Correction to: Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

The original version of the article unfortunately contained an error in the unit of the protein concentrations under 'Stereotactic Intraparenchymal Injections' subsection in 'Methods' section.

Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

Pigment epithelium-derived factor (PEDF) is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. In the brain, blood-brain barrier (BBB) function is essential for homeostasis. Its impairment plays a crucial role in the pathophysiology of many neurological diseases, including ischemic stroke. We investigated (a) whether PEDF counteracted vascular endothelial growth factor (VEGF)-induced BBB disruption in the mouse brain, (b) the time course and route of BBB permeability and the dynamics of PEDF expression after cerebral ischemia, and (c) whether intraventricular infusion of PEDF ameliorated brain ischemia by reducing BBB impairment. C57Bl6/N mice received intraparenchymal injections of CSF, VEGF, or a combination of VEGF and PEDF. PEDF increased paracellular but not transcellular BBB integrity as indicated by an increase in the tight junction protein claudin-5. In another group of mice undergoing 60-min middle cerebral artery occlusion (MCAO), transcellular BBB permeability (fibrinogen staining in the absence of a loss of claudin-5) increased as early as 6 h after reperfusion. PEDF immunofluorescence increased at 24 h, which paralleled with a decreased paracellular BBB permeability (claudin-5). PEDF after MCAO originated from the blood stream and endogenous pericytes. In the third experiment, the intraventricular infusion of PEDF decreased edema and cell death after MCAO, potentially mediated by the improvement of the paracellular route of BBB permeability (claudin-5) in the absence of an amelioration of Evans Blue extravasation. Together, our data suggest that PEDF improves BBB function after cerebral ischemia by affecting the paracellular but not the transcellular route. However, further quantitative data of the different routes of BBB permeability will be required to validate our findings.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

ATM activity in T cells is critical for immune surveillance of lymphoma in vivo.

The proximal DNA damage response kinase ATM is frequently inactivated in human malignancies. Germline mutations in the ATM gene cause Ataxia-telangiectasia (A-T), characterized by cerebellar ataxia and cancer predisposition. Whether ATM deficiency impacts on tumor initiation or also on the maintenance of the malignant state is unclear. Here, we show that Atm reactivation in initially Atm-deficient B- and T cell lymphomas induces tumor regression. We further find a reduced T cell abundance in B cell lymphomas from Atm-defective mice and A-T patients. Using T cell-specific Atm-knockout models, as well as allogeneic transplantation experiments, we pinpoint impaired immune surveillance as a contributor to cancer predisposition and development. Moreover, we demonstrate that Atm-deficient T cells display impaired proliferation capacity upon stimulation, due to replication stress. Altogether, our data indicate that T cell-specific restoration of ATM activity or allogeneic hematopoietic stem cell transplantation may prevent lymphomagenesis in A-T patients.

The Cdkn1aSUPER Mouse as a Tool to Study p53-Mediated Tumor Suppression.

Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression.

Two mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains an incurable disease. Two recurrent cytogenetic aberrations, namely del(17p), affecting TP53, and del(11q), affecting ATM, are associated with resistance against genotoxic chemotherapy (del17p) and poor outcome (del11q and del17p). Both del(17p) and del(11q) are also associated with inferior outcome to the novel targeted agents, such as the BTK inhibitor ibrutinib. Thus, even in the era of targeted therapies, CLL with alterations in the ATM/p53 pathway remains a clinical challenge. Here we generated two mouse models of Atm- and Trp53-deficient CLL. These animals display a significantly earlier disease onset and reduced overall survival, compared to controls. We employed these models in conjunction with transcriptome analyses following cyclophosphamide treatment to reveal that Atm deficiency is associated with an exquisite and genotype-specific sensitivity against PARP inhibition. Thus, we generate two aggressive CLL models and provide a preclinical rational for the use of PARP inhibitors in ATM-affected human CLL.ATM and TP53 mutations are associated with poor prognosis in chronic lymphocytic leukaemia (CLL). Here the authors generate mouse models of Tp53- and Atm-defective CLL mimicking the high-risk form of human disease and show that Atm-deficient CLL is sensitive to PARP1 inhibition.

Ercc1 Deficiency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Specific Manner.

KRAS-mutant lung adenocarcinoma is among the most common cancer entities and, in advanced stages, typically displays poor prognosis due to acquired resistance against chemotherapy, which is still largely based on cisplatin-containing combination regimens. Mechanisms of cisplatin resistance have been extensively investigated, and ERCC1 has emerged as a key player due to its central role in the repair of cisplatin-induced DNA lesions. However, clinical data have not unequivocally confirmed ERCC1 status as a predictor of the response to cisplatin treatment. Therefore, we employed an autochthonous mouse model of Kras-driven lung adenocarcinoma resembling human lung adenocarcinoma to investigate the role of Ercc1 in the response to cisplatin treatment. Our data show that Ercc1 deficiency in Tp53-deficient murine lung adenocarcinoma induces a more aggressive tumor phenotype that displays enhanced sensitivity to cisplatin treatment. Furthermore, tumors that relapsed after cisplatin treatment in our model develop a robust etoposide sensitivity that is independent of the Ercc1 status and depends solely on previous cisplatin exposure. Our results provide a solid rationale for further investigation of the possibility of preselection of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients might benefit from sequential cisplatin and etoposide chemotherapy. IMPLICATIONS: This study provides a solid rationale for the stratification of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients could benefit from sequential cisplatin and etoposide chemotherapy. Mol Cancer Res; 14(11); 1110-23. ©2016 AACR.

Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression.

Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G(1/)S transition through the suppression of Cdkn1a(P21) transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.

Influence of pigment epithelium-derived factor on outcome after striatal cerebral ischemia in the mouse.

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.

Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors.

When the integrity of the genome is threatened, cells activate a complex, kinase-based signaling network to arrest the cell cycle, initiate DNA repair, or, if the extent of damage is beyond repair capacity, induce apoptotic cell death. The ATM protein lies at the heart of this signaling network, which is collectively referred to as the DNA damage response (DDR). ATM is involved in numerous DDR-regulated cellular responses-cell cycle arrest, DNA repair, and apoptosis. Disabling mutations in the gene encoding ATM occur frequently in various human tumors, including lung cancer and hematological malignancies. We report that ATM deficiency prevents apoptosis in human and murine cancer cells exposed to genotoxic chemotherapy. Using genetic and pharmacological approaches, we demonstrate in vitro and in vivo that ATM-defective cells display strong non-oncogene addiction to DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Further, this dependence of ATM-defective cells on DNA-PKcs offers a window of opportunity for therapeutic intervention: We show that pharmacological or genetic abrogation of DNA-PKcs in ATM-defective cells leads to the accumulation of DNA double-strand breaks and the subsequent CtBP-interacting protein (CtIP)-dependent generation of large single-stranded DNA (ssDNA) repair intermediates. These ssDNA structures trigger proapoptotic signaling through the RPA/ATRIP/ATR/Chk1/p53/Puma axis, ultimately leading to the apoptotic demise of ATM-defective cells exposed to DNA-PKcs inhibitors. Finally, we demonstrate that DNA-PKcs inhibitors are effective as single agents against ATM-defective lymphomas in vivo. Together, our data implicate DNA-PKcs as a drug target for the treatment of ATM-defective malignancies.

Posttranscriptional regulation of gene expression-adding another layer of complexity to the DNA damage response.

In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components-a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases.