Deletion Of Men1 Research Articles

Combined deletion of MEN1, ATRX and PTEN triggers development of high-grade pancreatic neuroendocrine tumors in mice

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of tumors that exhibit an unpredictable and broad spectrum of clinical presentations and biological aggressiveness. Surgical resection is still the only curative therapeutic option for localized PanNET, but the majority of patients are diagnosed at an advanced and metastatic stage with limited therapeutic options. Key factors limiting the development of new therapeutics are the extensive heterogeneity of PanNETs and the lack of appropriate clinically relevant models. In that context, genomic sequencing of human PanNETs revealed recurrent mutations and structural alterations in several tumor suppressors. Here, we demonstrated that combined loss of MEN1, ATRX, and PTEN, tumor suppressors commonly mutated in human PanNETs, triggers the development of high-grade pancreatic neuroendocrine tumors in mice. Histopathological evaluation and gene expression analyses of the developed tumors confirm the presence of PanNET hallmarks and significant overlap in gene expression patterns found in human disease. Thus, we postulate that the presented novel genetically defined mouse model is the first clinically relevant immunocompetent high-grade PanNET mouse model.

Abstract 3586: A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors

Abstract Background: Neuroendocrine tumors (NETs) represent heterogenous malignancies whose origins and etiology remain poorly understood. Men1-driven reprogramming of neural crest-derived glial cells was recently implicated in NET development. In these studies, aberrant Sonic hedgehog (SHH) pathway activation known to pattern neural cell fate coincided with the neuroendocrine phenotype in mice. Here, we investigated the hypothesis that loss of menin encoded by the MEN1 gene drives SHH-dependent oncogenic reprogramming of NETs by modulating the neural crest cell fate. Methods: Glial cell specific Men1 deletion was accomplished by expressing Cre recombinase downstream of the human glial fibrillary acidic protein promoter (GFAPΔMen1). Hedgehog (HH) activation of Men1-deficient glial cells was blocked by deleting the gene encoding primary ciliary protein Kif3a required for transducing SHH signaling. The resulting GFAPΔMen1 mice were evaluated for NET development and dysregulated hormone profiles. To identify aberrant SHH activation leading to neuroendocrine reprogramming, we performed single cell RNA- and Assay for Transposase Accessible Chromatin sequencing on GFAPΔMen1 NETs and primary glial cultures. Hyperactivation of SHH in human gastroenteropancreatic (GEP)-NETs was confirmed by immunofluorescent staining and western blot. Lastly, primary mouse and human NET organoids were treated with an agonist and inhibitors of HH signaling and evaluated for ERK/AKT activation, proliferation, and transcript fluctuations indicative of neural crest cell reprogramming. Results: GFAPΔMen1 mice developed NETs in the pancreas, pituitary, and small intestine. Astonishingly, impaired SHH activation in GFAP+/Men1−/− cells abolished the development of NETs and restored hormone levels to that of wild type mice. GFAPΔMen1 NETs and glial cultures showed increased SHH signaling and chromatin accessibility of HH pathway genes, whereas additive Kif3a deletion blocked HH activation. Moreover, menin negative glial cells demonstrated a phenotypic shift in neuroglial lineages that suggested SHH-mediated reprogramming of neural crest-derived cells following the loss of menin. Consistent with hyperactive SHH activation in the GFAPΔMen1 mice, human GEP-NETs overexpressed SHH and downstream signaling targets. Functionally, SHH induced ERK/AKT activation, cell proliferation, and neuroendocrine transcript expression in human and mouse NET organoids, whereas pharmacological inhibition of HH signaling reversed these effects. Conclusions: Our observations implicate neural crest-derived glial cells as potential neuroendocrine cell precursors that are susceptible to transformation through increased HH signaling. These studies warrant future investigation into the delivery of Hedgehog inhibitors in the adjuvant setting for NET treatment. Citation Format: Suzann Duan, Ricky A. Sontz, Martin Deymier, Juanita L. Merchant. A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3586.

Menin Reduces Parvalbumin Expression and is Required for the Anti-Depressant Function of Ketamine.

Dysfunction of parvalbumin (PV) neurons is closely involved in depression, however, the detailed mechanism remains unclear. Based on the previous finding that multiple endocrine neoplasia type 1 (Protein: Menin; Gene: Men1) mutation (G503D) is associated with a higher risk of depression, a Menin-G503D mouse model is generated that exhibits heritable depressive-like phenotypes and increases PV expression in brain. This study generates and screens a serial of neuronal specific Men1 deletion mice, and found that PV interneuron Men1 deletion mice (PcKO) exhibit increased cortical PV levels and depressive-like behaviors. Restoration of Menin, knockdown PV expression or inhibition of PV neuronal activity in PV neurons all can ameliorate the depressive-like behaviors of PcKO mice. This study next found that ketamine stabilizes Menin by inhibiting protein kinase A (PKA) activity, which mediates the anti-depressant function of ketamine. These results demonstrate a critical role for Menin in depression, and prove that Menin is key to the antidepressant function of ketamine.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

Genetic Deletion of Menin in Mouse Mesenchymal Stem Cells: An Experimental and Computational Analysis.

ABSTRACTLoss‐of‐function mutations in the MEN1 tumor‐suppressor gene cause the multiple endocrine neoplasia type 1 syndrome. Menin, the MEN1 gene product, is expressed in many tissues, including bone, where its function remains elusive. We conditionally inactivated menin in mesenchymal stem cells (MSCs) using paired‐related homeobox 1 (Prx1)‐Cre and compared resultant skeletal phenotypes of Prx1‐Cre;Men1 f/f menin‐knockout mice (KO) and wild‐type controls using in vivo and in vitro experimental approaches and mechanics simulation. Dual‐energy X‐ray absorptiometry demonstrated significantly reduced bone mineral density, and 3‐dimensional micro‐CT imaging revealed a decrease in trabecular bone volume, altered trabecular structure, and an increase in trabecular separation in KO mice at 6 and 9 months of age. Numbers of osteoblasts were unaltered, and dynamic histomorphometry demonstrated unaltered bone formation; however, osteoclast number and activity and receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) mRNA profiles were increased, supporting increased osteoclastogenesis and bone resorption. In vitro, proliferative capabilities of bone marrow stem cells and differentiation of osteoblasts and mineralization were unaltered; however, osteoclast generation was increased. Gross femur geometrical alterations observed included significant reductions in length and in mid‐metaphyseal cross‐sectional area. Atomic force microscopy demonstrated significant decreases in elasticity of both cortical and trabecular bone at the nanoscale, whereas three‐point bending tests demonstrated a 30% reduction in bone stiffness; finite element analysis showed morphological changes of the femur microgeometry and a significantly diminished femur flexural rigidity. The biomechanical results demonstrated the detrimental outcome of the accelerated osteoclastic bone resorption. Our studies have a twofold implication; first, MEN1 deletion from MSCs can negatively regulate bone mass and bone biomechanics, and second, the experimental and computational biomechanical analyses employed in the present study should be applicable for improved phenotypic characterization of murine bone. Furthermore, our findings of critical menin function in bone may underpin the more severe skeletal phenotype found in hyperparathyroidism associated with loss‐of‐function of the MEN1 gene. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling

During decidualization in rodents, uterine stroma undergoes extensive reprograming into distinct cells, forming the discrete regions defined as the primary decidual zone (PDZ), the secondary decidual zone (SDZ) and the layer of undifferentiated stromal cells respectively. Here we show that uterine deletion of Men1, a member of the histone H3K4 methyltransferase complex, disrupts the terminal differentiation of stroma, resulting in chaotic decidualization and pregnancy failure. Genome-wide epigenetic profile reveals that Men1 binding in chromatin recapitulates H3K4me3 distribution. Further transcriptomic investigation demonstrates that Men1 directly regulates the expression of PTX3, an extra-cellular trap for FGF2 in decidual cells. Decreased Ptx3 upon Men1 ablation leads to aberrant activation of ERK1/2 in the SDZ due to the unrestrained FGF2 signal emanated from undifferentiated stromal cells, which blunt BMP2 induction and decidualization. In brief, our study provides genetic and molecular mechanisms for epigenetic rewiring mediated decidual regionalization by Men1 and sheds new light on pregnancy maintenance.

Menin regulates lipid deposition in mouse hepatocytes via interacting with transcription factor FoxO1.

Non-alcoholic fatty liver disease (NAFLD) is rapidly being recognized as the leading cause of chronic liver disease worldwide. Men1, encoding protein of menin, is a key causative gene of multiple endocrine neoplasia type 1 syndrome including pancreatic tumor. It is known that insulin that secretes by endocrine tissue pancreatic islets plays a critical role in hepatic metabolism. Mouse model of hemizygous deletion of Men1 was shown to have severe hepatic metabolism disorders. However, the molecular function of menin on lipid deposition in hepatocytes needs to be further studied. Transcriptome sequencing does show that expression suppression of Men1 in mouse hepatocytes widely affect signaling pathways involved in hepatic metabolism, such as fatty acid metabolism, insulin response, glucose metabolism and inflammation. Further molecular studies indicates that menin overexpression inhibits expressions of the fat synthesis genes Srebp-1c, Fas, and Acc1, the fat differentiation genes Pparγ1 and Pparγ2, and the fat transport gene Cd36, thereby inhibiting the fat accumulation in hepatocytes. The biological process of menin regulating hepatic lipid metabolism was accomplished by interacting with the transcription factor FoxO1, which is also found to be critical for lipid metabolism. Moreover, menin responds to insulin in hepatocytes and mediates its regulatory effect on hepatic metabolism. Our findings suggest that menin is a crucial mediation factor in regulating the hepatic fat deposition, suggesting it could be a potential important therapeutic target for NAFLD.

GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming.

Background & AimsEfforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive models that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a non-cell autonomous role for loss of menin in neuroendocrine cell specification, resulting in an induction of gastrin in enteric glia. Here, we investigated the hypothesis that cell autonomous Men1 inactivation in glial fibrillary acidic protein (GFAP)-expressing cells induced neuroendocrine differentiation and tumorigenesis.MethodsTransgenic GFAPΔMen1 mice were generated by conditional GFAP-directed Men1 deletion in GFAP-expressing cells. Cre specificity was confirmed using a tdTomato reporter. GFAPΔMen1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. Small interfering RNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel.ResultsGFAPΔMen1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAPΔMen1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Men1 deletion induced loss of glial-restricted progenitor lineage markers and an increase in neuroendocrine genes, suggesting a reprogramming of GFAP+ cells. Deleting Kif3a, a mediator of Hedgehog signaling, in GFAP-expressing cells attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate. Similar results in the pancreas were observed when Sox10 was used to delete Men1.ConclusionsGFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.

Abstract 728: Tissue-specific tumorigenesis in multiple endocrine neoplasia type 1

Abstract Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer syndrome caused by a germline mutation in the MEN1 gene. While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in specific tissues such as the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in mouse pancreatic exocrine tissue does not result in tumor formation, suggesting a tissue-specific mechanism. Loss of menin activates a menin-interacting protein retinoblastoma-binding-protein 5 (RBBP5). Since RBBP5 transcriptionally regulates DNA methyltransferase 1 (DNMT1), this causes global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. We hypothesize that while RBBP5 is ubiquitously expressed, it exclusively binds to the DNMT1 promoter in MEN1-target-tissues through its recruitment by tissue-specific factors. Using ChIP-PCR, we demonstrated that Rbbp5 is bound to the Dnmt1 promoter in MEN1-target-tissues, while not in non-target tissues. In the present study, we set out to identify and validate putative endocrine-specific factors involved in Rbbp5 binding to the Dnmt1 promoter. To investigate this tissue-specific occupancy of Rbbp5 at the Dnmt1 promoter, a ChIP-Seq analysis was carried out on DNA purified from pancreatic endocrine islet cells and pancreatic exocrine cells from wild type (WT) mice. These DNA samples were analyzed to identify differentially occupied DNA binding sites of menin and Rbbp5 by a high-throughput tissue-specific ChIP-seq assay. This analysis yielded a list of endocrine-specific genes regulated by both menin and Rbbp5. In order to further identify the tissue-specific factors that may alter menin-Rbbp5 binding to the Dnmt1 promoter, we performed tissue-specific RNA-Seq on endocrine and exocrine pancreas tissues from WT mice. Integrating tissue-specific ChIP-Seq and RNA-seq data, we identified 20 endocrine tissue-specific factors that may alter the interaction of the menin-Rbbp5 complex with the Dnmt1 promoter. By carrying out this unbiased genome-wide screen, we identified endocrine-specific candidate factors that may interact with the menin-Rbbp5 complex at the Dnmt1 promoter. After screening all candidates, the stand-out candidates were identified for further validation. Criteria to be met to establish stand-out candidates included: MEN1-target-tissue-specific expression through immunohistochemical staining and high mRNA expression by qRT-PCR analysis in WT mouse pancreatic islets on samples extracted by laser capture microscopy. In conclusion, we have identified candidates for Rbbp5 recruitment to the Dnmt1 promoter that must be tested further to determine their role in the observed tissue specificity of MEN1-related tumorigenesis. Citation Format: Juliet C. Gardiner. Tissue-specific tumorigenesis in multiple endocrine neoplasia type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 728.

Tissue-Specific Tumorigenesis in Multiple Endocrine Neoplasia Type I

While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in the entire pancreas of a mouse results in tumor formation only in the endocrine pancreas, not in the exocrine pancreas, suggesting an endocrine tissue-specific mechanism. The MEN1 gene encodes the menin protein, which interacts with chromatin associated protein complexes, therefore engaging in epigenetic control mechanisms. Recognizing menin’s participation in epigenetic regulation led to an investigation of whether the pathogenesis of MEN1 syndrome may be related to epigenetic changes in the affected endocrine tissues. Indeed, MEN1-associated endocrine cell types exhibit various menin-dependent epigenetic mechanisms. In fact, a significant increase in methylated DNA loci was observed in MEN1 human parathyroid tumors when compared to human parathyroid adenomas and carcinomas without known MEN1 mutations. Subsequent studies revealed that loss of menin results in increased activity of DNA methyltransferase 1 (Dnmt1). Our studies have shown that Dnmt1 is transcriptionally regulated by the menin-interacting protein Rbbp5. While menin normally functions to suppress Rbbp5 activity, loss of menin activates Rbbp5, thus upregulating Dnmt1 expression, causing global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. In order to assess the behavior of Rbbp5 in both MEN1-target tissues and non-target tissues, Rbbp5 protein expression was analyzed in both MEN1-target tissues (endocrine pancreas, anterior pituitary, parathyroid) and non-MEN1-target tissues (kidney, lung, liver, brain, heart) of wild-type (WT) mice. We confirmed that Rbbp5 protein expression is ubiquitous throughout all of these WT mouse tissues. Since Rbbp5 is a transcriptional activator responsible for enhanced Dnmt1 gene expression, and the loss of menin causes Dnmt1 overexpression solely in MEN1-target tissues, we assessed whether Rbbp5 binds preferentially in a tissue-specific manner to the Dnmt1 promoter. We determined the presence of Rbbp5 on the Dnmt1 promoter in MEN1-target tissues (WT mouse endocrine pancreas, normal human parathyroid, WT mouse pituitary) and the absence of Rbbp5 on the Dnmt1 promoter in non-MEN1-target tissues (WT mouse liver, WT mouse kidney, WT mouse lung). These results confirmed that Rbbp5 does exhibit MEN1-target-tissue-specific occupancy at the Dnmt1 promoter. This endocrine-specific localization of Rbbp5 to the Dnmt1 promoter suggests the presence of additional tissue-specific factors (with tissue-specific expression or interactions/activity) that must be validated and tested further.

Clinicopathologic and molecular characterization of NTRK-rearranged thyroid carcinoma (NRTC)

Primary thyroid neoplasms with actionable NTRK rearrangements are rare, and their clinical behavior, histologic characteristics, and molecular landscape are not well understood. We report an institutional series of eleven NTRK-rearranged thyroid carcinomas (NRTC) by performing clinicopathologic review and next-generation sequencing for targeted mutations and gene rearrangements. The NRTC encompass a histomorphologic spectrum of ten papillary thyroid carcinomas (PTC), including one with high-grade features, and one secretory carcinoma (SC), in ten adults and one adolescent. All NRTC were characterized by an unusual multinodular growth pattern, extensive lymphovascular invasion, and cervical lymph node metastases at initial presentation. Immunophenotypically, while most cases were positive for TTF1 and PAX8, the SC case was negative/weak for these markers and instead diffusely expressed GATA3, mammaglobin and S100. Observed gene rearrangements included ETV6-NTRK3 (n = 4, including the SC), TPR-NTRK1 (n = 2), RBPMS-NTRK3 (n = 2), SQSTM1-NTRK1 (n = 1), SQSTM1-NTRK3 (n = 1), and EML4-NTRK3 (n = 1). Mutation profiling revealed a concurrent TERT promotor mutation C228T in two (22%) patients and a novel frameshift MEN1 deletion in one. All patients received total thyroidectomy and radioactive iodine. Despite frequent development of persistent/recurrent disease (9 cases, 82%) and distant metastases (6 cases; 55%), no tumor-related death occurred over a median (range) follow-up of 44 (11 to 471) months. Three patients received NTRK inhibitor therapy, with the SC case showing complete resolution and two other patients experiencing 33% and 69.7% decrease of disease burden. Although the range of features is variable, NRTC appear to be clinically aggressive tumors with high metastatic rate but relatively low mortality with NTRK inhibitor therapy. The histologic findings of multinodular growth and extensive lymphovascular spread, seen in all NRTC, including PTC and SC, may serve as useful histomorphologic clues to prompt NTRK status testing. We also present the first report of concurrent TERT promotor activating mutation which did not appear to confer entrectinib resistance to NRTC.

Abstract 2080: Tissue-specific tumorigenesis in multiple endocrine neoplasia type I

Abstract While a germline heterozygous mutation in the Multiple Endocrine Neoplasia type 1 (MEN1) gene predisposes tumor formation in specific tissues such as the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in mouse pancreatic exocrine tissue does not result in tumor formation, suggesting a tissue-specific mechanism. Loss of menin activates a menin-interacting protein retinoblastoma-binding-protein 5 (RBBP5). Since RBBP5 transcriptionally regulates DNA methyltransferase 1 (DNMT1), this causes global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. We hypothesize that while RBBP5 is ubiquitously expressed, it exclusively binds to the DNMT1 promoter in MEN1-target-tissues through its recruitment by tissue-specific factors. Using chromatin immunoprecipitation, we demonstrated that Rbbp5 is bound to the Dnmt1 promoter in MEN1-target-tissues, while not in non-target tissues. Following a high-throughput genome-wide approach, we identified two candidate factors that may recruit Rbbp5 to the Dnmt1 promoter. Immunohistochemistry showed MEN1-target-tissue-specific expression of these target factors. Co-immunoprecipitation revealed MEN1-target-tissue-specific binding of Rbbp5 to the factors. In conclusion, Rbbp5 binds the Dnmt1 promoter in MEN1-target-tissues and we have identified candidates for Rbbp5 recruitment to the Dnmt1 promoter that must be tested further to determine their role in the observed tissue specificity of MEN1-related tumorigenesis. Citation Format: Juliet C. Gardiner, Steven K. Libutti. Tissue-specific tumorigenesis in multiple endocrine neoplasia type I [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2080.

OR06-04 Tissue-Specific Tumorigenesis in Multiple Endocrine Neoplasia Type I

While a germline heterozygous mutation in the Multiple Endocrine Neoplasia type 1 (MEN1) gene predisposes tumor formation in specific tissues such as the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in mouse pancreatic exocrine tissue does not result in tumor formation, suggesting a tissue-specific mechanism. Loss of menin activates a menin-interacting protein retinoblastoma-binding-protein 5 (RBBP5). Since RBBP5 transcriptionally regulates DNA methyltransferase 1 (DNMT1), this causes global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. We hypothesize that while RBBP5 is ubiquitously expressed, it exclusively binds to the DNMT1 promoter in MEN1-target-tissues through its recruitment by tissue-specific factors. Using chromatin immunoprecipitation, we demonstrated that Rbbp5 is bound to the Dnmt1 promoter in MEN1-target-tissues, while not in non-target tissues. Following a high-throughput genome-wide approach, we identified two candidate factors that may recruit Rbbp5 to the Dnmt1 promoter. Immunohistochemistry showed MEN1-target-tissue-specific expression of these target factors. Co-immunoprecipitation revealed MEN1-target-tissue-specific binding of Rbbp5 to the factors. In conclusion, Rbbp5 binds the Dnmt1 promoter in MEN1-target-tissues and we have identified candidates for Rbbp5 recruitment to the Dnmt1 promoter that must be tested further to determine their role in the observed tissue specificity of MEN1-related tumorigenesis.

Distinct pathways affected by menin versus MLL1/MLL2 in MLL-rearranged acute myeloid leukemia

Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.

Neuron-Specific Menin Deletion Leads to Synaptic Dysfunction and Cognitive Impairment by Modulating p35 Expression

Menin (MEN1) is a critical modulator of tissue development and maintenance. As such, MEN1 mutations are associated with multiple endocrine neoplasia type 1 (MEN1) syndrome. Although menin is abundantly expressed in the nervous system, little is known with regard to its function in the adult brain. Here, we demonstrate that neuron-specific deletion of Men1 (CcKO) affects dendritic branching and spine formation, resulting in defects in synaptic function, learning, and memory. Furthermore, we find that menin binds to the p35 promoter region to facilitate p35 transcription. As a primary Cdk5 activator, p35 is expressed mainly in neurons and is critical for brain development and synaptic plasticity. Restoration of p35 expression in the hippocampus and cortex of Men1 CcKO mice rescues synaptic and cognitive deficits associated with Men1 deletion. These results reveal a critical role for menin in synaptic and cognitive function by modulating the p35-Cdk5 pathway.

Abstract 1538: A mouse model that mimics human MEN1 tumor is effective in preclinical neuroendocrine tumor therapeutics

Abstract Conditional knockout of Men1 in β cells leads to development of pituitary adenomas and pancreatic neuroendocrine tumors (PanNETs) by the age of eight to twelve months. This suggests that other events, like loss of additional mutations, may be required for development of tumors in the absence of MEN1. Since it was reported that mutations in genes of PI3K/mTOR pathway are found in human neuroendocrine tumors, we hypothesized that double deletion of PTEN and MEN1 may accelerate neuroendocrine tumorigenesis. Mice with double deletion of PTEN and MEN1 in β cells were produced by crosses among mice with Cre gene under the control of Rat Insulin promoter (Rip-Cre), floxed allele Men1 (Men1fl/fl), and Pten (Ptenfl/fl). Then mice with the genotype of Men1fl/fl Ptenfl/fl Rip-Cre were monitored for development of tumors. Consistent with our hypothesis, mice with conditional deletion of PTEN and MEN1 leads to fully developed PanNETs and pituitary adenomas by three to four months old, much earlier than that with Men1 deletion alone. These tumors resemble human MEN1 tumors at a histological and molecular level. Activation of phospho-AKT was observed in these tumors and Rapamycin treatments lead to delayed PanNETs and pituitary adenomas for at least two months. This mouse model (Men1fl/fl Ptenfl/fl Rip-Cre) directly supports that PI3K/mTOR pathway plays a critical role in neuroendocrine tumorigenesis, and also provides a platform to study new therapeutic opportunities for PanNETs and neuroendocrine tumors of the pituitary through the targeting of PI3K/mTOR pathway and MEN1 pathway. This model represents an important tool for studying the biology and preclinical therapeutics of human neuroendocrine tumors. Citation Format: Chung Wong, Richard Clausen, Laura Tang, Christian Davidson, Evan Vosburgh, Arnold Levine, Eugenia Xu. A mouse model that mimics human MEN1 tumor is effective in preclinical neuroendocrine tumor therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1538. doi:10.1158/1538-7445.AM2017-1538

A Potential Role of Neuroendocrine Tumor Gene MEN1 in Reserve Intestinal Stem Cell (rISC) Proliferation

IntroductionNeuroendocrine Tumors (NETs) are rare, slow‐growing neoplasias that feature an excess of hormone‐producing endocrine cells. The occurrence of NETs has long been linked to loss‐of‐function mutations of the tumor suppressor gene, Men1, which encodes the transcription‐factor associating protein menin. Evidence suggests that cancers originate from stem cell populations that acquire driver mutations, resulting in tissue hyperplasia. In the intestine, the cell of origin and underlying mutations giving rise to Gastrointestinal NETs (GINETs) is unknown. The intestinal epithelium is renewed by two populations of stem cells (ISCs), one that actively proliferates (aISCs) and one that is a quiescent ‘reserve’ population (rISC). rISCs express high levels of the transcription factor, SOX9, and also exhibit stem cell as well as endocrine‐like gene expression profiles.HypothesisWe hypothesize that the Men1 gene suppresses stem cell proliferation in the intestinal crypt compartment, and a loss‐of‐function mutation in ISCs catalyzes GI‐NET tumorigenesis.ResultsKi67 immunohistochemistry staining of a tamoxifen‐induced Men1 conditional knockout from a Men1fl/fl:Sox9‐IRES‐CreERT2:Sox9EGFP mouse model trends towards an expansion of the proliferative zone. Interestingly, Pyronin Y flow cytometric analysis of Sox9high cells derived from Men1 knockouts indicates elevated levels of RNA consistent with an active cell cycle, supporting Men1‐dependent cycle re‐entry of the Sox9high population. A FACS (fluorescence activated cell sorting) cell cycle assay using single Sox9high (rISCs) dissociated from a Sox9EGFP mouse demonstrates that Sox9high cells in S/G2/M cell‐cycle state preferentially form organoids compared to the more numerous G0/G1 population. Future studies will evaluate whether the loss of Men1 drives Sox9high cells to preferentially enter S/G2/M, indicating an increase in proliferative potential.ConclusionsEarly evidence of enhanced proliferation within the intestinal crypt compartment suggests that the conditional deletion of Men1 in Sox9 expressing stem cell populations may lead to hyperplasia. While Pyronin Y expression indicates a ‘ramping‐up’ of RNA production prior to cell‐cycle, future studies will evaluate the role of Men1 in driving Sox9high cells into S/G2/M phase. This result would indicate that these cells have undergone a transformation in proliferative potential consistent with our observed increase in organoid formation efficiency of the rISC population. Longitudinal in vivo studies are in progress to evaluate NET formation due to Men1 deletion alone and in combination with other candidate tumor suppressor genes.Support or Funding InformationKevin was supported through funding by the American Physiological Society's Undergraduate Summer Research Fellowship. Additional funding was provided by the University Cancer Research Fund Development Grant. Special thanks for the support provided from the following: Magness Lab, UNC Gastrointestinal Stem Cell Group, UNC Advanced Analytics Core, UNC Zeiss Confocal Microscopy Core, and NIH T35‐DK007386.

Loss of menin in osteoblast lineage affects osteocyte\u2013osteoclast crosstalk causing osteoporosis

During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10 (CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data reveal a novel role for Men1 in osteocyte–osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.

Deletion of Men1 and somatostatin induces hypergastrinemia and gastric carcinoids

BackgroundGastric carcinoids are slow growing neuroendocrine tumours arising from enterochromaffin-like (ECL) cells in the corpus of stomach. Although most of these tumours arise in the setting of gastric atrophy and...

The Cell Death Inhibitor ARC Is Induced in a Tissue-Specific Manner by Deletion of the Tumor Suppressor Gene Men1, but Not Required for Tumor Development and Growth.

Multiple endocrine neoplasia type 1 (MEN1) is a genetic disorder characterized by tissue-specific tumors in the endocrine pancreas, parathyroid, and pituitary glands. Although tumor development in these tissues is dependent upon genetic inactivation of the tumor suppressor Men1, loss of both alleles of this gene is not sufficient to induce these cancers. Men1 encodes menin, a nuclear protein that influences transcription. A previous ChIP on chip analysis suggested that menin binds promoter sequences of nol3, encoding ARC, which is a cell death inhibitor that has been implicated in cancer pathogenesis. We hypothesized that ARC functions as a co-factor with Men1 loss to induce the tissue-restricted distribution of tumors seen in MEN1. Using mouse models that recapitulate this syndrome, we found that biallelic deletion of Men1 results in selective induction of ARC expression in tissues that develop tumors. Specifically, loss of Men1 in all cells of the pancreas resulted in marked increases in ARC mRNA and protein in the endocrine, but not exocrine, pancreas. Similarly, ARC expression increased in the parathyroid with inactivation of Men1 in that tissue. To test if ARC contributes to MEN1 tumor development in the endocrine pancreas, we generated mice that lacked none, one, or both copies of ARC in the context of Men1 deletion. Studies in a cohort of 126 mice demonstrated that, although mice lacking Men1 developed insulinomas as expected, elimination of ARC in this context did not significantly alter tumor load. Cellular rates of proliferation and death in these tumors were also not perturbed in the absence of ARC. These results indicate that ARC is upregulated by loss Men1 in the tissue-restricted distribution of MEN1 tumors, but that ARC is not required for tumor development in this syndrome.