Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double strand breaks (DSB) which co-localized with 'replication factories' and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell-specific target with significant clinical potential

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID

Characterizing the Role of the DNA Damage Response in Class switch recombination

B cells undergo secondary antibody diversification to elicit an effective immune response. Secondary antibody diversification generates antibodies with high affinity for the cognate antigen, and antibodies of different classes. Different classes of antibodies mediate different effector functions, enabling the humoral response to be fine-tuned to the infection at hand. The different classes are generated by a process called class switch recombination (CSR), which requires the generation and repair of double-stranded DNA breaks (DSBs) at the antibody heavy chain locus, and relies on the 53BP1-dependent DNA damage response (DDR). However, the molecular mechanism and function of DDR in CSR is not fully understood. To characterize the role of DDR in CSR, we first established an in vitro CH12F3-2 mouse B cell line/ RNA interference system and used this system to demonstrate that the DDR factors RNF8 and RN168, play a role in CSR. Using this system, we then developed and conducted a genome-wide screen to identify novel CSR factors, followed by a secondary screen to identify DDR factors among the CSR candidates. Based on this study, we selected one candidate, the Drosophila enhancer of yellow 2 homolog (Eny2), for further characterization. Eny2 is a structural component of the H2B-deubiquitination module of the SAGA complex. We found that Eny2 likely acts downstream of the DSBs generated for CSR, and that Eny2 might act with the SAGA complex in mediating H2B-deubiquitination to facilitate CSR. We also provide evidence that, in addition to its role in CSR, Eny2 is important for H2B-deubiquitination upon irradiation-induced DNA damage, and may be required for optimal DNAPK activation.Ph

The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.

International audienceThe stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH

Epigenetic Therapy for Solid Tumors: Highlighting the Impact of Tumor Hypoxia

In the last few decades, epigenetics has emerged as an exciting new field in development and disease, with a more recent focus towards cancer. Epigenetics has classically referred to heritable patterns of gene expression, primarily mediated through DNA methylation patterns. More recently, it has come to include the reversible chemical modification of histones and DNA that dictate gene expression patterns. Both the epigenetic up-regulation of oncogenes and downregulation of tumor suppressors have been shown to drive tumor development. Current clinical trials for cancer therapy include pharmacological inhibition of DNA methylation and histone deacetylation, with the aim of reversing these cancer-promoting epigenetic changes. However, the DNA methyltransferase and histone deacetylase inhibitors have met with less than promising results in the treatment of solid tumors. Regions of hypoxia are a common occurrence in solid tumors. Tumor hypoxia is associated with increased aggressiveness and therapy resistance, and importantly, hypoxic tumor cells have a distinct epigenetic profile. In this review, we provide a summary of the recent clinical trials using epigenetic drugs in solid tumors, discuss the hypoxia-induced epigenetic changes and highlight the importance of testing the epigenetic drugs for efficacy against the most aggressive hypoxic fraction of the tumor in future preclinical testing

The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1

KDM4A regulates HIF-1 levels through H3K9me3

Abstract Regions of hypoxia (low oxygen) occur in most solid tumours and cells in these areas are the most aggressive and therapy resistant. In response to decreased oxygen, extensive changes in gene expression mediated by Hypoxia-Inducible Factors (HIFs) contribute significantly to the aggressive hypoxic tumour phenotype. In addition to HIFs, multiple histone demethylases are altered in their expression and activity, providing a secondary mechanism to extend the hypoxic signalling response. In this study, we demonstrate that the levels of HIF-1α are directly controlled by the repressive chromatin mark, H3K9me3. In conditions where the histone demethylase KDM4A is depleted or inactive, H3K9me3 accumulates at the HIF-1α locus, leading to a decrease in HIF-1α mRNA and a reduction in HIF-1α stabilisation. Loss of KDM4A in hypoxic conditions leads to a decreased HIF-1α mediated transcriptional response and correlates with a reduction in the characteristics associated with tumour aggressiveness, including invasion, migration, and oxygen consumption. The contribution of KDM4A to the regulation of HIF-1α is most robust in conditions of mild hypoxia. This suggests that KDM4A can enhance the function of HIF-1α by increasing the total available protein to counteract any residual activity of prolyl hydroxylases

The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation

Summary: Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR. : Activation-induced deaminase induces class switch recombination by inducing DNA breaks at the immunoglobulin locus. Ramachandran et al. showed that the SAGA deubiquitination module is critical for this process. They showed that this complex is required for optimal γH2AX formation and functions upstream of various double-stranded DNA repair pathways

ssDNA Patch Formation and Negative Supercoiling in <i>E. coli</i>: Wild-Type BL21(DE3) versus TopA Deficient VS111(DE3).

<p>A) p-GEX5.3-hAID(T27N) plasmid topology was determined for uninduced and 1 hr IPTG-induced bacterial strains by chloroquine agarose gel analysis for plasmids derived from wildtype BL21(DE3) and TopA-deficient VS111(DE3) bacterial strains. Highly-negative supercoiled DNA (HNSC) runs faster than its less supercoiled counterpart. B) ssDNA frequencies in uninduced and IPTG-induced bacterial strains. White bars indicate no IPTG treatment, black bars indicated IPTG treatment. Statistical analysis was performed using the Student's t-test (* = P = 0.0446 comparing ssDNA frequencies in uninduced and IPTG-induced BL21 cells; ** = P = 0.0059 comparing ssDNA frequencies in uninduced and IPTG-induced VS111 cells; ** = P = 0.0018 comparing ssDNA frequencies in IPTG-induced BL21 and VS111). C) ssDNA patch lengths in non-IPTG induced (open symbols) and IPTG induced (closed symbols) BL21 and VS111 bacterial strains. D) The location, lengths and strand distribution of ssDNA patches revealed in the p-GEX5.3-hAID(T27N) plasmid by <i>in situ</i> bisulfite treatment in uninduced (grey) and IPTG-induced (black) BL21(DE3) and VS111(DE3)[TopA mutant]. E) Schematic of the p-GEX5.3-hAID(WT)KanS vector used for the kanamycin reversion analysis. Kanamycin sensitivity is rendered by a proline (CCA) at position 94 of the kanamycin protein. C to T mutation of either the first or second C of the codon results in a serine or a leucine amino acid, respectively, which confers kanamycin resistance. F) AID-induced mutation frequencies of the kanamycin gene in BL21, MG1655 and TopA-deficient VS111 bacterial strains. Values were calculated after subtracting background mutation frequencies obtained from the mutation analysis of the p-GEX5.3KanS vector controls. EV = empty vector control; AID = AID expression vector (E). Statistical analysis were conducted using the student's two tailed t-test (*** = P = 0.0001 comparing BL21 EV and BL21 AID; *** = P = 0.0006 comparing MG1655 AID and VS111 AID; *** = P = 0.0008 comparing VS111 EV and VS111 AID).</p