Ras-responsive Element Binding Protein Research Articles

A novel interaction between RNA m6A methyltransferase METTL3 and RREB1

Regulation of gene expression is achieved through the modulation of regulatory inputs both pre- and post-transcriptionally. Methyltransferase-like 3 (METTL3) is a key player in pre-mRNA processing, actively catalyzing N6-methyladenosine (m6A). Among the most enriched mRNA targets of METTL3 is the Ras Responsive Element Binding Protein 1 (RREB1), a transcription factor which functions to govern cell fate, proliferation and DNA repair. Here, we show a novel interaction between METTL3 and RREB1. Further examination of this interaction indicates that METTL3's N-terminus is the primary interacting domain. Our findings uncover a novel interacting partner of METTL3, providing further insights into METTL3's regulatory network.

TGF-β and RAS jointly unmask primed enhancers to drive metastasis

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor β (TGF-β) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-β and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1, priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-β. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-β gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth.

Loss of RREB1 reduces adipogenesis and improves insulin sensitivity in mouse and human adipocytes.

There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1 +/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1 +/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

A Proteomics Approach Identifies RREB1 as a Crucial Molecular Target of Imidazo-Pyrazole Treatment in SKMEL-28 Melanoma Cells.

Cutaneous melanoma is the most dangerous and deadly form of human skin malignancy. Despite its rarity, it accounts for a staggering 80% of deaths attributed to cutaneous cancers overall. Moreover, its final stages often exhibit resistance to drug treatments, resulting in unfavorable outcomes. Hence, ensuring access to novel and improved chemotherapeutic agents is imperative for patients grappling with this severe ailment. Pyrazole and its fused systems derived thereof are heteroaromatic moieties widely employed in medicinal chemistry to develop effective drugs for various therapeutic areas, including inflammation, pain, oxidation, pathogens, depression, and fever. In a previous study, we described the biochemical properties of a newly synthesized group of imidazo-pyrazole compounds. In this paper, to improve our knowledge of the pharmacological properties of these molecules, we conduct a differential proteomic analysis on a human melanoma cell line treated with one of these imidazo-pyrazole derivatives. Our results detail the changes to the SKMEL-28 cell line proteome induced by 24, 48, and 72 h of 3e imidazo-pyrazole treatment. Notably, we highlight the down-regulation of the Ras-responsive element binding protein 1 (RREB1), a member of the zinc finger transcription factors family involved in the tumorigenesis of melanoma. RREB1 is a downstream element of the MAPK pathway, and its activation is mediated by ERK1/2 through phosphorylation.

RREB1 regulates neuronal proteostasis and the microtubule network.

Transcription factors play vital roles in neuron development; however, little is known about the role of these proteins in maintaining neuronal homeostasis. Here, we show that the transcription factor RREB1 (Ras-responsive element-binding protein 1) is essential for neuron survival in the mammalian brain. A spontaneous mouse mutation causing loss of a nervous system-enriched Rreb1 transcript is associated with progressive loss of cerebellar Purkinje cells and ataxia. Analysis of chromatin immunoprecipitation and sequencing, along with RNA sequencing data revealed dysregulation of RREB1 targets associated with the microtubule cytoskeleton. In agreement with the known role of microtubules in dendritic development, dendritic complexity was disrupted in Rreb1-deficient neurons. Analysis of sequencing data also suggested that RREB1 plays a role in the endomembrane system. Mutant Purkinje cells had fewer numbers of autophagosomes and lysosomes and contained P62- and ubiquitin-positive inclusions. Together, these studies demonstrate that RREB1 functions to maintain the microtubule network and proteostasis in mammalian neurons.

Oncogenic KRAS effector USP13 promotes metastasis in non-small cell lung cancer through deubiquitinating β-catenin

KRAS mutations are frequently detected in non-small cell lung cancers (NSCLCs). Although covalent KRASG12C inhibitors have been developed to treat KRASG12C-mutant cancers, effective treatments are still lacking for other KRAS-mutant NSCLCs. Thus, identifying a KRAS effector that confers poor prognosis would provide an alternative strategy for the treatment of KRAS-driven cancers. Here, we show that KRAS drives expression of deubiquitinase USP13 through Ras-responsive element-binding protein 1 (RREB1). Elevated USP13 promotes KRAS-mutant NSCLC metastasis, which is associated with poor prognosis in NSCLC patients. Mechanistically, USP13 interacts with and removes the K63-linked polyubiquitination of β-catenin at lysine 508, which enhances the binding between β-catenin and transcription factor TCF4. Importantly, we identify 2-methoxyestradiol as an effective inhibitor for USP13 from a natural compound library, and it could potently suppress the metastasis of KRAS-mutant NSCLC cells invitro and invivo. These findings identify USP13 as a therapeutic target for metastatic NSCLC with KRAS mutations.

Moderate-intensity endurance training improves late phase β-cell function in adults with type 2 diabetes

Physical activity is important for type 2 diabetes treatment, yet the underlying mechanisms for these beneficial effects of exercise are not fully understood. Here, we investigated the effects of exercise training on biphasic β-cell insulin secretory function, a key factor regulating blood glucose. Adults with type 2 diabetes (7F/3M, age 49± 5 years, BMI 30± 3kg/m2) completed a 10-week moderate-intensity exercise program and multiple components of glucose homeostasis were measured. Training improved glycemic control, insulin sensitivity, and processing of proinsulin-to-insulin. Training increased late phase β-cell function by 38% (p= 0.01), which was correlated with changes in VO2peak suggesting training response-dependent effects. Ras-Responsive Element Binding Protein 1 (RREB1) concentrations, a protein postulated to increase type 2 diabetes risk, were inversely correlated with increases in training-induced late-phase disposition index, consistent with an inhibitory role of RREB1 on insulin secretion. Moderate-intensity exercise training improves late-phase β-cell function and glycemic control in adults with type 2 diabetes.

Abstract PR004: Mechanistic basis for TGF-β-induced fibrogenic EMTs in metastasis

Abstract The cytokine TGF-β is a central regulator of tissue homeostasis and regeneration through multiple coordinated effects on epithelial, immune, and mesenchymal stromal systems. Malfunctions of TGF-β signaling cause fibrosis, immune dysfunction, and cancer. As part of its multifunctional program, TGF-β induces epithelial-mesenchymal transitions (EMTs). Carcinoma cells use TGF-β to undergo EMT and adopt a highly plastic phenotype that facilitates tumor growth and metastasis. Notably, TGF-β-induced EMTs in epithelial progenitor cells are accompanied by the expression of fibrogenic factors that activate fibroblasts to produce and remodel the extracellular matrix. This not only occurs during wound healing but also in lung adenocarcinoma (LUAD) and pancreatic adenocarcinoma (PDAC) cells, suggesting that EMT and fibrogenesis are parts of an orchestrated program. We recently showed that the transcription factor RAS-Responsive Element-Binding Protein 1 (RREB1) activated by RAS-MAPK signaling synergizes with SMADs to coordinately activate the expression of EMT transcription factor Snai1 and fibrogenic genes in carcinoma progenitors (Su et al Nature 2020). Using metastasis transplantation models, we have now found that both arms of the RREB1-dependent TGF-β responses are important for metastasis. Knockout of individual fibrogenic genes in cancer cells inhibited metastasis without altering the induction of EMT by TGF-β. To address why these TGF-β target genes specifically require KRAS-activated RREB1 for transcriptional induction, we employed proteomics approaches coupled with a CRISPR-based genetic screen. With this approach we identified DXH9 and INO80 as RREB1-interacting factors essential for TGF-β activation of fibrogenic EMT programs in PDAC and LUAD cells. DXH9 and INO80 are helicases that interact and SMAD3 and RREB1 in response to TGF-β. Knockout of Dhx9 or Ino80 abolished the induction of Snai1 and fibrogenic genes by TGF-β, inhibited the LUAD metastasis, and decreased intratumoral fibrosis, thus phenocopying the knockout of Rreb1. The helicase domain of DHX9 is required for TGF-β gene responses and metastasis. Our evidence indicates that RREB1, DXH9, and INO80 function to remodel the chromatin at specific loci that constitute a TGF-β-dependent fibrogenic EMT program. This work illuminates a previously unknown cooperation between TGF-β and RAS-MAPK pathways during epithelial tissue regeneration and its cooption in carcinoma metastasis. Citation Format: Jun Ho Lee, Harihar Basnet, Francisco Sánchez-Rivera, Zhenghan Wang, Liangji Li, Joan Massagué. Mechanistic basis for TGF-β-induced fibrogenic EMTs in metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR004.

Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression

Aims/hypothesisGenome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk.MethodsA zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-βH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles.ResultsCRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-βH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-βH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function.Conclusions/interpretationTogether, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.Graphical abstract

A novel method to assess copy number variation in melanoma: Droplet digital PCR for precise quantitation of the RREB1 gene in formalin-fixed, paraffin-embedded melanocytic neoplasms, a proof-of-concept study.

Melanocytic neoplasms can be challenging to diagnose. One well-established diagnostic aid is the detection of copy number variation (CNV) in a few key genetic loci using conventional methods such as fluorescence in situ hybridization (FISH) and chromosomal microarray (CMA). Droplet digital polymerase chain reaction (ddPCR) is a novel, cost-effective, rapid, and automated method to detect CNV. We perform the first investigation of ddPCR to assay Ras-responsive element-binding protein-1 (RREB1), the most common CNV in melanoma using formalin-fixed, paraffin-embedded (FFPE) melanocytic lesion samples; CMA data are used as the gold standard. Archival samples from 2013 to 2021 were analyzed, including 153 data points from 39 FFPE samples representing 34 patients. Benign, borderline, malignant, and metastatic melanocytic neoplasms were examined. ddPCR showed a sensitivity and specificity of 93.8% and 95.7% using one reference gene, and 87.5% and 100% using a different reference gene for RREB1 gain detection. Here we show that ddPCR can provide inexpensive, rapid, and robust data on the commonest copy number alteration in melanoma. Future development and validation could provide a useful ancillary tool in the diagnosis of challenging melanocytic lesions.

244-OR: Moderate Intensity Endurance Exercise Training Improves Late-Phase ß-Cell Function in Humans with Type 2 Diabetes

Type 2 diabetes (T2D) is associated with β-cell dysfunction. Although exercise is considered beneficial in T2D, the effects of moderate intensity endurance exercise training on β-cell function in humans with T2D are not known. GWAS uncovered Ras-Responsive Element Binding Protein 1 (RREB1) as a risk factor for T2D and in vitro studies suggest RREB1 impairs β-cell function; no studies have investigated exercise effects on RREB1. Here, we determined the effects of moderate intensity exercise training on β-cell function, RREB1, insulin, glucagon, and amylin in humans with T2D (7F/3M, 49±5y, BMI 30±3kg/m2) . Subjects completed 10-weeks of supervised exercise (Wks 1-3: 20-30min/day; 3X/wk; ∼50% VO2peak and Wks 4-10: 45-60min/day; 4X/wk; ∼70% VO2peak) . Fasting blood samples for OGTT, HbA1c, and hormones were obtained pre- and post-training (48hr) . Serum RREB1 was measured pre- and post-training and also in response to a single bout of maximal exercise. Training decreased HbA1c (7.7% to 7.2%, P=0.01) , pro-insulin (-18%, P=0.03) , pro-insulin/c-peptide ratio (-8%, P=0.03) , and glucose (OGTT: 90min, -18%, P=0.01; 120min, -15%, P<0.001) . Both β-cell function (late-phase disposition index, +38%, P=0.01) and insulin sensitivity (Stumvoll Index, +25%, P=0.01) improved. Body weight and composition, VO2peak, glucagon, amylin, c-peptide, and insulin were unchanged. Baseline RREB1 negatively correlated with the exercise-induced increase in late-phase disposition index (R= -0.71, P=0.03) , consistent with an inhibitory role of RREB1. Training did not alter serum RREB1, but interestingly, acute exercise decreased RREB1 both before (-51%, P<0.01) and after training (-11%, P=0.04) . Thus, RREB1 is an exercise-regulated serum factor whose decrease may function to improve β-cell function with exercise. In conclusion, moderate intensity exercise training, in the absence of increased VO2peak, improves late phase β-cell function and glucose control in humans with T2D. Disclosure H.Zhang: None. R.Middelbeek: Research Support; Novo Nordisk. L.Simpson: None. N.P.Carbone: None. L.J.Goodyear: None. Funding National Institutes of Health (K23-DK114550, R01-DK112283, 5P30DK36836, 5P30DK057521-20)

Ras signaling and RREB1 are required for the dissociation of medial edge epithelial cells in murine palatogenesis

ABSTRACTCleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial–mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

MiR-26a enhances colorectal cancer cell growth by targeting RREB1 deacetylation to activate AKT-mediated glycolysis.

We previously reported the inhibitory effects of microRNA-26a (miR-26a) on the conversion of pyruvate to acetyl coenzyme A in glucose metabolism by directly targeting pyruvate dehydrogenase protein X component in colorectal cancer (CRC) cells (Chen B et al., BMC Cancer 2014). Here, using microRNA in situ hybridization, we confirmed that miR-26a levels were elevated in 77 human CRC tissue samples and further investigated the key miR-26a-mediated metabolic regulation elements and signaling pathways in CRC cells through quantitative proteomic dissection combined with cancer cell biology and biochemical loss-of-function analysis. We found that AKT transcription signaling was a target pathway via miR-26a-mediated deacetylation modification of Ras-responsive element-binding protein 1 (RREB1) at the Lys-60 residue. miR-26a improved the deacetylation level of RREB1, thus contributing to RREB1 binding to the AKT1 promoter to activate AKT transcription and its related signaling pathway in glycolysis. Moreover, miR-26a promoted CRC tumorigenesis in CRC cells and subcutaneous xenograft mice. Thus, miR-26a is a key regulator of CRC tumorigenesis that mediates the deacetylation modification of RREB1 to enhance AKT1 transcription and downstream target gene expression in glycolysis for CRC growth.

Knockdown of RREB1 inhibits cell proliferation via enhanced p16 expression in gastric cancer

ABSTRACT Gastric cancer (GC) is the most common gastrointestinal malignancy worldwide. However, the molecular mechanisms of the progression of GC are not fully understood. Ras-responsive element binding protein 1 (RREB1) is an oncogene in many types of cancer that is involved in various biological processes, such as DNA damage repair, cell growth and proliferation, cell differentiation, fat development, and fasting glucose balance. In this study, we demonstrate the role of RREB1 in gastric cancer. First, by immunohistochemistry staining (IHC) and bioinformatics analysis, we demonstrated the expression of RREB1 in gastric cancer and paired normal gastric tissues. Then, we established RREB1 overexpression and knockdown cell lines via lentiviral transfection and detected cell proliferation by using MTT, colony-forming, cell cycle and apoptosis assays in vitro. We demonstrated the effect of RREB1 on cell proliferation in vivo by using a subcutaneous xenograft tumor model in nude mice. Finally, by using Western blotting and IHC, we demonstrated the possible mechanism by which RREB1 affects cell proliferation. The IHC and bioinformatics analyses demonstrated that RREB1 was highly expressed in gastric cancer and showed that RREB1-expressing patients had a larger tumor size and more lymphovascular invasion than RREB1-negative patients. Knockdown of RREB1 inhibited cell proliferation in vivo and in vitro. Knockdown of RREB1 enhanced p16 expression in vivo and in vitro, and p16 expression was negatively related to RREB1 in gastric cancer tissue. RREB1 was highly expressed in gastric cancer, and knockdown of RREB1 inhibited cell proliferation via enhanced p16 expression.

Transcriptome analysis reveals hub genes in the hepatopancreas of Exopalaemon carinicauda in response to hypoxia and reoxygenation

Hypoxia is a common stressor in shrimp, but the molecular mechanisms underlying the adaptation of the ridgetail white prawn, Exopalaemon carinicauda, to hypoxia and reoxygenation remain poorly understood. In the present study, ridgetail white prawns were exposed to gradual changes in hypoxia for 6 h via oxygen consumption in a static respiration chamber and then rapidly aerated to allow reoxygenation for 8 h. The dissolved oxygen concentration after 6 h of hypoxia was 1.04 mg/L, and the survival rate of the shrimp was 47%. A transcriptomic analysis of hepatopancreas tissues after 0 (control), 3 (hypoxic starting point), and 6 (hypoxic lethal half point) h of exposure to hypoxia and after 1 and 8 h of reoxygenation was conducted using the Illumina HiSeqTM 4000 high-throughput sequencing platform. A total of 93,227 genes were obtained, and 4315 of these genes were identified as differentially expressed genes (DEGs) that respond to hypoxia and reoxygenation. A KEGG pathway enrichment analysis suggested that all identified DEGs were mostly enriched in ribosome biogenesis in eukaryotes, apoptosis, the longevity regulating pathway, the MAPK signaling pathway, and protein processing in the endoplasmic reticulum. In addition, 20,203 genes with RPKM values ≥1 were parsed into 20 modules through a weighted gene coexpression network (WGCNA), and three of these modules were related to hypoxia and reoxygenation. GO and KEGG enrichment analyses of these three modules were then performed, and three hub genes were identified based on their connectivity: RREB1 (Ras-responsive element-binding protein 1-like), UBE1 (ubiquitin-activating enzyme E1), and an unknown gene. This study might help further elucidate the hypoxia tolerance mechanism of E. carinicauda.

The transcription factor Rreb1 regulates epithelial architecture, invasiveness, and vasculogenesis in early mouse embryos.

Ras-responsive element-binding protein 1 (Rreb1) is a zinc-finger transcription factor acting downstream of RAS signaling. Rreb1 has been implicated in cancer and Noonan-like RASopathies. However, little is known about its role in mammalian non-disease states. Here, we show that Rreb1 is essential for mouse embryonic development. Loss of Rreb1 led to a reduction in the expression of vasculogenic factors, cardiovascular defects, and embryonic lethality. During gastrulation, the absence of Rreb1 also resulted in the upregulation of cytoskeleton-associated genes, a change in the organization of F-ACTIN and adherens junctions within the pluripotent epiblast, and perturbed epithelial architecture. Moreover, Rreb1 mutant cells ectopically exited the epiblast epithelium through the underlying basement membrane, paralleling cell behaviors observed during metastasis. Thus, disentangling the function of Rreb1 in development should shed light on its role in cancer and other diseases involving loss of epithelial integrity.

MBRS-12. A TRANSPOSON MUTAGENESIS SCREEN IDENTIFIES Rreb1 AS A DRIVER FOR GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB can be divided into four major subgroups – WNT, Sonic hedgehog (SHH), Group 3 (G3), and Group 4 (G4) – that exhibit distinct genetic alterations, gene expression profiles, and clinical outcomes. Patients with G3-MB have the worst prognosis, and a deeper understanding of this disease is critical for development of new therapies. Most G3-MBs express high levels of the MYC oncogene, suggesting that MYC plays an important role in tumorigenesis. To identify genes that cooperate with MYC to promote formation of G3-MB, we performed an in vivo mutagenesis screen using mice expressing the Sleeping Beauty (SB) transposon. Cerebellar stem cells from transposon/transposase-expressing mice were infected with viruses encoding Myc, and transplanted into the cerebellum of adult hosts. The resulting tumors were sequenced to identify transposon-targeted genes, and these genes were functionally analyzed to determine whether they could cooperate with Myc to drive G3-MB. These studies identified the transcription factor Ras-responsive element binding protein 1 (Rreb1) as a potent Myc-cooperating gene. Tumors driven by Myc and Rreb1 resemble G3-MB at a histological and molecular level. Moreover, RREB1 is overexpressed in human G3-MB, and knockdown of RREB1 impairs growth of G3-MB cell lines and patient-derived xenografts. Ongoing studies are aimed at identifying the mechanisms by which Rreb1 contributes to tumor growth. Our studies demonstrate an important role for RREB1 in G3-MB, and provide a new model that can be used to identify therapeutic targets and develop more effective therapies for medulloblastoma.

TMOD-30. IDENTIFYING NEW DRIVERS OF GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB can be divided into four major subgroups – WNT, Sonic hedgehog (SHH), Group 3 (G3-MB), and Group 4 (G4-MB) – that exhibit distinct genetic alterations, gene expression profiles, and clinical outcomes. Patients with G3-MB have the worst prognosis, and a deeper understanding of this form of the disease is critical for development of new therapies. Most G3-MBs express high levels of the MYC oncogene, suggesting that MYC plays an important role in tumorigenesis. However, MYC overexpression is not sufficient to drive tumor formation. To identify genes that cooperate with MYC to promote development of G3-MB, we performed an in vivo mutagenesis screen using mice expressing the Sleeping Beauty (SB) transposon. Cerebellar stem cells isolated from transposon/transposase-expressing transgenic mice were infected with viruses encoding Myc, and these cells were transplanted into the cerebellum of adult hosts. Tumors that arose were subjected to DNA and RNA sequencing to identify candidate genes, and these genes were subjected to functional analysis to determine whether they could cooperate with Myc to drive G3-MB. These studies identified the transcription factor Ras-responsive element binding protein 1 (Rreb1) as a potent Myc-cooperating gene. Tumors driven by Myc and Rreb1 (MR tumors) resemble G3-MB at a histological and molecular level. Moreover, RREB1 is overexpressed in human G3-MB, and knockdown of RREB1 expression impairs growth of G3-MB cell lines and patient-derived xenografts. Ongoing studies are aimed at identifying the molecular mechanisms by which Rreb1 contributes to tumor growth. Our studies demonstrate an important role for RREB1 in G3-MB, and provide a new model that can be used to identify therapeutic targets and develop more effective and less toxic therapies for this devastating pediatric brain tumor.

Haploinsufficiency of RREB1 causes a Noonan-like RASopathy via epigenetic reprogramming of RAS-MAPK pathway genes

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.

Functional Analysis of Two Zinc (Zn) Transporters (ZIP3 and ZIP8) Promoters and Their Distinct Response to MTF1 and RREB1 in the Regulation of Zn Metabolism.

ZIP (zinc-regulated transporters, iron-regulated transporter-like protein) family plays an important role in organism Zn balance. This research identified the promoter regions of ZIP3 and ZIP8, two members of ZIP family, from a freshwater teleost yellow catfish Pelteobagrus fulvidraco, characterized the binding sequences of the metal-responsive transcription factor-1 (MTF-1) and Ras responsive element binding protein 1 (RREB1) on their promoter regions. The present study cloned and obtained the 2027 bp of ZIP3 promoter and 1664 bp of ZIP8 promoter, and predicted several key elements on their promoters, such as the binding sites of CREB (cAMP-response element binding protein), KLF4 (Kruppel like factor 4), MTF-1 and RREB1. The sequence deletion from −361 bp to −895 bp down-regulated the luciferase activity of ZIP3 promoter, and the deletion from −897 bp to −1664 bp down-regulated the luciferase activity of ZIP8 promoter. Within different deletion plasmids, the relative luciferase activities of ZIP3 and ZIP8 promoters changes to Zn incubation in a Zn concentration-dependent manner. The site mutagenesis and EMSA (electrophoretic mobility shift assay) found that the −1327 bp/−1343 bp MTF-1 binding site and the −248 bp/−267 bp RREB1 binding site on the ZIP3 promoter, and the −1543 bp/−1557 bp MTF-1 binding site on the ZIP8 promoter are functional sites. Low Zn increased the binding capability between MTF-1 and its responsive site on the ZIP3 promoter, and high Zn increased the transcriptional activation ZIP3 by RREB1; Zn also promoted the binding ability between MTF-1 and its responsive element on the ZIP8 promoter. This study provides the first direct evidence for the response elements of MTF-1 and RREB1 on ZIP3 and MTF-1 on ZIP8 to Zn, which are very important for the evaluation of Zn nutrition and toxicity in vertebrates.