LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination.

Osteosarcoma, the most common pediatric bone tumor, is an aggressive heterogeneous malignancy defined by complex chromosomal aberrations. Overall survival rates remain at ~70%, but patients with chemoresistant or metastatic disease have extremely poor outcomes of <30%. A subgroup of tumors harbor amplification of chromosome 8q24.2 and increased expression of the oncogenic long noncoding RNA (lncRNA) Plasmacytoma Variant Translocation-1 (PVT-1), which is associated with an extremely poor clinical prognosis. This study demonstrates that PVT-1 is critical for osteosarcoma tumor-initiation potential. Chromatin Hybridization by RNA Purification analysis identified Tripartite-Motif Containing Family 28 (TRIM28) as a novel PVT-1 binding partner. Mechanistically, co-immunoprecipitation studies showed the PVT-1/TRIM28 complex binds and increases SUMOylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34), which leads to enhanced ubiquitination and degradation of tumor suppressor complex 2 (TSC2), thus contributing to increased self-renewal and stem cell phenotypes. Furthermore, we identified that osteosarcoma cells with increased PVT-1 have enhanced sensitivity to the SUMOylation inhibitor, TAK-981. Altogether, this study elucidated a role for PVT-1 in the enhancement of cancer stem-like behaviors, including migration and invasion, in osteosarcoma, and identified the novel PVT-1/TRIM28 axis signaling cascade as a potential therapeutic target for osteosarcoma treatment.

Oncogenic Orphan Nuclear Receptor NR4A3 Interacts and Cooperates with MYB in Acinic Cell Carcinoma.

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4;9), t(9;12), t(8;9) or t(2;4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

Pediatric myeloid sarcoma: a single institution clinicopathologic and molecular analysis.

Myeloid sarcoma (MS) is a neoplastic condition composed of immature myeloid cells involving an extramedullary site. We investigated underlying chromosomal and molecular alterations to assess potential molecular markers of prognosis and outcome in this rare pediatric disease. We conducted a retrospective review of clinicopathologic and cytogenetic data from 33 pediatric patients with MS (ages 1 month-18 years) at our institution over a 32 year period (1984-2016). Tissue-based cancer microarray and targeted next-generation sequencing analysis were performed on six cases. The median age at diagnosis was 2.8 years with a male-to-female ratio of 2.6:1. MS is commonly presented with concomitant marrow involvement (n = 12, 36.4%) or as a recurrence of acute myeloid leukemia (AML; n = 14, 42.4%). The skin (n = 18, 54.5%) and soft tissue (n = 9, 27.3%) were the most common sites of involvement. Twenty-one of 25 samples (84.0%) harbored chromosomal aberrations; KMT2A alterations (n = 10, 40.0%) or complex cytogenetics (n = 7, 28.0%) were most frequent. Mutations in RAS, tyrosine kinase, cell signaling, and chromatin remodeling genes were detected. When compared to pediatric patients with AML without extramedullary involvement (EMI), inferior overall survival (OS) was observed (18.8 months vs. 89.3 months, p = .008). Pediatric patients with MS with non-favorable cytogenetics [abnormalities other than t(8;21), inv(16)/t(16;16), or t(15;17)] had a significantly lower OS compared to patients with AML with non-favorable cytogenetics and no extramedullary involvement (8.0 months vs. 28.1 months, p < .001). Pediatric MS is a rare disease with diverse clinical presentations. Non-favorable cytogenetics may be a poor prognostic marker for pediatric patients with MS and molecular diagnostics can assist with risk stratification and identify potentially actionable targets.

Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation.

The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro.

Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing.

Genetic mutations in TP53 contribute to multiple human cancers. Here we report the generation of a H1-p53(R248W/R248W) human embryonic stem cell line harboring a homozygous TP53 R248W mutation created by TALEN-mediated precise gene editing. The H1-p53(R248W/R248W) cell line maintains a normal karyotype, robust pluripotency gene expression, and the potential to differentiate to the three germ layers.

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers.

Establishment and genomic characterization of primary salivary duct carcinoma cell line.

OBJECTIVES: To develop and characterize in vitro salivary duct carcinoma as a surrogate for functional studies. MATERIALS AND METHODS: Cells were dispersed from tumor tissue fragments under sterile conditions in RPMI media. Disassociated cells were cultivated, immortalized with hTERT and propagated for more than 100 passages. Morphologic, linage, cytogenetic and genomic analyses were performed on different passages of cell line and primary tumor. Soft agar growth was performed. RESULTS: Analysis of cytomorphologic features, growth characteristics and lineage specific markers expression confirmed the epithelial derivation and the neoplastic nature of the cell line. DNA STRs analysis showed identical match of both cell line and primary tumor. Cultivated cells expressed Androgen Receptor (AR), PTEN, and EFGR proteins and the AR-V7 isoform transcript. Comparative exome-sequencing identified common mutated genes in both cell line and primary tumor. In-vitro colony formation of late passages is established. CONCLUSION: We report the development of the first human salivary duct carcinoma cell line (MDA-SDC-04) that retains critical biological and genomic features of the donor tumor.

SV-STAT accurately detects structural variation via alignment to reference-based assemblies.

BACKGROUND: Genomic deletions, inversions, and other rearrangements known collectively as structural variations (SVs) are implicated in many human disorders. Technologies for sequencing DNA provide a potentially rich source of information in which to detect breakpoints of structural variations at base-pair resolution. However, accurate prediction of SVs remains challenging, and existing informatics tools predict rearrangements with significant rates of false positives or negatives. RESULTS: To address this challenge, we developed 'Structural Variation detection by STAck and Tail' (SV-STAT) which implements a novel scoring metric. The software uses this statistic to quantify evidence for structural variation in genomic regions suspected of harboring rearrangements. To demonstrate SV-STAT, we used targeted and genome-wide approaches. First, we applied a custom capture array followed by Roche/454 and SV-STAT to three pediatric B-lineage acute lymphoblastic leukemias, identifying five structural variations joining known and novel breakpoint regions. Next, we detected SVs genome-wide in paired-end Illumina data collected from additional tumor samples. SV-STAT showed predictive accuracy as high as or higher than leading alternatives. The software is freely available under the terms of the GNU General Public License version 3 at https://gitorious.org/svstat/svstat. CONCLUSIONS: SV-STAT works across multiple sequencing chemistries, paired and single-end technologies, targeted or whole-genome strategies, and it complements existing SV-detection software. The method is a significant advance towards accurate detection and genotyping of genomic rearrangements from DNA sequencing data.

Novel t(1;3)(q21,p21) translocation in a basal cell adenocarcinoma of the parotid gland: potential association with tumorigenesis.

We report a rare translocation involving chromosomes 1q23 and 3p21 regions in a basaloid salivary carcinoma. Our case together with a previously reported instance of translocation involving chromosome 1q 21-24 region defines a specific chromosomal segment that may house a gene associated with the development of a subset of basaloid salivary tumors.

Translocation t(7;12) as the sole chromosomal abnormality resulting in ACTB-GLI1 fusion in pediatric gastric pericytoma.

We hereby report an unusual gastric tumor arising from the pyloric wall of the stomach in a 9-year-old child harboring the exceptionally rare translocation t(7;12) resulting in ACTB-GLI1 gene fusion. This tumor has been previously classified as pericytoma with t(7;12) and described in 6 patients, 2 of them children. We discuss the challenges in recognizing this rare entity and the importance of the molecular studies in establishing the correct diagnosis. Our case is the first report of this type arising in the stomach of a child.

Novel MYBL1 Gene Rearrangements with Recurrent MYBL1-NFIB Fusions in Salivary Adenoid Cystic Carcinomas Lacking t(6;9) Translocations.

PURPOSE: Adenoid cystic carcinoma (ACC) is an indolent salivary gland malignancy, characterized by t(6;9) translocations and MYB-NFIB gene fusions in approximately 50% of the tumors. The genetic alterations underlying t(6;9)-negative and t(6;9)-positive/MYB-NFIB fusion-negative ACC remain unknown. To uncover the genetic alterations in ACC lacking the canonical translocation and fusion transcript and identify new abnormalities in translocation positive tumors. EXPERIMENTAL DESIGN: We performed whole-genome sequencing in 21 salivary ACCs and conducted targeted molecular analyses in a validation set (81 patients). Microarray gene-expression data were also analyzed to explore the biologic differences between fusion positive and negative tumors. RESULTS: We identified a novel MYBL1-NFIB gene fusion as a result of t(8;9) translocation and multiple rearrangements in the MYBL1 gene in 35% of the t(6;9)-negative ACCs. All MYBL1 alterations involved deletion of the C-terminal negative regulatory domain and were associated with high MYBL1 expression. Reciprocal MYB and MYBL1 expression was consistently found in ACCs. In addition, 5'-NFIB fusions that did not involve MYB/MYBL1 genes were identified in a subset of t(6;9)-positive/fusion-negative tumors. We also delineated distinct gene-expression profiles in ACCs associated with the length of the MYB or MYBL1 fusions, suggesting a biologic importance of the C-terminal part of these fusions. CONCLUSIONS: Our study defines new molecular subclasses of ACC characterized by MYBL1 rearrangements and 5'-NFIB gene fusions.

Coamplification of Myc/Pvt1 and homozygous deletion of Nlrp1 locus are frequent genetics changes in mouse osteosarcoma.

Osteosarcomas (OSs) are characterized by high levels of genomic instability (GI). To gain insights into the GI and its contribution toward understanding the genetic basis of OS, we characterized 19 primary and 13 metastatic mouse tumors in a genetically engineered novel mouse model of OS by a combination of genomic techniques. Through the bone-specific deletion of the wild-type Trp53 locus or activation of a metastatic-promoting missense R172Hp53 allele, C57BL/6 mice developed either localized or metastatic OS. Subsequent tumors were isolated and primary cultures created from primary bone and/or distal metastatic lesions, for example, lung and liver. These tumors exhibited high levels of GI with complex chromosomal rearrangements, amplifications, and deletions comparable to human OS. The combined genomic approaches identified frequent amplification of chromosome 15D1 and loss of 11B4 by CGH and/or SKY. Both 15D1 and 11B4 have homology with frequently altered chromosomal bands 8q24 and 17p13 in human OS, respectively. Subsequent array CGH, FISH, and qRT-PCR analysis identified coamplification and overexpression of Myc/Pvt1 transcripts from the 15D1 amplicon and loss and decreased expression of the Nlrp1b from 11B4. The Nlrp1 gene is the key mediator of apoptosis and interacts strongly with caspase 2.

TP53 loss creates therapeutic vulnerability in colorectal cancer.

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Alterations associated with androgen receptor gene activation in salivary duct carcinoma of both sexes: potential therapeutic ramifications.

PURPOSE: To investigate the molecular events associated with the activation of androgen receptor (AR) as a potential therapeutic target in patients with salivary duct carcinoma (SDC). EXPERIMENTAL DESIGN: Comprehensive molecular and expression analysis of the AR gene in 35 tumor specimens (20 males and 15 females) and cell lines derived from SDC using Western blotting and RT-PCR, FISH analysis, and DNA sequencing was conducted. In vitro and in vivo animal studies were also performed. RESULTS: AR expression was detected in 70% of the tumors and was mainly nuclear and homogenous in both male and female SDCs, although variable cytoplasmic and/or nuclear localization was also found. We report the identification of ligand-independent AR splice variants, mutations, and extra AR gene copy in primary untreated SDC tumors. In contrast to prostate cancer, no AR gene amplification was observed. In vitro knockdown of AR in a female derived SDC cell line revealed marked growth inhibition in culture and in vivo androgen-independent tumor growth. CONCLUSIONS: Our study provides new detailed information on the molecular and structural alterations associated with AR gene activation in SDC and sheds more light on the putative functional role of AR in SDC cells. On the basis of these data, we propose that patients with SDC (male and female) can be stratified for hormone-based therapy in future clinical trials.

Notch activation as a driver of osteogenic sarcoma.

Osteogenic sarcoma (OS) is a deadly skeletal malignancy whose cause is unknown. We report here a mouse model of OS based on conditional expression of the intracellular domain of Notch1 (NICD). Expression of the NICD in immature osteoblasts was sufficient to drive the formation of bone tumors, including OS, with complete penetrance. These tumors display features of human OS; namely, histopathology, cytogenetic complexity, and metastatic potential. We show that Notch activation combined with loss of p53 synergistically accelerates OS development in mice, although p53-driven OS is not Rbpj dependent, which demonstrates a dual dominance of the Notch oncogene and p53 mutation in the development of OS. Using this model, we also reveal the osteoblasts as the potential sources of OS.

Amplification and over-expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells.

Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers. An integrative bioinformatics analysis of this region nominated the MAP3K3 gene as a potential therapeutic target in breast cancer. This gene encodes mitogen-activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), which has not yet been reported to be associated with cancer-causing genetic aberrations. We found that MAP3K3 was amplified in approximately 8-20% of breast cancers. Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 non-amplified breast cancer cells. Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFα and TRAIL, as well as doxorubicin, VP-16 and fluorouracil, three commonly used chemotherapeutic drugs for treating breast cancer. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells (MCF-10A). Combined, these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy. Therefore, MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers, and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy.

MMTV-Espl1 transgenic mice develop aneuploid, estrogen receptor alpha (ERα)-positive mammary adenocarcinomas.

Separase, a protease encoded by the ESPL1 gene, cleaves the chromosomal cohesin during mitosis. Separase protein and transcripts are overexpressed in a wide range of human cancers. To investigate the physiological consequence of Separase overexpression in animals, we have generated a transgenic MMTV-Espl1 mouse model that overexpresses Separase protein in the mammary glands. MMTV-Espl1 mice in a C57BL/6 genetic background develop aggressive, highly aneuploid and estrogen receptor alpha-positive (ERα+) mammary adenocarcinomas with an 80% penetrance. The mammary tumors caused by overexpression of Separase, alone or combined with p53 heterozygosity, in mammary epithelium mimic several aspects of the most aggressive forms of human breast cancer, including high levels of genetic instability, cell cycle defects, poor differentiation, distant metastasis and metaplasia. Histopathologically, MMTV-Espl1 tumors are highly heterogeneous showing features of both luminal as well as basal subtypes of breast cancers, with aggressive disease phenotype. In addition to aneuploidy, Separase overexpression results in chromosomal instability (CIN) including premature chromatid separation (PCS), lagging chromosomes, anaphase bridges, micronuclei, centrosome amplification, multinucleated cells, gradual accumulation of DNA damage and progressive loss of tumor suppressors p53 and cadherin gene loci. These results suggest that Separase-overexpressing mammary cells are not only susceptible to chromosomal missegregation-induced aneuploidy but also other genetic instabilities including DNA damage and loss of key tumor suppressor gene loci, which in combination can initiate tumorigenesis and disease progression.

Detailed genome-wide SNP analysis of major salivary carcinomas localizes subtype-specific chromosome sites and oncogenes of potential clinical significance.

The molecular genetic alterations underlying the development and diversity of salivary gland carcinomas are largely unknown. To characterize these events, comparative genomic hybridization analysis was performed, using a single-nucleotide polymorphism microarray platform, of 60 fresh-frozen specimens that represent the main salivary carcinoma types: mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and salivary duct carcinoma (SDC). The results were correlated with the clinicopathologic features and translocation statuses to characterize the genetic alterations. The most commonly shared copy number abnormalities (CNAs) in all types were losses at chromosomes 6q23-26 and the 9p21 region. Subtype-specific CNAs included a loss at 12q11-12 in ACC and a gain at 17q11-12 in SDC. Focal copy number losses included 1p36.33-p36-22 in ACC, 9p13.2 in MEC, and 3p12.3-q11-2, 6q21-22.1, 12q14.1, and 12q15 in SDC. Tumor-specific amplicons were identified at 11q23.3 (PVRL1) in ACC, 11q13.3 (NUMA1) in MEC, and 6p21.1 (CCND3), 9p13.2 (PAX5), 12q15 (CNOT2/RAB3IP), 12q21.1 (GLIPR1L1), and 17q12 (ERBB2/CCL4) in SDC. A comparative CNA analysis of fusion-positive and fusion-negative ACCs and MECs revealed relatively lower CNAs in fusion-positive tumors than in fusion-negative tumors in both tumor types. An association between CNAs and high grade and advanced stage was observed in MECs only. These findings support the pathogenetic segregation of these entities and define novel chromosomal sites for future identification of biomarkers and therapeutic targets.