Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

Despite being the first homolog of the bacterial RecQ helicase to be identified in humans the function of RECQL1 remains poorly characterised. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here we identify two families with a novel genome instability disorder, named RECON (RECql ONe) Syndrome caused by biallelic mutations in the RECQL gene. The affected individuals exhibit short stature, progeroid facial features, a hypoplastic nose, xeroderma and skin photosensitivity. Affected individuals were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser) located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase and fork restoration activity, whilst its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.

No abstract

Glioblastoma (GBM) is the most frequent malignant brain tumor. It represents the most aggressive astrocytoma with an overall survival of 14 months. Despite improvements in surgery techniques, radio-and chemotherapy, most patients present treatment resistance, recurrence and disease progression. Therefore, development of effective alternative therapies is essential to overcome treatment failure. The purpose of the study was to evaluate the antitumoral activity of the synthetic compound LQB-118, in vitro. Monolayer and three-dimensional (3D) cell culture systems of human-derived GBM cell lines were used to evaluate the effect of LQB-118 on cell viability, cell death and migration. LQB-118 reduced cell viability as determined by MTT and trypan blue exclusion assays and promoted apoptosis in monolayer cell lines with an intrinsic temozolomide (TMZ)-resistance profile. In 3D culture models, LQB-118 reduced cell viability as evaluated by APH assay and inhibited cell migration while the TMZ resistance profile was maintained. Moreover, LQB-118 reduced p38 and AKT expression and phosphorylation, whereas it reduced only the phosphorylated ERK1/2 form. LQB-118 reduced p38 and NRF2 expression, an axis that is associated with TMZ resistance, revealing a mechanism to overcome resistance. LQB-118 also demonstrated an additional effect when combined with ionizing radiation and cisplatin. In conclusion, the present data demonstrated that LQB-118 maintained its effectiveness in a 3D cell conformation, which shares more similarities with the tumor mass. LQB-118 is a promising agent for GBM treatment as monotherapy and associated with radiotherapy or cisplatin. Its effect is associated with inhibition of GBM-related survival signaling pathways.

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.

No abstract

Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in early-stage, localized tumors and their surrounding normal tissues is poorly characterized. To investigate this phenomenon, here we developed a method for spatially resolved single-cell CNA profiling and applied it to characterize the CNA landscape in 10,007 nuclei extracted from 70 tumor and normal tissue regions (~125 mm3 tissue cubes) from prostatectomies performed in six patients with localized prostate cancer. We identified two distinct groups of cells with abnormal karyotype, one mainly consisting of sparse alterations (‘pseudo-diploid’ cells) and the other characterized by genome-wide karyotypic changes (‘monster’ cells). Pseudo-diploid cells displayed high clonal diversity and formed numerous small sized clones ranging from highly spatially localized to broadly spread clones, whereas monster cells were singular events detected throughout the prostate. We observed a remarkable correlation between the fraction of the genome affected by CNAs and the number of tissue regions in which pseudo-diploid cells were found. Highly localized pseudo-diploid clones were enriched in tumor regions and carried deletions of known or putative tumor suppressors, including APC, CDKN1B, FOXO1, FOXP1, and RB1. Spatially resolved targeted deep sequencing of 523 cancer genes detected non-synonymous mutations in both normal and tumor regions, including mutations in FOXA1, FOXP1, and SPOP genes previously implicated in prostate cancer. Strikingly, in two regions in which targeted deep sequencing detected a point mutation affecting the DNA-binding activity of the FOXA1 transcription factor, we also found a co-deletion of FOXO1 and FOXO3 genes in cells from two different pseudo-diploid clones, implicating combinatorial perturbations of Forkhead transcription factors as an early driver of prostate carcinogenesis. Our study reveals that CNAs and mutations are widespread across normal and tumor regions in the prostate glands of patients with localized prostate cancer and suggests that a subset of alterations—most likely small deletions causing the loss of key tumor suppressors—confer a fitness advantage and channel cells towards tumorigenesis.