A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway

BACKGROUND: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. RESULTS: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21(CIP1). CONCLUSIONS: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer

Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors

Subsequent Malignant Neoplasms in Retinoblastoma Survivors

Retinoblastoma (Rb) is a pediatric malignant eye tumor. Subsequent malignant neoplasms (SMNs) and trilateral Rb (TRb) are the leading cause of death in heritable Rb patients in developed countries. The high rate of SMNs in heritable Rb patients is attributed to the presence of a mutation in the RB1 tumor suppressor gene. In addition, Rb therapy choices also influence SMN incidence in this patient group. The incidence rates and age of occurrence for the most frequent SMNs and TRb will be discussed. In addition, the impact of genetic predisposition and Rb treatments on the development of SMNs will be evaluated. Furthermore, screening and other prevention methods will be reviewed

Subsequent Malignant Neoplasms in Retinoblastoma Survivors

Retinoblastoma (Rb) is a pediatric malignant eye tumor. Subsequent malignant neoplasms (SMNs) and trilateral Rb (TRb) are the leading cause of death in heritable Rb patients in developed countries. The high rate of SMNs in heritable Rb patients is attributed to the presence of a mutation in the RB1 tumor suppressor gene. In addition, Rb therapy choices also influence SMN incidence in this patient group. The incidence rates and age of occurrence for the most frequent SMNs and TRb will be discussed. In addition, the impact of genetic predisposition and Rb treatments on the development of SMNs will be evaluated. Furthermore, screening and other prevention methods will be reviewed

High-Level MYCN-Amplified RB1-Proficient Retinoblastoma Tumors Retain Distinct Molecular Signatures

Purpose: Retinoblastomas are malignant eye tumors diagnosed in young children. Most retinoblastomas are genetically characterized by biallelic inactivation of the RB1 gene. However, 1.5% of tumors demonstrate high-level amplification of the proto-oncogene MYCN. Patients with MYCN-amplified RB1-proficient retinoblastoma receive a diagnosis at an earlier age and show a clinically and histologically more malignant phenotype. This study aimed to identify genome-wide molecular features that distinguish this subtype from other retinoblastomas. Design: Cohort study. Participants: Forty-seven retinoblastoma tumors, comprising 36 RB1–/–, 4 RB1+/–, and 7 RB1+/+ tumors. In total, 5 retinoblastomas displayed high-level MYCN amplification, with 3 being RB1+/+, 1 being RB1+/–, and 1 being RB1–/–. Methods: Integrated analysis, based on gene expression, methylation, and methylation-expression correlations, was performed to identify distinct molecular components of MYCN-amplified RB1-proficient retinoblastomas compared with other retinoblastoma subtypes. The methylation and methylation-expression correlation analysis was initially conducted within a subset of samples (n = 15) for which methylation profiles were available. The significant findings were cross-validated in the entire cohort (n = 47) and in publicly available data. Main Outcome Measures: Differentially expressed genes/pathways, differentially methylated genes, and methylation-driven differential gene expression. Results: A large number of genes (n = 3155) were identified with distinct expression patterns in MYCN-amplified RB1-proficient retinoblastomas. The upregulated and downregulated genes were associated with translation and cell-cycle processes, respectively. Methylation analysis revealed distinct methylated patterns in MYCN-amplified RB1-proficient tumors, many of which showing significant impact on gene expression. Data integration identified a 40-gene expression signature with hypermethylated state resulting in a significant downregulation in MYCN-amplified RB1-proficient retinoblastomas. Cross-validation using the entire cohort and the public domain expression data verified the overall lower expression of these genes not only in retinoblastomas with a MYCN-amplified RB1-proficient background, but also in MYCN-amplified neuroblastomas. These include the metabolism-associated TSTD1 gene and the cyclin-dependent kinase inhibitor gene CDKN2C. Conclusions: MYCN-amplified RB1-proficient retinoblastomas display significantly distinct molecular features compared with other retinoblastomas, including a set of 40 hypermethylation-driven downregulated genes. This gene set can give insight into the biology of MYCN-amplified retinoblastomas and may help us to understand the more aggressive clinical behavior

Knockdown of ARNTL, TNIP1 and RBCK1 prevents p21^CIP1induction in BJtsLT cells.

<p>BJtsLT cells were infected at 32°C and shifted to 39°C for colony formation. After 14 days of culturing at 39°C cells were harvested, protein lysates were prepared and subjected to western blot for p53, CDK4 (control) and p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4a–c</a>). Additionally, total RNA was isolated and used for QRT-PCR for p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4d</a>).</p

shRNA barcode screen identifies mediators of the p53 dependent cell cycle arrest.

<p>a) Schematic outline of the BJtsLT genetic screen. BJtsLT cells were infected with the NKI shRNA library and were either left at 32°C or shifted to 39°C. After 7 days the cells at 32°C had reached confluency and were harvested. Cells at 39°C were harvested after 23 days after which they had formed visible colonies. b) Analysis of the relative abundance of shRNAs recovered from the BJtsLT barcode experiment. Data are normalized and plotted as <i>M</i>, the ²log (ratio Cy5/Cy3), versus <i>A</i> (²log(√intensity Cy3×Cy5)). The data are the average of two independent hybridization experiments performed in duplicate with reversed colour. A red box is drawn around the top 100 enriched shRNAs at 39°C. c) Schematic overview of selection criteria used to select hits from the shRNA barcode screen for further validation.</p

Colony formation ARNTL, RBCK1 and TNIP1 shRNA vectors.

<p>Cells were infected with shRNA vectors targeting ARNTL, RBCK1 and TNIP1 and control shRNA vectors targeting GFP, p53, 53BP1 and p21. Cells were infected at 32°C and shifted to 39°C 2 days after infection. After three weeks culture at 39°C, the cells were fixed and stained.</p

Barcode identified shRNA vectors suppress protein and mRNA levels of their targets.

<p>a) QRT-PCR for ARNTL in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. b) QRT-PCR for RBCK1 in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. c) QRT-PCR for TNIP1 in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. d) Flag-ARNTL together with the shRNA vectors targeting ARNTL were transiently transfected in Phoenix cells. Extracts were immunoblotted using Flag and CDK4 (control) antibodies. e) BJ cells were infected with the indicated shRNA vectors and Extracts were immunoblotted using TNIP1 and CDK4 (control) antibodies.</p

ARNTL regulates p21^CIP1 expression.

<p>a) Knockdown of ARNTL inhibits radiation induced p21^CIP1 induction. U2OS cells were infected with the shRNA vectors as indicated. Cells were seeded and irradiated with 20 Gy of γ-radiation. After o/n incubation cells were lysed and lysates were subjected to western blot using antibodies for p53, CDK4 (control) and p21^CIP1. b) Knockdown of ARNTL can also rescue a p19-induced cell cycle arrest. U2OS cells were infected with the indicated shRNA vectors followed by a super-infection with p19ARF-RFP virus. Cells were seeded and incubated for three weeks. After three weeks the infected cells were fixed and stained. c) Knockdown of ARNTL in U2OS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">Fig 5b</a>) was quantified by QRT-PCR. d) ARNTL knockdown is not involved in p53 independent p21^CIP1 induction. HCT116 wt and p53^−/− cells were infected with knockdown vectors targeting p53, p21^CIP1 and ARNTL. Cells were treated with 0.5 µM PXD101 for 16 hrs. Cells were then lysed and lysates were subjected to western analysis for p53, CDK4 (control) and p21^CIP1. e) Quantification of p21 protein levels in the western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">figure 5c</a> using IMAGE J software.</p