Author Correction: EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma.

In this Letter, the sentence beginning "This work was funded…." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.

EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR.

Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain.

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.

E6 proteins from diverse papillomaviruses self-associate both in vitro and in vivo.

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.

Attenuation-corrected 99mTc-MIBI SPECT in overweight patients with chronic ischaemic dysfunction: a comparison to NH3 PET and implications for the diagnosis of myocardial viability.

OBJECTIVE: We determined the value of attenuation correction (AC) of myocardial perfusion estimation with (99m)Tc-MIBI SPECT in overweight patients by comparison of uncorrected (filtered back-projection (FBP) and corrected (an iterative algorithm with a measured attenuation coefficients map (FL-AC)) (99m)Tc-MIBI relative uptake to perfusion data obtained in the same patients with NH3 PET. In addition, the impact of attenuation correction for the assessment of myocardial viability with (99m)Tc-MIBI SPECT was determined using FDG PET as the reference method. METHODS: Thirty consecutive overweight patients (BMI=28+/-4) with left ventricular dysfunction underwent a resting (99m)Tc-MIBI SPECT and a PET study (NH3 and FDG). (99m)Tc-MIBI SPECT scans were reconstructed without attenuation correction (FBP) and with attenuation correction (FL-AC). The left ventricle was divided into 16 segments, in which the relative uptake was quantified using circumferential profiles. A relative uptake > or = 60% was considered consistent with viable myocardium for FDG and MIBI. RESULTS: The absolute difference between (99m)Tc-MIBI SPECT and NH3 PET uptakes was less pronounced in the inferior (12+/-10% vs. 17+/-12%, P<0.001), anteroseptal (12+/-11% vs. 16+/-12%, P=0.009) and septal (15+/-12% vs. 18+/-14%, P=0.003) regions (FL-AC vs. FBP, respectively). The sensitivity of MIBI for diagnosing myocardial viability increased from 83 to 100% (P=0.034), without loss in specificity. CONCLUSION: Attenuation correction improves myocardial perfusion estimation by (99m)Tc-MIBI SPECT in the inferior, anteroseptal and septal regions and increases its sensitivity for the diagnosis of myocardial viability.

Direct comparison between 2-dimensional and 3-dimensional PET acquisition modes for myocardial blood flow absolute quantification with O-15 water and N-13 ammonia.

BACKGROUND: Positron emission tomography scanners with retractable septa allow both 3-dimensional (3D) and 2-dimensional (2D) acquisition modes. The study aim was to directly compare 2D and 3D acquisition modes for the evaluation of absolute myocardial blood flow (MBF) over a wide range of flow values. METHODS AND RESULTS: Instrumentation was used in 4 dogs to reduce the left circumflex artery lumen by greater than 75%. During infusion of adenosine, MBF was measured with both 2D and 3D dynamic acquisition and both oxygen 15 water and nitrogen 13 ammonia. Injected activities were 333 MBq and 111 MBq for 2D acquisition and 3D acquisition, respectively. Data were reconstructed by analytic methods, and MBF was assessed by use of an 18-segment model. MBF values ranged from 0.4 to 5.8 mL x g(-1) x min(-1) with O-15 water and from 0.3 to 3.9 mL x g(-1) x min(-1) with N-13 ammonia. No significant differences were observed in absolute MBF values obtained with the 2 acquisition modes, regardless of the flow tracer used. Two-dimensionally and three-dimensionally derived MBF values were significantly strongly correlated by use of both O-15 water (y = 0.98x + 0.18, r = 0.87, P < .001) and N-13 ammonia (y = 0.99x + 0.09, r = 0.95, P < .001). CONCLUSION: Quantification of MBF in dogs with 3D positron emission tomography provides results similar to those obtained with the 2D technique, despite a lower activity being injected.