Meningioma 1 Expression Research Articles

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Intrinsically disordered Meningioma-1 stabilizes the BAF complex to cause AML

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.

Menin is necessary for long term maintenance of meningioma-1 driven leukemia

Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A–Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A–Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.

Prognostic significance of combined BAALC and MN1 gene expression level in acute myeloid leukemia with normal karyotype.

Acute myeloid leukemia with normal karyotype (AML-NK) is the largest group of AML patients with very heterogeneous disease outcome. In order to ensure more precise risk stratification new molecular markers have been introduced, like expression level for BAALC (Brain and Acute Leukemia, Cytoplasmic) and MN1 (Meningioma 1) genes. In this study, we investigated expression level of both genes in 111 adult AML-NK at diagnosis and examined their prognostic potential. BAALC and MN1 expression were detected in about one third of the patients, and positive correlation between these two genes was found. The BAALC+ /or MN1+ status was not associated with the presence of FLT3-ITD mutations, but exhibited strong correlation with NPM1wt status (P<.001). Therefore, among BAALC+ /or MN1+ patients the most frequent ones were FLT3-ITD- /NPM1- double negative patients with intermediate prognosis. When BAALC+ /or MN1+ patients were divided into BAALChigh /BAALClow (21/21) and MN1high /MN1low (21/22) groups, we detected that BAALChigh /or MN1high patients had a tendency toward lower complete remission rate. Also, survival analysis showed that BAALChigh /or MN1high patients had shorter disease-free survival and overall survival (OS). The most pronounced influence on prognosis was detected in FLT3-ITD- /NPM1- group of patients that are lacking reliable prognostic markers, where OS in BAALChigh /or MN1high was only 5months vs 25months in BAALClow /or MN1low . These findings indicate that BAALC and MN1 expression level could be used for more precise risk stratification of AML-NK patients and especially FLT3-ITD- /NPM1- patients, transforming this intermediate-risk group, into a group with an adverse prognosis.

MN1 overexpression with varying tumor grade is a promising predictor of survival of glioma patients.

Gliomas have substantial mortality to incidence rate ratio and a dismal clinical course. Newer molecular insights, therefore, are imperative to refine glioma diagnosis, prognosis and therapy. Meningioma 1 (MN1) gene is a transcriptional co-regulator implicated in other malignancies, albeit its significance in glioma pathology remains to be explored. IGFBP5 is regulated transcriptionally by MN1 and IGF1 and is associated with higher glioma grade and shorter survival time, prompting us to ascertain their correlation in these tumors. We quantified the expression of MN1, IGFBP5 and IGF1 in 40 glioma samples and examined their interrelatedness. MN1 mRNA-protein inter-correlation and the gene’s copy number were evaluated in these tumors. Publicly available TCGA datasets were used to examine the association of MN1 expression levels with patient survival and for validating our findings. We observed MN1 overexpression correlated with low-grade (LGGs) and not high-grade gliomas and is not determined by the copy number alteration of the gene. Notably, gliomas with upregulated MN1 have better overall survival (OS) and progression-free survival (PFS). IGFBP5 expression associated inversely with MN1 expression levels in gliomas but correlated positively with IGF1 expression in only LGGs. This suggests a potential grade-specific interplay between repressive and activating roles of MN1 and IGF1, respectively, in the regulation of IGFBP5. Thus, MN1 overexpression, a promising predictor of OS and PFS in gliomas, may serve as a prognostic biomarker in clinical practice to categorize patients with survival advantage.

Allogeneic stem cell transplantation mitigates the adverse prognostic impact of high diagnostic BAALC and MN1 expression in AML

For most acute myeloid leukemia (AML) patients, an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of sustained remissions and long-term survival. At diagnosis, high expression of the AML-associated genes BAALC (brain and acute leukemia, cytoplasmic) and MN1 (meningioma-1) were repeatedly linked to inferior outcomes in patients consolidated with chemotherapy while data for patients receiving HSCT remain limited. Using clinically applicable digital droplet PCR assays, we analyzed the diagnostic BAALC/ABL1 and MN1/ABL1 copy numbers in 302 AML patients. High BAALC/ABL1 and MN1/ABL1 copy numbers associated with common adverse prognostic factors at diagnosis. However, while high diagnostic copy numbers of both genes associated with shorter event free survival (EFS) and overall survival (OS) in patients receiving chemotherapy, there was no prognostic impact in patients undergoing HSCT. Our data suggests that the adverse prognostic impact of high BAALC and MN1 expression are mitigated by allogeneic HSCT. But preHSCT BAALC/ABL1 and MN1/ABL1 assessed in remission prior to HSCT remained prognosticators for EFS and OS independent of the diagnostic expression status. Whether allogeneic HSCT may improve survival for AML patients with high diagnostic BAALC or MN1 expression should be investigated prospectively and may improve informed decisions towards individualized consolidation options in AML.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

MN1, FOXP1 and hsa-miR-181a-5p as prognostic markers in acute myeloid leukemia patients treated with intensive induction chemotherapy and autologous stem cell transplantation

BackgroundThe meningioma-1 (MN1) gene is expressed in hematopoietic CD34+ cells and down-regulated during myeloid differentiation. MN1 overexpression has been linked to shorter overall and disease free survival in AML patients treated with intensive induction chemotherapy. MN1 overexpression may still be an adverse prognostic marker in AML patients treated with autologous stem cell transplant (auto-SCT) after intensive induction chemotherapy. MethodsWe retrospectively analysed 54 peripheral blood mononuclear cell (PBMC) samples of AML patients who received auto-SCT at remission (CR1) after intensive induction chemotherapy. MN1 and putative MN1-associated mRNAs, as well as MN1-associated micro-RNAs were assessed at diagnosis in peripheral blood mononuclear cells using Taqman gene expression assays. ResultsAML patients with elevated MN1 or FoxP1 gene expression at diagnosis had a significantly shorter progression-free and overall survival after intensive induction chemo-therapy and auto-SCT. The presence of the favourable risk NPM1 mutation associated with reduced MN1 gene expression. In contrast to MN1 and FOXP1, elevated expression of the putative tumor suppressive micro-RNA hsa-miR-181a-5p was predictive for positive outcome. Correlation analysis of MN1 with myeloid gene expression levels revealed association of MN1 and BMI-1, CD34, FOXP1 and MDM2 expression. Analysis of non-coding RNAs revealed an inverse correlation of MN1 with hsa-miR-20a-5p and hsa-miR-181b-5p expression. ConclusionsMN1, FOXP1 and hsa-miR-181a-5p are prognostic markers in AML patients treated with intensive induction chemotherapy and auto-SCT. While MDM2 is a validated therapeutic target, the transcription factors MN1 and FOXP1, and the chromatin modulator BMI-1 are potential therapeutic targets in the treatment of AML. The tumor suppressor hsa-miR-181a-5p may be a candidate miRNA mimic for therapeutic use.

MN1 Expression Is an Indepedent Prognostic Marker in FLT3-Mutated Acute Myeloid Leukemia and Is Involved in the Resistance to FLT3 Inhibitors

Introduction: Meningioma 1 (MN1) gene was described as a prognostic marker for AML patients with normal karyotype (Carturan et al. Oncotarget 2016). In addition, MN1 high expression was linked to RA resistance and was shown to be necessary and sufficient to transform common myeloid progenitors in a MEIS1/AbdB-like HOX protein complex-dependent manner. However, the relevance of the copresence of MN1 overexpression in AML patients with mutations of the FLT3 and/or NPM1 genes is unknown. Moreover, it is also unknown the functional effects of MN1 in the biology of leukemic blasts harboring FLT3 mutations and if it may modulate the response to FLT3 inhibitors, such as quizartinib (AC220) and midostaurin (PKC). Aims: Herein, we investigated the prognostic impact of MN1 expression across multiple transcriptomic platforms and AML data sets. Additionally, we transduced different AML cell lines to evaluate the impact of MN1 on cell survival and differentiation. Methods: Three different AML series (1th: GSE6891, 240 patients, 2nd: TCGA, 113 patients and 3rd: BeatAML, 139 patients) were used. All patients presented similar gender distribution, were above 18y and treated by the 3+7 scheme. All genes from the RNAseq (TCGA) were pre-ranked according to their differential expression comparing tumors with high and low expression of MN1, using their median expression rate as the cutoff. GSEA was performed using the Reactome, KEGG and Hallmarks databases. Additionally, we transduced 07 AML cell lines with the MN1 gene and the control. For those cells, clonogenicity and proliferation rate was evaluated to identify which cell lines are sensitive and resistant to MN1. Drug induced apoptotic rate was assessed for FLT3-ITD- cell lines when treated with AraC (10 nM) and FLT3-ITD+ cell lines (MOLM13/MV411) upon treatment with PKC and AC220 over a time period of 24, 48 and 72 h. Apoptosis was further confirmed by cleaved Caspase-3/PARP detection. The myeloid differentiation in response to PMA treatment (100 ng/ml) was determined by the surface levels of CD11b, CD11c, CD14, CD15, CD61 and HLADR. Results: In all three AML series, high MN1 levels higher than the median (hereafter called High MN1) were associated with lower frequency of FLT3 and NPM1 mutations (P&amp;lt;.01 for all comparisons). Baseline features were similar between patients with low and high MN1 levels in all the cohorts. The median 5y OS rates were 33%, 29% and 41% in the GSE6891, TCGA and BeatAML, respectively. Multivariate analysis showed that patients carrying the FLT3 mutation who expressed high MN1 levels exhibited increased OS rates in the GSE6891 (50%), TCGA (50%) and BeatAML (71%) cohorts in comparison with patients with lower MN1 levels taking ELN, sex and age as confounders (P&amp;lt;.05). Regarding the patients with NPM1 and IDH1/2 mutations, high MN1 levels was associated with lower OS rates in all the cohorts. The analysis to predict genetic interactors in AML samples (BeatAML) revealed that MN1 is associated with cellular mediators of myeloid differentiation, such as FOXM1, CEBPA and MEIS1, and drug-resistance (CYP26A1). Additionally, GSEA indicated that the MN1 expression was associated with biological processes such as apoptosis, STAT5 signaling and Interferon response. In AML cell lines, induced MN1 overexpression strongly reduced growth, colony formation (basal and under treatment with PKC/AC220) and increased drug-induced apoptosis in both FLT3-ITD cells (P&amp;lt;.01 for all). Forced MN1 expression in FLT3-ITD cells lead to decreased cytokine mediated resistance to FLT3 inhibitors (PKC/AC220; P&amp;lt;.01 for all). Using a nonlinear regression analysis, IC50 for PKC and AC220 was significantly lower for MN1-expressing cells (PKC: 8.4 and 4.9 nM; AC220: 0.69 and 0.17 nM for MV411 and MOLM13, respectively) in comparison to control cells (PKC: 13.3 and 8.5 nM; AC220: 0.62 and 0.38 nM, respectively). 3 out of 7 cell lines showed increased cell proliferation when MN1 gene was overexpressed (P&amp;lt;.05). In accordance, MN1 overexpression reduced the drug-induced apoptosis rate in those AML cells with increased proliferation in a time-dependent manner (P&amp;lt;.05). Phorbol induced myeloid differentiation was decreased for all MN1 transduced cell lines (P&amp;lt;.01 for all), except for the THP1 cells. Conclusion: In summary, our results demonstrated that MN1 overexpression decreases cell viability and proliferation in FLT3-ITD+ cells and decreased resistance to FLT3 inhibitors. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding.

Myeloid Neoplasms with t(12;22)(p13;q12) and MN1 Ectopia Are Refractory to Chemotherapy and Can Benefit from Hematopoietic Stem Cell Transplantation

Translocation of t(12;22)(p13;q12) is a rare but recurrent chromosomal (chr) abnormality in hematologic malignancies involving ETS variant 6 (ETV6) and meningioma 1 (MN1) genes. The pathogenic mechanism of t(12;22)(p13;q12) and the fact that nearly half of the cases lack fusion genes remain mysterious. We focused here on AML cases with t(12;22)(p13;q12) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). A total of eight cases with t(12;22)(p13;q12) and additionally one with t(12;17;22)( p13;q21;q13) (six males and three females) were detected in 2782 newly diagnosed AML and MDS patients by G-banding karyotyping (Table 1). The median age of onset was 45 years (range 4-60 years). The cases include three AML-M5, one AML-M2, one AML progressed from MDS (MDS-AML), one MDS with excess blasts 2 (MDS-EB-2), one chronic myelomonocytic leukemia (CMML), and two myeloid/T mixed-phenotype acute leukemia (MPAL) (Table 1). All cases underwent standard or intensive chemotherapy for myeloid malignancies, and six patients underwent allo-HSCT (Fig. 1a). MN1-ETV6, ETV6-MN1, and MN1 expression were analyzed by RT-PCR. Mutations of 58 commonly mutated genes in hematologic malignancies were analyzed by next-generation sequencing in seven cases (Table 1). Fluorescence in situ hybridization (FISH) was performed using the MN1 dual-color break-apart probe on cases P4 and P9, who were negative for MN1-ETV6. 30 × whole-genome sequencing (WGS) was performed on diagnostic bone marrow (BM) samples from the four cases which were negative for MN1-ETV6, and structure variations including translocations (Fig. 1b) were analyzed. 5/9 cases were positive for both MN1-ETV6 and ETV6-MN1fusions (all type I), and the expression of MN1 was significantly up-regulated in 3/4 fusion negative cases (Table 1). FISH analysis showed split signals in both P4 and P9, indicating a fracture within or adjacent to MN1. WGS analysis showed ectopia of intact MN1 in case P1, P3, and P4 (Fig. 1b), including translocated adjacent to the super enhancers (SEs) of the cracked ETV6. A novel MN1-STAT3 fusion was identified and confirmed by RT-PCR in case P9. We observed overexpression of MN1 in MN1-ETV6 negative cases in our study, which strongly indicates that the contribution of the SEs within ETV6 made on the overexpression of MN1 or aberrant activation of the chimeric MN1-ETV6. Both type I/II MN1-ETV6, and the novel MN1-STAT3 in this study retains the main functional domains of MN1 (Fig. 1c). The MN1-STAT3 contains only 69 amino acids of the STAT3 residual without functional domains, and STAT3-MN1 was absent due to the triadic translocation. Thus, the fusion protein will retain the primary function of MN1 but not STAT3 in case P9, which also indicates that it is the functionally retained MN1 within MN1-ETV6 rather than the fusion, that plays the essential role in leukemogenesis. The genomic evidence of ETV6 disruption was observed in 8/9 cases in our study. ETV6 is a tumor suppressor which is essential in hemopoietic stem cell differentiation and multilineage blood cell development. It is conceivable that the MN1 overactivation and the ETV6 disruptive haploinsufficiency synthetically contribute to the leukemogenesis in t(12;22)(p13;q12) malignancies. This scenario is partially similar to the mechanism of GATA2-MECOM in inv(3)(q21q26) AML, within which that the central pathogenesis is the ectopia of the GATA2 SE that activates MECOM expression and confers the GATA2 haploinsufficiency simultaneously. The present study confirmed that myeloid neoplasms with t(12;22)(p13;q12) were rare and responded poorly to conventional chemotherapy, and provide evidence that these patients can benefit from allo-HSCT. Our investigation especially highlights the evidence of a SE-associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms in a spatiotemporally specific manner, rather than the conventional thought of MN1-ETV6/ETV6-MN1 fusion formation. Figure 1 Disclosures No relevant conflicts of interest to declare.

SWI/SNF Dysregulation through a Prion-like Domain Causes AML

Meningioma-1 (MN1) was first described in a sporadic t(4;22) translocation in meningioma. Rare MN1 fusions to the ETS factors ETV6 (t(12;22)) and FLI1 (cryptic) also occur in AML. t(12;22) translocations have been described in two different patterns:Type 1: MN1 exon 1, is fused to a C-terminal fragment of ETV6. The first exon codes for almost all of the protein and has been shown previously to be sufficient to induce AML in a mouse model. Type 2: The entire MN1 coding frame, including the stop codon, is fused to a portion of ETV6. This results in a fusion on a DNA level, but on a protein level only MN1 is expressed. In both cases the fusion results in very high MN1 expression, either alone (type 2) or as a fusion protein (type 1). We performed ChIP-seq for H3K4me2/H3K27ac/Med1 on cell lines and a primary patient sample, and identified a large putative enhancer within and downstream of the ETV6 locus. Type 2 translocations suggest that hijacking of this enhancer is the critical oncogenic event. Besides the rare fusions, a subgroup of AML patients have very high MN1 expression without evident fusions. We speculate that some of these patients may have cryptic enhancer fusions. Importantly, several independent studies show that MN1 overexpression confers a poor prognosis. The survival rate 2 years after diagnosis is at only 20-30% reflecting the aggressiveness of this leukemia. In mice MN1 overexpression induces one of the most aggressive leukemias known as a single hit. These leukemias are Hoxa9 high and transcriptionally resemble KMT2A-rearranged leukemias. Despite its clear contribution to aggressive AML, it is not understood how MN1 functions on a molecular level. MN1 has no identified classic structural domains and lacks sequence homology with any other protein. Therefore, no predictions about structure or possible binding partners exist, and only few binding partners have been shown experimentally. This severely limits therapy options for patients and potential future drug development. Therefore, we aimed to define the MN1 interaction partner(s) and mechanism of leukemogenesis. To identify the MN1 interactome in AML we used two complementary methods, co-immunoprecipitation (CoIP) and proximity-dependent labeling (BioID), followed by Mass Spectrometry. As top hit in both screens we identified the mSWI/SNF complex, including the ATPase Smarca4, as an interactor of MN1. mSWI/SNF is a multisubunit complex with cell context- and function- dependent variable members. This complex is responsible for chromatin remodeling and plays an important role in gene expression and lineage determination. Its role in various forms of cancer is well established, where subunits are deleted, mutated, or misrecruited. We find co-sedimentation of MN1 with identified mSWI/SNF members in glycerol gradients using murine and human cells with MN1 overexpression. ChIP-seq data indicates a high overlap in DNA occupancy between MN1 and Smarca4. Using a conditional Smarca4 KO mouse model we show that Smarca4 is indispensable for MN1 driven leukemia. Together, these experiments substantiate a critical interaction between the oncogenic driver MN1 and the epigenetic modifier complex mSWI/SNF. MN1 contains a long polyQ stretch encoded by 28 CAG repeats. Such glutamine rich regions have been recognized as domains facilitating transcriptional activation, stabilizing protein-protein interactions, and being important components in higher order complex formation. PolyQ domains belong to the family of prion-like domains, which have roles in SWI/SNF recruitment. We show that the deletion of MN1's polyQ stretch abolishes differentiation block and allows the cells to differentiate. This is reflected in poor replating efficiency in Methylcellulose assays compared to full length MN1 driven leukemia cells. In vivo, MN1s' polyQ stretch is important for AML initiation. On a molecular level, we find that polyQ deletion reduces the affinity of the mSWI/SNF complex to chromatin in comparison to full length MN1, and fails to maintain the expression of key MN1 target genes such as the later Hoxa cluster, Meis1 and Flt3. In conclusion, our data support a model wherein MN1's oncogenic function is mediated by mSWI/SNF dysregulation, via the MN1 polyQ stretch. Disclosures Bernt: Glaxo-Smith-Kline: Other: Family member working for GSK; Agios: Consultancy; Epizyme: Other: applied for joint patent.

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 &lt;1% engraftment in all ten mice, whereas mice transplanted with MLL‐AF9/Mn1 wt cells showed &gt;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.

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.

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.