Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the ...administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Hereditary breast cancers are frequently caused by germline
BRCA1 mutations. The
BRCA1
C61G
mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 ...heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the
Brca1
C61G
mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the
Brca1
C61G
mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the
Brca1
C61G
mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.
► BRCA1 RING function is dispensable for therapy resistance ► BRCA1 RING mutant tumors have an altered DNA damage response ► Cisplatin-resistant tumors do not show genetic reversion of the
Brca1
C61G
mutation ► Functional assays for HRD may be more predictive than genomic classifiers
Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically ...well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of
, and
gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X-ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node-negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy.
.
In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently ...induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.
BackgroundNeoantigens expressed and presented by tumor cells can be recognized by the immune system and facilitate destruction of tumors by cytotoxic CD8+ T cells. The main source of neoantigens has ...been single nucleotide variations with only one amino acid difference from a wildtype protein. We have instead focused on a class of neoantigens that arise from insertions and deletions (indels) or other gene re-arrangements, leading to formation of neo-open reading frame peptides (NOPs). NOPs are stretches of novel amino acid sequences containing numerous potentially highly immunogenic epitopes and therefore provide a valuable source of neoantigens with the capability to improve efficacy of cancer vaccines.MethodsThe analysis of The Cancer Genome Atlas (TCGA) database and in-house sequenced human tumor samples was used to predict expression of shared neoantigens resulting from indels and other gene re-arrangements. Several selected candidates underwent thorough in vitro experimental validation to confirm tumor cell presentation and immunogenicity of NOPs. Additionally, we tested immunogenicity of mouse surrogate NOPs identified via the same pipeline in the melanoma cell line B16F10 by encoding ten different NOP sequences in an mRNA vaccine.ResultsWe identified several different NOP sequences with predicted expression in human tumor samples that were selected for further validation. From each tested NOP, one or multiple epitopes were binding in vitro to either HLA-A*02:01, HLA-A*01:01, or HLA-A*24:02 allele. Presentation of epitopes on the tumor cell surface was confirmed by eluting the epitopes from HLA alleles followed by mass spectrometry analysis. We also detected presence of antigen specific CD8+ T cells in blood of healthy donors by tetramer staining. In vitro expanded NOP specific CD8+ T cell clones were able to recognize target positive tumor cell lines as measured by increased activation and degranulation markers. In vivo immunogenicity assessment after vaccination of mice with a mRNA vaccine encoding B16F10-derived NOPs showed large number (up to 60%) of polyfunctional CD8+ T cells, and to lower extent also CD4+ T cells after in vitro re-stimulation of splenocytes with NOP peptide pools. Deconvolution confirmed that 3/10 of NOPs induced CD8+ T cell responses and 2/10 NOPs mounted CD4+ T cells responses.ConclusionsThe neo-open reading frame peptides represent a class of experimentally validated, highly immunogenic neoantigens, suitable to induce robust in vivo immune responses when encoded in an mRNA vaccine format.
The objective of this study was to investigate the volatile flavor compounds of nkui, a Cameroonian food, using solid phase microextraction (SPME) and a two‐dimensional gas chromatography time of ...flight mass spectrometry GC×GC‐TOF‐MS system. Using SPME, volatile compounds were extracted from nkui and analyzed by GC×GC‐TOF‐MS. The data retrieved revealed the presence of flavor volatiles including acids (20%), alcohols (4%), aldehydes (10%), aromatic compounds (4%), esters (7%), furans (4%), ketones (11%), terpenes and terpernoids (27%). Although the terpene compounds were the most predominant, an ester (linalyl acetate) had the highest percentage of 19%, conferring a sweet, green and citrus flavor. Results obtained from this study suggest that the characteristic flavor of nkui was due to the combination of different volatile flavor compounds, which contributed to its aroma. Considering the medicinal importance of these compounds, their presence positions nkui as a vital food source with health benefits and medicinal properties.
The volatile flavor components of Nkui, a Cameroonian food was investigated using SPME‐GC×GC‐TOF‐MS. Results indicated that the characteristic flavor of Nkui was due to the combination of different volatile flavor compounds, which contributed to its aroma.
Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired ...recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous ...recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.
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