c-Myc Accelerates S-Phase and Requires WRN to Avoid Replication Stress

c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a “replication stress” pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy

An unbiased in vivo screen reveals multiple transcription factors that control HPV E6-regulated hTERT in keratinocytes

AbstractActivation of telomerase by human papillomavirus 16 (HPV16) E6 is a critical step for cell immortalization and transformation in human foreskin keratinocytes (HFKs). Multiple transcription factors have been identified as being involved in E6-induced hTERT expression. Here, we adapted an unbiased in vivo screen using a LacO–LacI system in human cells to discover hTERT promoter-interacting regulators. This approach allowed us to identify a novel hTERT repressor, Maz, which bound the hTERT promoter. E6 expression reduced Maz binding and correspondingly increased Sp1 binding at the hTERT promoter. Knockdown of Maz further increased histone acetylation, as well as hTERT expression in the presence of E6. Overall, these data indicate the utility of a novel screen for promoter-interacting and transcription-regulating proteins. These data also highlight multiple factors that normally regulate hTERT repression in HFKs, and therefore are targeted by E6 for hTERT expression

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer

Sudden Unexpected Deaths and Vaccinations during the First Two Years of Life in Italy: A Case Series Study

Background The signal of an association between vaccination in the second year of life with a hexavalent vaccine and sudden unexpected deaths (SUD) in the two days following vaccination was reported in Germany in 2003. A study to establish whether the immunisation with hexavalent vaccines increased the short term risk of SUD in infants was conducted in Italy. Methodology/Principal Findings The reference population comprises around 3 million infants vaccinated in Italy in the study period 1999–2004 (1.5 million received hexavalent vaccines). Events of SUD in infants aged 1–23 months were identified through the death certificates. Vaccination history was retrieved from immunisation registries. Association between immunisation and death was assessed adopting a case series design focusing on the risk periods 0–1, 0–7, and 0–14 days after immunisation. Among the 604 infants who died of SUD, 244 (40%) had received at least one vaccination. Four deaths occurred within two days from vaccination with the hexavalent vaccines (RR = 1.5; 95% CI 0.6 to 4.2). The RRs for the risk periods 0–7 and 0–14 were 2.0 (95% CI 1.2 to 3.5) and 1.5 (95% CI 0.9 to 2.4). The increased risk was limited to the first dose (RR = 2.2; 95% CI 1.1 to 4.4), whereas no increase was observed for the second and third doses combined. Conclusions The RRs of SUD for any vaccines and any risk periods, even when greater than 1, were almost an order of magnitude lower than the estimates in Germany. The limited increase in RRs found in Italy appears confined to the first dose and may be partly explained by a residual uncontrolled confounding effect of age

Abstract A65: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT

Abstract Intratumoral hypoxia is well known as a hallmark of most solid tumors. Here, we studied expression level of about 200 microRNAs in a panel of cancer cell lines from different tissues, and identified a microRNA miR210, its expression was prominently up-regulated by hypoxia. Over expression of miR210 in cancer cell lines partially reversed the hypoxic gene expression signature and allowed cells to escape from hypoxia-induced cell-cycle arrest. We then identified MNT, a known MYC antagonist, as a key miR-210 target. miR-210 directly down-regulated MNT transcript and protein via recognition sites in the transcript 3′ UTR. Consistent with a role for MNT as a miR-210 target, RNA interference-mediated knockdown of MNT phenocopied miR-210 over expression cell cycle effect. Furthermore, loss of MYC abolished the ability of miR-210 to override hypoxia-induced cell-cycle arrest. Thus, miR-210 could function as a modulator in cancer cells in response to hypoxic conditions through Myc network. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A65.</jats:p

Abstract A37: Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas

Abstract Overview: Synthetic lethality in cancer has been investigated for the last decade, but only recently do we have the convergence of high throughput RNA interference screening capabilities with large genomic and molecular data sets derived from primary tumor samples that allows for more comprehensive characterization of putative therapeutic targets against multiple cancers. Accurately relating cancer cell line RNA interference synthetic lethal screening data to primary human tumors has proven to be challenging, yet critically important, for improving validation rates and therapeutic efficacy. To this end, we have combined siRNA synthetic lethal screening hits in MYC amplified cell lines [Toyoshima et al. PNAS 2012] with genomic and molecular data from twelve tumor types in The Cancer Genome Atlas (TCGA) to identify and prioritize cross-cancer therapeutic targets with direct evidence for synthetic lethal relationships in both cell line and primary tumor samples. Approach: In order to relate TCGA data to synthetic lethal siRNA screening in cell lines, we began by integrating all data types produced by TCGA, including: clinical data, sample characteristics, somatic mutations, gene expression, DNA methylation, miRNA expression, reverse-phase protein array data, and somatic copy number variation. From these data, feature matrices have been constructed which combine all available information regarding patients and samples for each given tumor type. For each such heterogeneous feature matrix, we have performed statistical analysis to infer associations in the data between MYC amplification and all other molecular features. Hits from MYC synthetic lethal cell line screening were evaluated for statistical significance in pairwise analysis of twelve TCGA tumor types, classes of pairwise data-derived associations as well as properties of network-level topologies were characterized, and relationships between genomic data and clinical features were investigated where possible. Results: Evidence for synthetic lethal relationships between MYC amplification and molecular features (e.g., gene expression and DNA methylation) for numerous experimentally validates hits was present across multiple TCGA tumor types. For example, statistically significant associations between MYC amplification and decreased CSNK1E methylation, indicative of increased expression, were present in head and neck squamous cell carcinoma, cutaneous melanoma, and stomach adenocarcinoma, corroborating the experimental findings of Toyoshima et al. Similarly, PES1 gene expression is increased in breast carcinoma, colon adenocarcinoma, and head and neck squamous cell carcinoma samples with MYC amplification. Synthetic lethal hits had differential expression profiles reflective of both increases and decreases in correlation with MYC amplification. Based on these preliminary findings, the intersection of siRNA cell line screening hits with accompanying statistical associations in primary tumor sample data may enable prioritization of preclinical models of MYC driven cancers. While the present approach has been applied to MYC amplification, synthetic lethal relationships are present for a multitude of significantly mutated, copy number aberrated, or epigenetically silenced genes in TCGA. Citation Format: Brady Bernard, Silvia Cermelli, Carla Grandori, Ilya Shmulevich. Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A37.</jats:p