Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies

Tumours with mutations in the BRCA1/BRCA2 genes have impaired double-stranded DNA break repair, compromised replication fork protection and increased sensitivity to replication blocking agents, a phenotype collectively known as 'BRCAness'. Tumours with a BRCAness phenotype become dependent on alternative repair pathways that are error-prone and introduce specific patterns of somatic mutations across the genome. The increasing availability of next-generation sequencing data of tumour samples has enabled identification of distinct mutational signatures associated with BRCAness. These signatures reveal that alternative repair pathways, including Polymerase θ-mediated alternative end-joining and RAD52-mediated single strand annealing are active in BRCA1/2-deficient tumours, pointing towards potential therapeutic targets in these tumours. Additionally, insight into the mutations and consequences of unrepaired DNA lesions may also aid in the identification of BRCA-like tumours lacking BRCA1/BRCA2 gene inactivation. This is clinically relevant, as these tumours respond favourably to treatment with DNA-damaging agents, including PARP inhibitors or cisplatin, which have been successfully used to treat patients with BRCA1/2-defective tumours. In this review, we aim to provide insight in the origins of the mutational landscape associated with BRCAness by exploring the molecular biology of alternative DNA repair pathways, which may represent actionable therapeutic targets in in these cells

Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors

Multimodality in galaxy clusters from SDSS DR8: substructure and velocity distribution

We search for the presence of substructure, a non-Gaussian, asymmetrical velocity distribution of galaxies, and large peculiar velocities of the main galaxies in galaxy clusters with at least 50 member galaxies, drawn from the SDSS DR8. We employ a number of 3D, 2D, and 1D tests to analyse the distribution of galaxies in clusters: 3D normal mixture modelling, the Dressler-Shectman test, the Anderson-Darling and Shapiro-Wilk tests and others. We find the peculiar velocities of the main galaxies, and use principal component analysis to characterise our results. More than 80% of the clusters in our sample have substructure according to 3D normal mixture modelling, the Dressler-Shectman (DS) test shows substructure in about 70% of the clusters. The median value of the peculiar velocities of the main galaxies in clusters is 206 km/s (41% of the rms velocity). The velocities of galaxies in more than 20% of the clusters show significant non-Gaussianity. While multidimensional normal mixture modelling is more sensitive than the DS test in resolving substructure in the sky distribution of cluster galaxies, the DS test determines better substructure expressed as tails in the velocity distribution of galaxies. Richer, larger, and more luminous clusters have larger amount of substructure and larger (compared to the rms velocity) peculiar velocities of the main galaxies. Principal component analysis of both the substructure indicators and the physical parameters of clusters shows that galaxy clusters are complicated objects, the properties of which cannot be explained with a small number of parameters or delimited by one single test. The presence of substructure, the non-Gaussian velocity distributions, as well as the large peculiar velocities of the main galaxies, shows that most of the clusters in our sample are dynamically young.Comment: 15 pages, 11 figures, 2 online tables, accepted for publication in Astronomy and Astrophysic

Ex vivo assays to predict enhanced chemosensitization by hyperthermia in urothelial cancer of the bladder

Introduction Bladder cancer (urothelial carcinoma) is a common malignancy characterized by high recurrence rates and intense clinical follow-up, indicating the necessity for more effective therapies. Current treatment regimens include intra-vesical administration of mitomycin C (MMC) for non-muscle invasive disease and systemic cisplatin for muscle-invasive or metastatic disease. Hyperthermia, heating a tumor to 40–44C, enhances the efficacy of these chemotherapeutics by various modes of action, one of which is inhibition of DNA repair via homologous recombination. Here, we explore whether ex vivo assays on freshly obtained bladder tumors can be applied to predict the response towards hyperthermia. Material and methods The cytochrome C release assay (apoptosis) and the RAD51 focus formation assay (DNA repair) were first established in the bladder cancer cell lines RT112 and T24 as measurements for hyperthermia efficiency, and subsequently tested in freshly obtained bladder tumors (n = 59). Results Hyperthermia significantly increased the fraction of apoptotic cells after cisplatin or MMC treatment in both RT112 and T24 cells and in most of the bladder tumors (8/10). The RAD51 focus formation assay detected both morphological and numerical changes of RAD51 foci upon hyperthermia in the RT112 and T24 cell lines. In 64% of 37 analyzed primary bladder tumor samples, hyperthermia induced similar morphological changes in RAD51 foci. Conclusion The cytochrome C assay and the RAD51 focus formation assay are both feasible on freshly obtained bladder tumors, and could serve to predict the efficacy of hyperthermia together with cytotoxic agents, such as MMC or cisplatin

Heat-induced BRCA2 degradation in human tumours provides rationale for hyperthermia-PARP-inhibitor combination therapies

Purpose: Hyperthermia (40–44 °C) effectively sensitises tumours to radiotherapy by locally altering tumour biology. One of the effects of heat at the cellular level is inhibition of DNA repair by homologous recombination via degradation of the BRCA2-protein. This suggests that hyperthermia can expand the group of patients that benefit from PARP-inhibitors, a drug exploiting homologous recombination deficiency. Here, we explore whether the molecular mechanisms that cause heat-mediated degradation of BRCA2 are conserved in cell lines from various origins and, most importantly, whether, BRCA2 protein levels can be attenuated by heat in freshly biopted human tumours. Experimental design: Cells from four established cell lines and from freshly biopsied material of cervical (15), head- and neck (9) or bladder tumours (27) were heated to 42 °C for 60 min ex vivo. In vivo hyperthermia was studied by taking two biopsies of the same breast or cervical tumour: one before and one after treatment. BRCA2 protein levels were measured by immunoblotting. Results: We found decreased BRCA2-levels after hyperthermia in all established cell lines and in 91% of all tumours treated ex vivo. For tumours treated with hyperthermia in vivo, technical issues and intra-tumour heterogeneity prevented obtaining interpretable results. Conclusions: This study demonstrates that heat-mediated degradation of BRCA2 occurs in tumour material directly derived from patients. Although BRCA2-degradation may not be a practical biomarker for heat deposition in situ, it does suggest that application of hyperthermia could be an effective method to expand the patient group that could benefit from PARP-inhibitors

Enhancement of contact line mobility by means of infrared laser illumination. II. Numerical simulations

A droplet that moves on a solid substrate with a velocity higher than a certain critical velocity disintegrates, i.e., leaves behind residual droplets.Infrared laserillumination can be used to increase the droplet mobility and suppress the shedding of droplets. By means of two-dimensional numerical simulations, we studied the effect of a non-uniform temperature distribution on the dynamics of straight receding contact lines. A streamfunction-vorticity model is used to describe the liquid flow in the vicinity of the receding contact line. The model takes into account the thermocapillary shear stress and the temperature-dependent liquid viscosity and density. A second, coupled model describes the laser-induced displacement of the contact line. Our results show that the reduction of the liquid viscosity with increasing temperature is the dominant mechanism for the increase of the critical velocity. Thermocapillary shear stresses are important primarily for low substrate speeds

The Sloan Great Wall. Morphology and galaxy content

We present the results of the study of the morphology and galaxy content of the Sloan Great Wall (SGW). We use the luminosity density field to determine superclusters in the SGW, and the fourth Minkowski functional V_3 and the morphological signature (the K_1-K_2 shapefinders curve) to show the different morphologies of the SGW, from a single filament to a multibranching, clumpy planar system. The richest supercluster in the SGW, SCl~126 and especially its core resemble a very rich filament, while another rich supercluster in the SGW, SCl~111, resembles a "multispider" - an assembly of high density regions connected by chains of galaxies. Using Minkowski functionals we study the substructure of individual galaxy populations determined by their color in these superclusters. We assess the statistical significance of the results with the halo model and smoothed bootstrap. We study the galaxy content and the properties of groups of galaxies in two richest superclusters of the SGW, paying special attention to bright red galaxies (BRGs) and to the first ranked galaxies in SGW groups. About 1/3 of BRGs are spirals. The scatter of colors of elliptical BRGs is smaller than that of spiral BRGs. About half of BRGs and of first ranked galaxies in groups have large peculiar velocities. Groups with elliptical BRGs as their first ranked galaxies populate superclusters more uniformly than the groups, which have a spiral BRG as its first ranked galaxy. The galaxy and group content of the core of the supercluster SCl~126 shows several differences in comparison with the outskirts of this supercluster and with the supercluster SCl~111. Our results suggest that the formation history and evolution of individual neighbour superclusters in the SGW has been different.Comment: Comments: 26 pages, 20 figures, accepted for publication in Ap

Balancing mass and momentum in the Local Group

In the rest frame of the Local Group (LG), the total momentum of the Milky Way (MW) and Andromeda (M31) should balance to zero. We use this fact to constrain new solutions for the solar motion with respect to the LG centre-of-mass, the total mass of the LG, and the individual masses of M31 and the MW. Using the set of remote LG galaxies at $>350$ kpc from the MW and M31, we find that the solar motion has amplitude $V_{\odot}=299\pm 15 {\rm ~km~s^{-1}}$ in a direction pointing toward galactic longitude $l_{\odot}=98.4^{\circ}\pm 3.6^{\circ}$ and galactic latitude $b_{\odot}=-5.9^{\circ}\pm 3.0^{\circ}$. The velocities of M31 and the MW in this rest frame give a direct measurement of their mass ratio, for which we find $\log_{10} (M_{\rm M31}/M_{\rm MW})=0.36 \pm 0.29$. We combine these measurements with the virial theorem to estimate the total mass within the LG as $M_{\rm LG}=(2.5\pm 0.4)\times 10^{12}~{\rm M}_{\odot}$. Our value for $M_{\rm LG}$ is consistent with the sum of literature values for $M_{\rm MW}$ and $M_{\rm M31}$. This suggests that the mass of the LG is almost entirely located within the two largest galaxies rather than being dispersed on larger scales or in a background medium. The outskirts of the LG are seemingly rather empty. Combining our measurement for $M_{\rm LG}$ and the mass ratio, we estimate the individual masses of the MW and M31 to be $M_{\rm MW}=(0.8\pm 0.5)\times 10^{12}~{\rm M}_{\odot}$ and $M_{\rm M31}=(1.7\pm 0.3)\times 10^{12}~{\rm M}_{\odot}$, respectively. Our analysis favours M31 being more massive than the MW by a factor of $\sim$2.3, and the uncertainties allow only a small probability (9.8%) that the MW is more massive. This is consistent with other properties such as the maximum rotational velocities, total stellar content, and numbers of globular clusters and dwarf satellites, which all suggest that $M_{\rm M31}/M_{\rm MW}>1$.Comment: 16 pages, 11 figures, 3 tables. Accepted for publication in MNRA