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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.

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.

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.

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.

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.

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

Chromosomal abnormalities like deletion, amplification and translocation are hallmarks of almost all the cancers. Amplification of human 8q24, a locus that contains the proto-oncogene C-MYC has been implicated in many human cancers, particularly in solid tumors. Importantly, this amplification is often associated with poor progonosis of breast cancer. Mouse models for studying the effects of c-Myc overexpression have been helpful in elucidating its in neoplastic transformation. However, the mammary tumors arising in these transgenic animals are focal and form with variable latency, strongly suggesting the need for additional genetic lesion to mirror the highly penetrant and aggressive tumors associated with C-MYC amplification. Moreover, in many cancers, C-MYC is co-amplified with the neighboring genetic elements which have also been implicated in tumorigenesis in multiple studies. We argue that overexpression of C-MYC alone is not sufficient to result in the poor prognosis seen in the breast cancer patients with 8q24 amplification and hypothesize that additional genes in the 8q24 amplicon contribute substantially to the mammary tumor initiation and/or aggressive progression of neoplasia. To test this hypothesis, we are modeling this genetic lesion in mice by using chromosome engineering to generate an altered allele containing a 4.5 Mb amplification in mouse chromosome 15 syntenic to Hu 8q24. The tumor susceptibility of these mice are compared with the ones that harbor an amplification of c-myc gene alone. Also, human breast tumors arising from BRCA1 mutations have been reported to have higher incidence of 8q24 amplifications (53%) than are usually found in sporadic breast cancers (10-15%). We hypothesize that a 4.5 Mb amplification of 8q24 has deleterious effects on BRCA1 linked breast cancer, resulting in poor outcome in these patients. To test this hypothesis, we will cross the mice carrying amplification of the 4.5 Mb c-myc locus with brca1 mutant mice. The effects of these amplifications on mammary tumorigenesis will be assessed by scoring the latency of mammary tumors arising in these mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3272.