates self-renewal and differentiation through distinct domains

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia, with high expression linked to all-trans retinoic acid (ATRA) resistance and poor overall and relapse-free survival. MN1 is also a potent and sufficient oncogene in murine leukemia, able to block myeloid differentiation, promote leukemic stem cell self-renewal and transform cells to the common myeloid progenitor level. Yet despite its established leukemic role, little is known about its mechanism of action.

The clinical characteristics and prognostic significance of MN1 gene and MN1-associated microRNA expression in adult patients with de novo acute myeloid leukemia

This study aimed to determine the clinical characteristics and prognostic significance of the meningioma 1 (MN1) gene and MN1-associated microRNA expression in Chinese adult de novo acute myeloid leukemia (AML) patients. The expression level of MN1, microRNA-20 (miR-20a), and microRNA-181b (miR-181b) in bone marrow mononuclear cells was measured in 158 newly diagnosed AML patients and 20 cases of normal healthy donors by real-time quantitative reverse transcriptase polymerase chain reaction. All AML patients significantly overexpressed MN1 at the level of 0.01983 (P < 0.001) compared with normal controls. High MN1 expression was associated with spleen involvement (P = 0.037), NPM1 wild type (P = 0.001), lower miR-20a expression levels (P = 0.015), and higher miR-181b expression levels (P = 0.035). MiR-20a (P = 0.029) and miR-181b (P = 0.017) overexpressed in the bone marrow cells of patients with certain subtypes of AML compared with healthy donors. High MN1 expressers had lower complete remission (CR) rates and shorter overall survival (OS) within the Southwest Oncology Group classification. In multivariable models, high MN1 expression was associated with worse CR rates (P = 0.01), relapse-free survival (RFS; P = 0.02), and OS (P = 0.02); high miR-20a expression was associated with higher CR rates (P = 0.008) and longer OS (P = 0.04), whereas high miR-181b expression was associated with lower CR rates (P = 0.03), and shorter RFS (P = 0.045) and OS (P = 0.017). High MN1 expression confers worse prognosis in Chinese adult patients with de novo AML. MN1 gene and MN1-associated microRNAs provide clinical prognosis of AML patients and may refine their molecular risk classification.

Microdeletion del(22)(q12.2) encompassing the facial development-associated gene, MN1 (meningioma 1) in a child with Pierre-Robin sequence (including cleft palate) and neurofibromatosis 2 (NF2): a case report and review of the literature

BackgroundPierre-Robin sequence (PRS) is defined by micro- and/or retrognathia, glossoptosis and cleft soft palate, either caused by deformational defect or part of a malformation syndrome. Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome caused by mutations in the NF2 gene on chromosome 22q12.2. NF2 is characterized by bilateral vestibular schwannomas, spinal cord schwannomas, meningiomas and ependymomas, and juvenile cataracts. To date, NF2 and PRS have not been described together in the same patient.Case presentationWe report a female with PRS (micrognathia, cleft palate), microcephaly, ocular hypertelorism, mental retardation and bilateral hearing loss, who at age 15 was also diagnosed with severe NF2 (bilateral cerebellopontine schwannomas and multiple extramedullary/intradural spine tumors). This is the first published report of an individual with both diagnosed PRS and NF2. High resolution karyotype revealed 46, XX, del(22)(q12.1q12.3), FISH confirmed a deletion encompassing NF2, and chromosomal microarray identified a 3,693 kb deletion encompassing multiple genes including NF2 and MN1 (meningioma 1).Five additional patients with craniofacial dysmorphism and deletion in chromosome 22-adjacent-to or containing NF2 were identified in PubMed and the DECIPHER clinical chromosomal database. Their shared chromosomal deletion encompassed MN1, PITPNB and TTC28. MN1, initially cloned from a patient with meningioma, is an oncogene in murine hematopoiesis and participates as a fusion gene (TEL/MN1) in human myeloid leukemias. Interestingly, Mn1-haploinsufficient mice have abnormal skull development and secondary cleft palate. Additionally, Mn1 regulates maturation and function of calvarial osteoblasts and is an upstream regulator of Tbx22, a gene associated with murine and human cleft palate. This suggests that deletion of MN1 in the six patients we describe may be causally linked to their cleft palates and/or craniofacial abnormalities.ConclusionsThus, our report describes a NF2-adjacent chromosome 22q12.2 deletion syndrome and is the first to report association of MN1 deletion with abnormal craniofacial development and/or cleft palate in humans.

Expression and clinical significance of MN1 and PTEN gene in patients with acute myeloid leukemia

The aim of our study was to explore the correlations between both the genes, evolution and prognosis of the disease by detecting the expression of MN1 (meningioma 1) gene and PTEN (phosphatase and tensin homolog) gene in patients with acute myeloid leukemia (AML). MN1 and PTEN mRNA were detected by reverse transcription-PCR in mononuclear cells of 38 patients with AML and 13 patients with normal bone marrow. Positive rates of MN1 and PTEN genes were 76.3% and 60.5% respectively in bone marrow mononuclear cells. The expression level of MN1 mRNA for the de novo group increased comparing with that for normal control group (P < 0.05), the expression level of PTEN mRNA for de novo group decreased obviously comparing with that for normal control group (P < 0.01), MN1 mRNA level decreased in remission group, while PTEN mRNA level increased comparing with that in de novo group (P < 0.05). MN1 mRNA level increased and PTEN mRNA decreased in relapsed group comparing with that in control group, and their difference was statistical significance (P < 0.05). The two genes’ levels had negative correlations in acute myeloid leukemia (r = −0.314, P < 0.05). There is close correlations between expression of MN1 and PTEN genes and the prognosis and occurrence of acute myeloid leukemia, and their expression can be taken as significant indexes to access the de novo, relapse and prognosis of acute myeloid leukemia.

MN1 Inhibits Myeloid Differentiation by Transcriptional Repression of EGR2

AbstractAbstract 229Overexpression of MN1 (meningioma 1) is a negative prognostic factor in acute myeloid leukemia (AML) patients with normal cytogenetics, and induces a rapidly lethal AML in mice. We have shown previously that MN1, a transcription cofactor of retinoic acid receptor alpha (RARA), increases resistance to all-trans retinoic acid (ATRA) by greater than 3000-fold in an in-vitro differentiation model. We investigated the molecular mechanisms involved in the MN1-induced myeloid differentiation block by fusing potent transcriptional activation or repression domains to MN1, conducting a structure-function analysis of MN1, gene expression profiling, ChIP-on chip experiments, and functional validation of MN1 target genes. We found that (1) MN1 inhibits myeloid differentiation through transcriptional repression; (2) the C-terminal domain of MN1 is critical for induction of resistance to ATRA; (3) EGR2 is a putative direct target of MN1 and RARA that is repressed in MN1 leukemias; and (4) that constitutive upregulation of EGR2 in MN1 leukemias permits differentiation and prevents engraftment of transplanted cells. To investigate whether MN1 impacts on myeloid differentiation through transcriptional activation or repression we fused a strong transcriptional activation domain (VP16) or repression domain (M33) to MN1. MN1VP16 immortalized murine bone marrow cells, however, these cells could differentiate to mature granulocytes, and succumbed to cell cycle arrest upon treatment with ATRA. Mice receiving transplants of MN1VP16 cells had a median survival of 143 days (n=16) compared to 35 days in mice receiving MN1-transduced cells (n=18; p<.001). Morphologic analysis of bone marrow mostly showed mature granulocytes with less than 20 percent immature forms consistent with a diagnosis of myeloproliferative-like disease. Conversely, mice receiving transplants with cells transduced with the fusion of MN1 to the transcriptional repression domain of M33 (n=7) developed leukemia with a similar latency and phenotype as mice receiving transplants from MN1-transduced cells (survival, P=.6). These data suggest that MN1 inhibits myeloid differentiation by transcriptional repression rather than activation of its target genes. A structure-function analysis was performed to identify the domain(s) of MN1 required to inhibit myeloid differentiation. Consecutive stretches of 200 amino acids of MN1 were interrogated The deletion constructs were subsequently transduced into bone marrow cells immortalized by NUP98-HOXD13 (ND13). ND13 cells are very sensitive to ATRA-induced differentiation and cell cycle arrest with an IC50 of 0.1 μ M, whereas overexpression of MN1 increases resistance greater than 3000-fold. Interestingly, deletion of the 200 C-terminal amino acids of MN1 restored ATRA sensitivity of ND13 cells compared to full-length MN1, suggesting that the C-terminus of MN1 is required for inhibition of myeloid differentiation. To identify MN1-regulated genes important for the myeloid differentiation block we performed gene expression profiling of MN1- and MN1VP16-transduced bone marrow cells. To further identify genes that might be directly regulated by MN1 we performed ChIP-on-chip using anti-MN1 and anti-RARA antibodies. EGR2, CCL5, CMAH, among others, were identified as targets of both MN1 and RARA whose gene expression was low in MN1 but high in MN1VP16 cells. Overexpression of these genes in MN1-transduced leukemic cells was used to validate their function. Blast percentage of in vitro cultured bone marrow cells was 93, 58, 83, and 41 percent in MN1+CTL cells, MN1+EGR2, MN1+CCL5, and MN1+CMAH cells, respectively. MN1+EGR2 cell engraftment in peripheral blood of mice declined from 2.2 percent at 4 weeks to undetectable levels at 8 weeks (n=4), whereas MN1+CCL5 and MN1+CMAH cell engraftment was 23 (n=4) and 26 (n=4) percent at 4 weeks, and 14 and 30 percent at 8 weeks, respectively. At time of death, EGR2 was not detectable in mice whereas leukemias of mice receiving MN1+CCL5 or MN1+CMAH- transduced cells were positive for CCL5 or CMAH, respectively.In conclusion, our data suggest that MN1 inhibits myeloid differentiation by transcriptional repression of a subset of its target genes, and that re-expression of EGR2, a zinc-finger transcription factor, may prevent outgrowth of MN1 leukemias in mice. Pharmacologic activation of EGR2 may become a novel antileukemic strategy. Disclosures:No relevant conflicts of interest to declare.

Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

AbstractAbstract 4836Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment.In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures:No relevant conflicts of interest to declare.