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

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

Advanced Unilateral Retinoblastoma: The Impact of Ophthalmic Artery Chemosurgery on Enucleation Rate and Patient Survival at MSKCC.

PURPOSE:To report on the influence of ophthalmic artery chemosurgery (OAC) on enucleation rates, ocular and patient survival from metastasis and impact on practice patterns at Memorial Sloan Kettering for children with advanced intraocular unilateral retinoblastoma. PATIENTS AND METHODS:Single-center retrospective review of all unilateral retinoblastoma patients with advanced intraocular retinoblastoma treated at MSKCC between our introduction of OAC (May 2006) and December 2014. End points were ocular survival, patient survival from metastases and enucleation rates. RESULTS:156 eyes of 156 retinoblastoma patients were included. Primary enucleation rates have progressively decreased from a rate of >95% before OAC to 66.7% in the first year of OAC use to the present rate of 7.4%. The percent of patients receiving OAC has progressively increased from 33.3% in 2006 to 92.6% in 2014. Overall, ocular survival was significantly better in eyes treated with OAC in the years 2010-2014 compared to 2006-2009 (p = 0.023, 92.7% vs 68.0% ocular survival at 48 months). There have been no metastatic deaths in the OAC group but two patients treated with primary enucleation have died of metastatic disease. CONCLUSION:OAC was introduced in 2006 and its impact on patient management is profound. Enucleation rates have decreased from over 95% to less than 10%. Our ocular survival rate has also significantly and progressively improved since May 2006. Despite treating more advanced eyes rather then enucleating them patient survival has not been compromised (there have been no metastatic deaths in the OAC group). In our institution, enucleation is no longer the most common treatment for advanced unilateral retinoblastoma