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The present electrochemical study deals with the evaluation of a complexation process involving different metal and ligands and its physicochemical properties to find out the ligand- metal ratio of complex in solution. For the determination of complexing nature “Monovariation method” have been used to ascertain the ligand metal ratio in the complex. The stability constant of the formed complexes were calculated by conductance measurement using Modified Job’s method (Turner Anderson Method). The analysis had been carried out by using conductometric principle and the final outcome of the experiment may influenced by temperature and with the ionic concentration of metal and ligand solution. Free energy change values were determined to find out the feasibility of the complexation process. One of the method was validated statistically by using system and method precision parameters. Lastly the complexes was been assayed by using a specific chromogenic agent with a specific spectroscopic technique that may give biological action in future.

The identification of cancer maintenance genes—driver genes essential to tumor survival—is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentialityin vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, andKcnb2knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

Differentiation of T and B cells to effector and memory cell fates are associated with extensive metabolic changes which are accompanied by altered mitochondrial dynamics. However, whether alterations in mitochondrial structure and function plays an active role in regulating effector versus memory cell fate decisions during immune responses remains unclear. Our studies here characterize changes in mitochondrial dynamics in activated B cells and show that increased mitochondrial mass and activity is a distinct feature of memory B cell lineage commitment in vivo. Using a directed screen of mitochondrial modulators, we identify mitochondrial fission inhibitor, Mdivi-1 as an agent that could enhance mitochondrial mass and function leading to augmented memory B cell differentiation. The enhanced memory B cell responses mediated by Mdivi-1, translated to more robust recall responses upon secondary antigen exposures. Moreover, Mdivi-1 when used in combination with subunit (SARS-CoV2) and inactivated (H1N1 influenza) vaccines led to remarkably improved vaccine efficacies and protection from lethal viral (H1N1) challenge. Single-cell transcriptomics revealed enhanced commitment to memory lineage differentiation in B cells following Mdivi-1 treatment. We propose that mitochondrial modulators such as Mdivi-1 are a novel class of “immune enhancers” that specifically reinforces immunological memory and could be broadly applied to improve the fidelity of immune responses and vaccine efficacies.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs—clusters of around 10–100 ecDNAs within the nucleus—enable intermolecular enhancer–gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer–gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy. Extrachromosomal DNA (ecDNA) congregates in clusters called ecDNA hubs that promote intermolecular interactions between gene-regulatory regions and thereby amplify the expression of oncogenes such as MYC in cancer cell lines.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of AML driven by the most common AF10 fusion proteins, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a comprehensive map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent anti-oncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.STATEMENT OF SIGNIFICANCEGene fusions of AF10/MLLT10 are recurrent in acute myeloid and lymphoid leukemia and are associated with extremely poor survival outcomes. We show that the JAK1 kinase is required for activation of the AF10 fusion oncotranscriptome and for leukemogenesis. Since a number of JAK/STAT pathways inhibitors are in clinical development or approved for use, our studies may help develop a therapeutic strategy for AF10-rearranged leukemias.

Background & Aims Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. Methods We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography–mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. Results EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. Conclusions HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Glutamine deficient pancreatic cancer cells upregulate classic EMT regulator Slug, providing a link between nutrient stress and metastasis., In this issue of JEM, Recouvreux et al. (https://doi.org/10.1084/jem.20200388) describe the role of nutrient sensing in the induction of epithelial–mesenchymal transition. Glutamine-deficient pancreatic cancer cells up-regulate classic EMT regulator Slug, providing a link between nutrient stress and metastasis.

Enzymatic activity of Telomerase Reverse Transcriptase (TERT) is important in maintaining the telomere length and has been implicated in cancer and aging related pathology. Since cancer susceptibility as well as longevity of dogs vary between breeds, this study involved sequencing the entire TERT gene of Canis familiaris from DNA samples obtained from forty dogs, with ten dogs each of four breeds: Shih Tzu, Dachshund, Irish Wolfhound, and Newfoundland, each with different life expectancies and susceptibility to cancer.We compared the sequences of all forty individuals amongst one another and with the published sequence of canine TERT, and analyzed relationships between members of the same or different breeds. Two separate phylogenetic trees were generated and analyzed from these individuals. Polymorphisms were found most frequently in intronic regions of the gene, although exonic polymorphisms also were observed. In many locations genotypes were observed that were either homozygous for the reference sequence or heterozygous, but the variant homozygous genotype was not observed.We propose that these homozygous variants are likely to have adverse effects in dogs. It was also found that the polymorphisms did not segregate by breed. Because the four breeds chosen come from geographically and physiologically distinct backgrounds, it can be inferred that the polymorphic diversification of TERT preceded breed derivation.

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1, BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin-LacZ and Gremlin1-LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin-LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1-LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671-1682, 2017.

Over the last several decades, multiple recurrent chromosomal amplifications and deletions have been detected in a large number of cancers. These regions of amplification and deletion can encompass a few to several hundred genes. Determining which of these genes is causing the outgrowth of the cancer is difficult. Complicating the analysis is the fact that several genes within the affected chromosomal region may cooperate to promote tumorigenesis. In this protocol we describe a method of chromosomal engineering in mice that allows modeling of chromosomal duplications and deficiencies. This method faithfully recapitulates several aspects of chromosomal loss and gain in human cancers and can reveal cancer drivers difficult to identify by other means.

Over the last several decades, multiple recurrent chromosomal amplifications and deletions have been detected in a large number of cancers. These regions of amplification and deletion can encompass a few to several hundred genes. Determining which of these genes is causing the outgrowth of the cancer is difficult. Complicating the analysis is the fact that several genes within the affected chromosomal region may cooperate to promote tumorigenesis. In this protocol we describe a method of chromosomal engineering in mice that allows modeling of chromosomal duplications and deficiencies. This method faithfully recapitulates several aspects of chromosomal loss and gain in human cancers and can reveal cancer drivers difficult to identify by other means.

An UV spectrophotometric method using simultaneous equation was developed for the simultaneous determination of ranitidine and itopride in a binary mixture. In the proposed method, the signals were measured at 315.0 and 258.0 nm corresponding to absorbance maxima of ranitidine and itopride hydrochloric acid (HCl) in double distilled water, respectively. Linearity range was observed in the concentration range of 2 to 20 µg/ml for both drugs. Concentration of each drug was obtained by using the absorptivity values calculated for both drugs at two wavelengths, 315.0 and 258.0 nm and solving the simultaneous equation. Developed method was applied to laboratory mixture. The method was validated statistically and recovery study was performed to confirm the accuracy of the method. The method was found to be rapid, simple, accurate and precise. Key words: Itopride, simultaneous equation, spectrophotometry, ranitidine.

Pain is a hallmark feature of sickle cell disease (SCD). Recurrent and unpredictable acute pain due to vaso-oclussive crises (VOC) is unique to SCD; and can be superimposed on chronic pain. To examine the mechanisms underlying pain in SCD, we performed RNA sequencing of dorsal root ganglion (DRG) of transgenic sickle mice and their age-matched control mice expressing normal human hemoglobin A, at 2 and 5 months of age. Sickle and control mice of both ages were equally divided into hypoxia/reoxygenation (to simulate VOC) and normoxia treatment, resulting in eight groups of mice. Each group had at least six mice. RNA isolated from the DRG was sequenced and paired-end 50 bp sequencing data were generated using Illumina’s HiSeq 2000. This large dataset can serve as a resource for examining transcriptional changes in the DRG that are associated with age and hypoxia/reoxygenation associated signatures of nociceptive mechanisms underlying chronic and acute pain, respectively.

Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p Disclosures Gupta: 1910 Genetics: Research Funding.

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1, BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin-LacZ and Gremlin1-LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin-LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1-LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671-1682, 2017.

Copy number gain of the 8q24 region including the v-myc avian myelocytomatosis viral oncogene homolog (MYC) oncogene has been observed in many different cancers and is associated with poor outcomes. While the role of MYC in tumor formation has been clearly delineated, we have recently shown that co-operation between adjacent long non-coding RNA plasmacytoma variant transcription 1 (PVT1) and MYC is necessary for tumor promotion. Chromosome engineered mice containing an increased copy of Myc-Pvt1 (Gain Myc-Pvt1) accelerates mammary tumors in MMTV-Neu mice, while single copy increase of each is not sufficient. In addition, mammary epithelium from the Gain Myc-Pvt1 mouse show precancerous phenotypes, notably increased DNA replication, elevated -H2AX phosphorylation and increased ductal branching. In an attempt to capture the molecular signatures in pre-cancerous cells we utilized RNA sequencing to identify potential targets of supernumerary Myc-Pvt1 cooperation in mammary epithelial cells. In this extra view we show that an extra copy of both Myc and Pvt1 leads to increased levels of Rspo1, a crucial regulator of canonical β-catenin signaling required for female development. Human breast cancer tumors with high levels of MYC transcript have significantly more PVT1 transcript and RSPO1 transcript than tumors with low levels of MYC showing that the murine results are relevant to a subset of human tumors. Thus, this work identifies a key mechanism in precancerous and cancerous tissue by which a main player in female differentiation is transcriptionally activated by supernumerary MYC and PVT1, leading to increased premalignant features, and ultimately to tumor formation.

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing1–4. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression5–10. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.

Individuals with 22q11.2 microdeletions show behavioral and cognitive deficits and are at high risk of developing schizophrenia. We analyzed an engineered mouse strain carrying a chromosomal deficiency spanning a segment syntenic to the human 22q11.2 locus. We uncovered a previously unknown alteration in the biogenesis of microRNAs (miRNAs) and identified a subset of brain miRNAs affected by the microdeletion. We provide evidence that the abnormal miRNA biogenesis emerges because of haploinsufficiency of the Dgcr8 gene, which encodes an RNA-binding moiety of the 'microprocessor' complex and contributes to the behavioral and neuronal deficits associated with the 22q11.2 microdeletion.

Genomic analyses of late-stage human cancers have uncovered deletions encompassing 1p36, thereby providing an extensive body of literature supporting the idea that a potent tumor suppressor resides in this interval. Although several genes have been proposed as 1p36 candidate tumor suppressors, convincing evidence that their encoded products protect from cancer has been scanty. A recent functional study identified chromodomain helicase DNA-binding protein 5 (CHD5) as a novel tumor suppressor mapping to 1p36. Here, we discuss evidence supporting the tumor-suppressive role of CHD5. Together, these findings suggest that strategies designed to enhance CHD5 activity could provide novel approaches for treating a broad range of human malignancies. [Cancer Res 2008;68(8):2551–6]

INTRODUCTION: Medulloblastoma is the most frequent malignant pediatric brain tumor with an incidence of 0.5-0.8/100,000 children. Up to 40% of tumors have chromosome 17p deletions and 17q duplications, usually in the context of isochromosome 17q (i17q). The high frequency of i17q in medulloblastoma is hypothesized to be secondary to a role in tumor initiation and progression. To test this hypothesis we have chromosome engineered ‘17p’ deletions and ‘17q’duplications in the syntenic region of mouse chromosome 11 to evaluate the role of chromosome 17 aberrations in medulloblastoma. METHOD: The Mutagenic Insertion and Chromosome Engineering Resource (MICER) was used to chromosome engineer ‘17p’ deletions and ‘17q’ duplications. loxP sites were sequentially targeted to sites flanking the syntenic 17p or 17q region of mouse chromosome 11. Cre-recombinase deleted or duplicated the chromosome 17 target regions in mouse ES cells and recombinant clones were isolated with HAT selection. Florescent In-situ Hybridization confirmed the 18 megabase deletion of ‘17p’ and the 45 megabase duplication of 17q from mouse ES cells. Mice with Cre inducible floxed (Cis) alleles or constitutive deletion/duplication (Trans) alleles were generated. RESULTS: Chimeric founder mice with constitutive deletion/duplication (Trans) alleles were generated from mouse ES cells for both‘17p’ and ‘17q’. Offspring inherited either the desired ‘17p’ deletion allele or a ‘17p’ duplication allele. However, experimentally only ‘17p’ duplication pups were born, indicating the constitutive ‘17p’ deletion and ‘17q’ deletion/duplication alleles are likely embryonic lethal. Conditional 17p-Cis deletion mice have been bred to Nestin-cre/Ptc + /−, these mice develop medulloblastoma harboring the 18 Mbp chromosome ‘17p’ equivalent deletion in 15% of tumours. CONCLUSION: Herein we have generated a sporadic and faithful murine model of the most common cytogenetic aberration present in human medulloblastoma. This model will serve as an invaluable tool for further biological discovery and therapeutic modeling of most cases of high risk medulloblastoma.

The non-long-terminal-repeat (non-LTR) retrotransposons (also called long interspersed repetitive elements [LINEs]) are among the oldest retroelements. Here we describe the properties of such an element from a primitive protozoan parasite, Entamoeba histolytica , that infects the human gut. This 4.8-kb element, called EhLINE1, is present in about 140 copies dispersed throughout the genome. The element belongs to the R4 clade of non-LTR elements. It has a centrally located reverse transcriptase domain and a restriction enzyme-like endonuclease (EN) domain at the carboxy terminus. We have cloned and expressed a 794-bp fragment containing the EN domain in Escherichia coli. The purified protein could nick supercoiled pBluescript DNA to yield open circular and linear DNAs. The conserved PDX 12-14 D motif was required for activity. Genomic sequences flanking the sites of insertion of EhLINE1 and the putative partner short interspersed repetitive element (SINE), EhSINE1, were analyzed. Both elements resulted in short target site duplications (TSD) upon insertion. A common feature was the presence of a short T-rich stretch just upstream of the TSD in most insertion sites. By sequence analysis an empty target site in the E. histolytica genome, known to be occupied by EhSINE1, was identified. When a 176-bp fragment containing the empty site was used as a substrate for EN, it was prominently nicked on the bottom strand at the precise point of insertion of EhSINE1, showing that this SINE could use the LINE-encoded endonuclease for its insertion. The nick on the bottom strand was toward the right of the TSD, which is uncommon. The lack of strict target site-specificity of the restriction enzyme-like EN encoded by EhLINE1 is also exceptional. A model for retrotransposition of EhLINE1/SINE1 is presented.

Although autism spectrum disorders (ASDs) share a core set of nosological features, they exhibit substantial genetic heterogeneity. A parsimonious hypothesis posits that dysregulated epigenetic mechanisms represent common pathways in the etiology of ASDs. To investigate this hypothesis, we generated a novel mouse model resulting from brain-specific deletion of chromodomain helicase DNA-binding 5 (Chd5), a chromatin remodeling protein known to regulate neuronal differentiation and a member of a gene family strongly implicated in ASDs. RNA sequencing of Chd5-/- mouse forebrain tissue revealed a preponderance of changes in expression of genes important in cellular development and signaling, sociocommunicative behavior and ASDs. Pyramidal neurons cultured from Chd5-/- cortex displayed alterations in dendritic morphology. Paralleling ASD nosology, Chd5-/- mice exhibited abnormal sociocommunicative behavior and a strong preference for familiarity. Chd5-/- mice further showed deficits in responding to the distress of a conspecific, a mouse homolog of empathy. Thus, dysregulated chromatin remodeling produces a pattern of transcriptional, neuronal and behavioral effects consistent with the presentation of ASDs.

Background Enzymatic activity of Telomerase Reverse Transcriptase (TERT) is important in maintaining the telomere length and has been implicated in cancer and aging related pathology. Since cancer susceptibility as well as longevity of dogs vary between breeds, this study involved sequencing the entire TERT gene of Canis familiaris from DNA samples obtained from forty dogs, with ten dogs each of four breeds: Shih Tzu, Dachshund, Irish Wolfhound, and Newfoundland, each with different life expectancies and susceptibility to cancer. Results We compared the sequences of all forty individuals amongst one another and with the published sequence of canine TERT, and analyzed relationships between members of the same or different breeds. Two separate phylogenetic trees were generated and analyzed from these individuals. Polymorphisms were found most frequently in intronic regions of the gene, although exonic polymorphisms also were observed. In many locations genotypes were observed that were either homozygous for the reference sequence or heterozygous, but the variant homozygous genotype was not observed. Conclusions We propose that these homozygous variants are likely to have adverse effects in dogs. It was also found that the polymorphisms did not segregate by breed. Because the four breeds chosen come from geographically and physiologically distinct backgrounds, it can be inferred that the polymorphic diversification of TERT preceded breed derivation.

The protozoan parasite Entamoeba histolytica is the causative agent of amoebiasis. The genome organization of this organism is not well understood. We had earlier reported the presence of a multicopy sequence, HMc, in E. histolytica. Subsequent analysis showed that HMc is a member of a retrotransposon family that we have named the E. histolytica retrotransposon-like element (EhRLE). Four other members of this family have been characterized. The EhRLE family is distributed across all chromosomes of the parasite. There are 140 copies, which show minor sequence variation with respect to one another (2--4% from the consensus sequence). From a sequence analysis of five members of the EhRLE family, the complete EhRLE unit is estimated to be 4086 bp in length. It has a 27-mer inverted repeat at its ends. A pairwise comparison with sequences in the database showed a highly significant match of a part of EhRLE with reverse transcriptases (RT), especially those encoded by non-long terminal repeat retrotransposons. There are stop codons in all the five EhRLEs, but a continuous open reading frame of 464 amino acids could be reconstructed by comparing the sequences of several EhRLEs. The reconstructed sequence showed a much better identity with RT as compared with any of the original EhRLE sequences. The non-pathogenic species, Entamoeba dispar, also contains this element, with 85% sequence identity with EhRLE. The data suggest that EhRLE may be a retrotransposon, but many of its members are probably nonfunctional due to the accumulation of mutations.

SerpinA3N, a serine protease inhibitor, has been shown to ameliorate neuropathic pain by inhibiting leukocyte elastase activity (Vicuña et al., Nature Med 2015, 21:518-523). Patients with sickle cell disease (SCD) have activated leukocytes and experience neuropathic pain. We observed that SerpinA3N protein expression is significantly reduced in the dorsal root ganglion (DRG) of 5 month old homozygous HbSS-BERK sickle mice, compared to age-matched HbAA-BERK control mice expressing normal human hemoglobin A (p < 0.05). Since HbSS-BERK sickle mice demonstrate tonic hyperalgesia (Kohli et al., Blood 2010, 116:456-465), we hypothesize that decreased expression of SerpinA3N contributes to hyperalgesia by increasing elastase activity in these mice. We examined our hypothesis by using both genders of HbSS-BERK sickle and HbAA-BERK control mice, treated intravenously with and without sivelestat, a small molecule inhibitor of elastase. Mechanical, thermal and musculoskeletal/deep tissue hyperalgesia were analyzed before (considered baseline, BL) and after treatments. Elastase activity was determined using a fluorescent elastase substrate MeOSuc-AAPV-AMC (EMD Milipore). We observed that elastase activity is significantly increased in the DRG and lungs of male and female sickle mice compared to gender and age-matched control mice (p < 0.05 and < 0.005 for female and male DRG, respectively; p < 0.001 and < 0.05 for female and male lungs, respectively). Thus, decreased SerpinA3N expression perhaps contributes to increased elastase activity in sickle mice. Treatment of sickle mice with a single dose of 2 mg/Kg sivelestat led to a significant decrease in mechanical, heat and cold hyperalgesia for up to 24 hours compared to BL (p < 0.05), but no effect was noted in deep tissue hyperalgesia. Increasing the single dose to 4.5 mg/Kg led to a significant decrease in mechanical and thermal as well as deep tissue hyperalgesia for up to 24 hours in both genders compared to BL or saline treated mice (p < 0.05). Continuation of treatment with sivelestat 4.5 mg/Kg/day for 3 days led to significantly reduced hyperalgesia for up to 6 days in both genders of mice. These findings suggest that elastase activity contributes to nociceptive mechanisms, since hyperalgesia remained reduced for up to 3 days even after discontinuation of sivelestat. Reduced hyperalgesia following 3 days of sivelestat (4.5 mg/Kg/day) treatment was accompanied by a significant decrease in elastase activity in the DRG (p < 0.005) and lungs (p < 0.05) of both genders of sickle mice compared to saline treatment, but no histopathological changes were observed in lungs, kidney, liver, spleen and heart. Activating transcription factor 3 (ATF3), a marker of neuropathic pain, is elevated in the DRG of sickle as compared to control mice (Vincent et al., Blood 2013, 122:1853-1862). Sivelestat treatment significantly reduced ATF3-immunoreactivity compared to saline treatment in the DRG of sickle mice (p < 0.05), suggesting a decrease in neuronal injury which underlies neuropathic pain. None of the treatments had any effect on control mice. Sivelestat treatment showed no significant effect on serum tryptase or cutaneous mast cell degranulation in sickle or control mice. Thus, sivelestat specifically inhibits elastase activity. However, both elastase and tryptase activate protease-activated receptor-2 (PAR2) in the peripheral nervous system, stimulating the release of pro-nociceptive neuropeptides and activation of transient receptor potential vanilloid (TRPV) channels, leading to hyperalgesia. Since PAR-2 and TRPV channels are activated in sickle mice, it is likely that sivelestat treatment has an inhibitory effect on these mechanisms. It has been suggested that leukocytes, including activated neutrophils, contribute to acute lung injury (a common complication of SCD) and the pathogenesis of SCD (Zhang et al., Blood 2016, 127:801-809). Our observations that decreased SerpinA3N protein expression leads to increased elastase activity in DRG and lung in sickle mice suggest that this mechanism may contribute to sickle pathobiology and pain. In Japan, sivelestat is already approved for treatment of acute lung injury. Therefore, pharmacological inhibition of elastase may offer the advantage of treating chronic pain while concomitantly ameliorating one of the pathogenic mechanisms of SCD. Disclosures No relevant conflicts of interest to declare.

Triggering the immune system to attack the cancerous cells is the overarching goal of cancer immunotherapy. The key aspect of cancer immunosurveillance theory argues that our immune system acts as sentinels in recognizing and destroying the newly transformed cells that are continuously arising in vivo. Immediately after an oncogenic assault, the cell fires up various intrinsic cellular mechanisms in response to the genotoxic stress. This includes activation of DNA damage response proteins, senescence programs and tumor suppressor genes. These intrinsic responses have to be linked to the extrinsic mechanisms leading to activation of different components of the immune system. This intimate relationship between the intrinsic and the extrinsic mechanisms help to maintain tissue homeostasis and prevent further deleterious effect to the organ. However, very little is known about the critical molecular players that touch off the activation of immune system following the intrinsic response elicited by a genotoxic stress, for example, activation of tumor suppressor. These key molecular intermediaries should ideally possess the following features: 1) their expression should be upregulated in response to cellular stress 2) their upregulation should lead to a distinct immune response in vivo. We have recently identified a molecule that fits both of these criteria. We discovered a murine endogenous retrovirus (ERV) whose expression is dependent on the activity of p53. We found that components of this ERV are sufficient to induce changes in cell viability. It is characterized by a domain which is found in human T-cell leukemia virus type 1, a retrovirus found in blood cancer patients and are known to cause chronic inflammation. We found that this protein domain family is endogenized in the genomes of multiple vertebrates, including humans, potentially highlighting the evolutionary and functional significance of this element. Functional analysis of the murine ERV and homologous human ERV components suggest that these endogenous retroviral elements are early transducers of oncogenic stress, which brokers an immune response at the earliest stage of cellular transformation. We propose that these ERV components play a critical role in developing the immunogenicity against cancer, and may illuminate the mechanistic details of the first few events leading to activation of immune response following an oncogenic assault. Till recently, ERVs have been relegated as a part of the so-called ‘junk DNA’. Only recently have researchers begun revisiting this field to find the significance of ERVs in vertebrate biology. Our work sheds new light on the potential role of ERV remnants in response to neoplastic transformation. Citation Format: Kojiro Tashiro, Robyn Leary, Anindya Bagchi. p53 responsive endogeneous retrovirus as early mediators of immunosurveillance [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B025.

Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), the CCDC26 gene candidate and gasdermin C GSDMC. Whether copy number gain of one or more of these genes drives neoplasia is not known. We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced to test the change in the latency in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc (but not with an extra copy of Myc or Pvt1, Ccdc26, Gsdmc) developed adenocarcinomas with reduced latency, suggesting that while an extra copy of Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors, and in SK-BR-3 and MDA-MB-231, two human breast cancer cell lines with 8q24 amplification. Ablation of PVT1 markedly decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. Analysis of the cancer genome atlas suggested 99% of tumors with increased MYC copy-number also harbor co-gain of PVT1. Tissue microarray analysis revealed that PVT1 RNA and MYC protein expression are correlated in primary human tumors. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR mediated deletion of PVT1 in colorectal cell line HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. We further confirmed the oncogenic potential of PVT1 in cancers even without supernumerary 8q24. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We propose that the dependence of high levels of MYC on lncRNA PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition. Citation Format: Kojiro Tashiro, Robert Klink, Jon Zetterval, Veronica Diedrich, Yuen-Yi Tseng, Anindya Bagchi. Insights into the oncogenic basis of long non-coding RNA PVT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2663.

Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), the CCDC26 gene candidate and gasdermin C GSDMC. Whether copy number gain of one or more of these genes drives neoplasia is not known. We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced to test the change in the latency in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc (but not with an extra copy of Myc or Pvt1, Ccdc26, Gsdmc) developed adenocarcinomas with reduced latency, suggesting that while an extra copy of Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors, and in SK-BR-3 and MDA-MB-231, two human breast cancer cell lines with 8q24 amplification. Ablation of PVT1 markedly decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. Analysis of the cancer genome atlas suggested 99% of tumors with increased MYC copy-number also harbor co-gain of PVT1. Tissue microarray analysis revealed that PVT1 RNA and MYC protein expression are correlated in primary human tumors. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR mediated deletion of PVT1 in colorectal cell line HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. We further confirmed the oncogenic potential of PVT1 in cancers even without supernumerary 8q24. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We propose that the dependence of high levels of MYC on lncRNA PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition. Citation Format: Anindya Bagchi. It takes two to tango: The PVT1-MYC alliance in human cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA13.

Many long non-coding RNAs (lncRNAs) that regulate the capacity of cells to undergo oncogenic transformation still remain unknown. However, their involvement in chromatin organization has been an emerging principle in cancer epigenomics. This recent concept has been documented through recent studies of the gene -poor chromosomal region 8q24 surrounding the C-MYC locus to understand the mechanisms enforcing or suppressing tumor cell growth from C-MYC transcription levels. In light of such examples informing us of the DNA elements and the epigenomic composition that become integrated into molecular programs that regulate C-MYC, some lncRNAs in non-genic regions are considered landmarks that enforce chromatin state changes to accommodate C-MYC expression. Here, we use an integrative genomic and proteomic approach to gain insight of how an endogenously tagged lncRNA PCAT-1 utilizes the chromatin and biochemical context to shape C-MYC locus topology to promote tumor cell growth, survival and fitness. Our results now find that WDR74 forms an integrative hub involving recognition of the m7G cap of lncRNA transcripts along with, RNMT, RAM, BRD4, β catenin, EZH2 that assist in a program that enforce C-MYC transcription and stabilize transcripts through utilizing a comprehensive mixture of cis-acting DNA elements, lncRNAs and trans functioning transcriptional and RNA capping complexes for prostate tumor cell growth. Our results now place the lncRNA PCAT-1 and the novel interacting WD40 repeat protein WDR74 at the axis along with β catenin, BRD4 and EZH2, and additional RBPs as a non -canonical chromatin complex to shape the chromosomal topology that benefit prostate cancer cell growth. Citation Format: Fan Zhang, Won-min Song, SiDe Li, Vashisht Ajay, Francesca Aguilo, Anindya Bagchi, James A. Wohlschlegel, Bin Zhang, Martin Walsh. The enhancer landscape involves a core noncoding RNA protein interaction network for C-MYC expression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR06.

Copy number gain of the 8q24.21 chromosomal region have been a prominent mutation in many human cancers and are associated with poor prognosis. The well-characterized MYC oncogene, which resides in the 8q24.21 region, has been, conventionally, the attractive candidate driving these cancers. However, MYC is often co-gained with an adjacent ”gene desert” region. This “gene desert” often encompasses the long non-coding RNA gene PVT1, the CCDC26 gene candidate, and the GSDMC gene. Whether low copy number gain of one or more of these genes drives neoplasia is not known. We used chromosome engineering in mice to develop strains containing an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced into these three strains to test for changes in latency and penetrance in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc, (but not those with an extra copy of Myc or Pvt1,Ccdc26,Gsdmc), developed adenocarcinomas with reduced latency, suggesting that while an additional copy of the Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the mouse mammary tumors, as well as in two human breast cell lines with 8q24 amplification (SK-BR-3 and MDA-MB-231). Silencing of PVT1 markedly decreased MYC protein levels, while no effect was seen in MYC transcript levels, suggesting a PVT1-dependence of MYC protein even in high MYC- amplification cancer cells. Analysis of the TCGA and Progenetix databases suggested that 18% of 45,922 human tumors queried harbor MYC copy number increases, and in >99% of MYC-copy-increase cancers, copy number of PVT1 was co-increased .Tissue microarray analysis using in situ hybridization for PVT1 and immunohistochemistry for MYC revealed that PVT1 RNA and MYC protein expression correlated in primary human tumors. These data together suggested that PVT1 can potentiate MYC in human cancer. CRISPR mediated deletion of PVT1 in colorectal cancer cell line HCT116 either partially or fully ablated tumor formation in xenografts and significantly reduced MYC protein levels by ~50%. We propose that the dependence of high MYC protein levels on PVT1 lncRNA provides a much-needed therapeutic target against MYC protein, which has been refractory to small molecule inhibition. Citation Format: Yuen-Yi Tseng, Anindya Bagchi. Long noncoding RNA PVT1 potentiates MYC in human cancers with 8q24 gain. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR01.

Gain of 8q24, harboring the avian myelocytomatosis viral oncogene homolog (MYC), is a frequent mutation in cancers. Although MYC is the usual suspect in these cancers, the role of other co-gained loci remains mostly unknown. We have recently found that MYC partners with the adjacent long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1), which stabilizes MYC protein and potentiates its activity.

SummaryCancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19Arf/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.

Estimation of genome size of Entamoeba histolytica by different methods has failed to give comparable values due to the inherent complexities of the organism, such as the uncertain level of ploidy, presence of multinucleated cells and a poorly demarcated cell division cycle. The genome of E. histolytica has a low G+C content (22.4%), and is composed of both linear chromosomes and a number of circular plasmid-like molecules. The rRNA genes are located exclusively on some of the circular DNAs. Karyotype analysis by pulsed field gel electrophoresis suggests the presence of 14 conserved linkage groups and an extensive size variation between homologous chromosomes from different isolates. Several repeat families have been identified, some of which have been shown to be present in all the electrophoretically separated chromosomes. The typical nucleosomal structure has not been demonstrated, though most of the histone genes have been identified. Most Entamoeba genes lack introns, have short 3′ and 5′ untranslated regions, and are tightly packed. Promoter analysis revealed the presence of three conserved motifs and several upstream regulatory elements. Unlike typical eukaryotes, the transcription of protein coding genes is α-amanitin resistant. Expressed Sequence Tag analysis has identified a group of highly abundant polyadenylated RNAs which are unlikely to be translated. The Expressed Sequence Tag approach has also helped identify several important genes which encode proteins that may be involved in different biochemical pathways, signal transduction mechanisms and organellar functions.

The early latency and poor prognosis of human breast cancer is often associated with the gain of extra copy of 8q24 locus containing the c-Myc oncogene. Evaluation of aberrations at the 8q24 locus by high-resolution SNP array identified a 2.1 Mb region which only includes the c-Myc, Pvt1, and Gsdmc genes. Interestingly, the transgenic c-myc mouse has shown that the overexpression of c-myc alone is not sufficient to develop the mammary adenocarcinoma, suggesting that the overexpression of c-myc may not truly reflect breast cancer patients who were diagnosed with low copy-number gain of 8q24. It also implies that the Pvt1 or Gsdmc may play an important role with the c-Myc during tumor progression. However, functional analysis of this 2.1 Mb interval of 8q24 has been limited by a lack of suitable mouse models. Here, we report a novel mouse model, namely c-myc-gsdmcdp/+ (3N), containing the extra copy-number gain of 2.1 Mb interval as observed in breast cancer patients. To demonstrate the effect of the 3N gain, we have performed in vitro mammary epithelium experiments and in vivo mammary gland analyses in female virgin mice at 6 and 10 weeks of age. Interestingly, the mouse mammary epithelium with the 3N gain displays above 3 fold expression of c-Myc and Pvt-1 and significantly larger acinar structures in three-dimensional (3D) culture compared to the wild type (wt). Moreover, we found that mammary glands from 3N mice show excessive side-branching as early as 6 weeks. In 10 weeks, mammary glands from 3N mice display not only excessive side-branching but also precocious lobulo-alveolar development and contain numerous epithelial cells that are hyperproliferative as demonstrated by BrdU incorporation analysis when compared to wt. Using combined stem cell markers, the mammary stem cells (MaSCs)/progenitor cells-enriched population was expanded in mammary tissue from 3N mice, suggesting that a higher number of MaSCs deregulates a branching network between ducts and lobulo-alveolar structures similarly to other premalignant mouse models. Strikingly, further molecular mechanistic investigation shows that increased expression of c-myc and pvt1 promotes upregulation of epithelial-mesenchymal transition markers in mammary glands from 3N mice. Previous studies have reported that ∼40% of HER2+ breast cancers have a low copy-number gain of 8q24. Therefore, we crossed our 3N mice with MMTVneu transgenic mice. In these double-mutation mice, we observed accelerated malignant properties in 3D culture and migratory analyses. These results suggest that low copy number-gain of 8q24 may collaborate with Her2 oncogenic pathway to accelerate malignant transformation. Thus, our 3N mouse model establishes a new milestone in explaining how the low copy-number gain of 8q24 participates breast carcinogenesis and will be further analyzed to determine how aggressive tumor phenotypes are linked to poor clinical outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3288. doi:1538-7445.AM2012-3288