Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.development ͉ filamentous fungi F ungal fruit bodies are sexual reproduction structures that generate meiotic spores. The model mold Aspergillus nidulans develops a closed spherical fruit body (cleistothecium) including different tissue types: Hülle cells surround and nurse the growing cleistothecium, pericarp cells develop the protecting wall, and inner ascogenous cells mature into sexual spores (1, 2). Massive reconstruction of vegetative hyphae is required to build the complex three-dimensional fruit body. The regulation of this development is hardly understood in any fungus (3). A genetic screen recently identified csnD and csnE resembling genes for subunits of the COP9 signalosome (CSN) of animals and plants to be essential for fruit body formation of A. nidulans (4).CSN is a multiprotein complex composed of proteins containing PCI and MPN interaction domains (5, 6). Csn5/Jab1 is the only subunit conserved in all eukaryotes, and it carries an MPNϩ domain containing the JAMM motif conferring metalloprotease (deneddylation) activity (6, 7). CSN controls by its MPNϩ domain the activity of cullin-RING E3 ligases by cleaving the ubiquitin-like protein Nedd8/Rub1 from the cullin (8, 9). Neddylated E3 ubiquitin ligases are key mediators of posttranslational labeling of proteins for the proteasome (10). The CSN thus controls eukaryotic ubiquitin-dependent protein degradation.The complete eight-subunit CSN, composed of six PCI and two MPN domain proteins, was described for eukaryotes as humans (11), mice (12), plants (13), flies (14), and Dictyostelium (15). In fungi, definitive evidence for an eight-subunit CSN is lacking so far. CSN complex purification from Neurospora crassa revealed subunits 1-7, but subunit 8 was identified neither in the purification experiment nor in the genome sequence by bioinformatics means (16). In fission yeast subunits 6 and 8 have not been identified yet (17), and in the CSN-related complex of Saccharomyces cerevisiae only subunit Csn5 (yeast Rri1p) is well conserved (18).The fungal CSN complexes known to date are not essential for viability but are involved in cellu...
SummaryThe COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.
Cand1 can be separated into two functional polypeptides. C-terminal Cand1 binds first at the cullin adaptor site. N-terminal Cand1 blocks the neddylation site subsequently. Defects in the split fungal Cand1 impair development more than defects in CSN.
Mutations in the tumor suppressor BRCA1 and BRCA2 genes can cause cell damage that significantly increases the risk of developing breast, ovarian, prostate, and pancreatic cancers. Large genomic rearrangements (LGRs) are defined as deletions, duplications or insertions; often involving a significant portion of an exon. Usually pathogenic, they have been reported to account for up to 27% of the BRCA1 and 5% of BRCA2 disease-causing mutations with a strong founder effect accounting for about 1/3 of all cancer diagnoses in some populations. Accurate detection of a BRCA1/2 pathogenic variants has immense impact on clinical management of disease including eligibility for PARP inhibitor therapy. However, these LGRs are frequently missed by PCR-based methods and NGS assays that do not detect partial or complete exon losses or gains. Given the difficulty in detecting LGRs, there is a need for improvement of BRCA1/2 testing algorithms including reference materials that incorporate challenging LGRs to support NGS assays that analyze for these mutations. Biosynthetic DNA constructs bearing clinically relevant BRCA1/2 variants including deletions up to 500 bp spanning 2 exons and duplications up to 170 bp, were mixed with purified genomic DNA from the GM24385 human reference cell line at 10% and 50% variant allele frequency (VAF) to represent somatic and inherited BRCA disease states, respectively. The same variants were engineered into GM24385 cells at the desired VAF (>5%) and the cells were formalin fixed and paraffin embedded using a proprietary method to mimic preserved tumor biopsies. In both materials, VAF was measured using digital PCR. The prepared purified gDNA and FFPE materials were sent to multiple clinical laboratories and analyzed using various NGS assays to assess variant detection performance and ensure that the product was compatible with clinical testing workflows. Digital PCR confirmed the presence of all 20 BRCA1 and BRCA2 variants within a 20% range of the target VAF in the purified gDNA material and between 11-23% in FFPE. NGS results were variable but confirmed the presence of most variants. A few variants were detected at very low levels but reported as not detected by the bioinformatic software due to limit of detection. We have successfully developed a reference material to support both BRCA1 and BRCA2 inherited and somatic genetic testing, providing laboratories and assay developers with a tool to challenge and optimize their assay’s detection and quantitation of pathogenic variants by NGS Provided as full process in FFPE format or in purified gDNA, these reference materials include 11 challenging LGRs, which are useful in evaluating the ability of different NGS platforms to detect such variants. This study has highlighted the need for reference materials that contain these challenging genomic alterations to improve genetic testing and ultimately, support PARP inhibitor treatment selection. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Maria Cowen, Matthew G. Butler, Yves Konigshofer, Melissa Berenger, Krystyna Nahlik, Dianren Xia, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6109.
Homologous recombination deficiency (HRD) arises when defects in DNA repair pathways occur, leading to genomic instability. HRD status is an emerging therapeutic biomarker; NGS assays that measure it can be used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials, and PARP inhibitor- and platinum-based therapies. Seraseq FFPE HRD reference materials were developed to cover a range of genomic instability scores (GIS) to help NGS HRD assay validation and development. Here we discuss a multi-site evaluation across various platforms of our high-positive, low-positive, and negative HRD reference materials. Tumor cell lines were characterized by sequencing and evaluated in collaboration with several IVD partners. Three breast cancer cell lines (along with their SNP-matched normal cell lines) were selected based on their GIS. Tumor cells were blended with their SNP-matched normal cells to achieve ~65% tumor content. Biosynthetic DNA containing mutations in homologous recombination repair (HRR) genes (ATM, BRIP1, RAD51C, RAD51D) were added to the high-positive and negative reference materials targeted at >5% variant allele frequency (VAF) and measured by digital PCR. Formalin fixed paraffin embedded (FFPE) blocks were made and each block was tested for yield per curl using both the Qiagen QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit for extraction and Qubit dsDNA HS kit for concentration analysis. DNA quality was assessed using an Agilent gDNA ScreenTape Assay for the TapeStation. Whole genome shotgun sequencing was performed on extracted DNA using LGC NxSeq AmpFREE Low DNA Library Kit and Roche KAPA UDI adapters and Illumina NextSeq 2000 P1 flow cell. HRD status was evaluated by external collaborators with multiple NGS and microarray assays, including the Illumina TSO500 HRD RUO assay, the SOPHiA DDM HRD Solution and the OncoScan CNV Array A breast cancer cell line with a GIS of ~75 was selected as the HRD high-positive reference material, a second breast cancer cell line with a GIS of ~60 was selected as HRD low-positive, and a third breast cancer cell line with a GIS of ~30 was selected as HRD negative. Representative DNA yields per 10-micron section (determined using the HRD positive FFPE curls extracted by Qiagen QIAamp method) were 165 ± 28 ng/curl. Digital PCR confirmed the presence of the 8 HRR mutations (in 4 genes) at levels >5% VAF. GIS varied for each material across assays, but HRD status was consistent, confirming the wide applicability of the new reference materials We have developed the Seraseq HRD reference materials to meet the needs of laboratories looking to analyze HRD in cancer patient samples. These reference materials facilitate standardization and quality control in HRD testing by clinical labs for current and new PARP inhibitor treatment stratification in expanded patient populations that may include those with WT BRCA1/2 genes Citation Format: Dana Ruminski Lowe, Robert M. Whiting, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Indira Chivukula, Krystyna Nahlik, Dianren Xia, Russell Garlick, Bharathi Anekella. Multi-site evaluation of FFPE homologous recombination deficiency reference materials. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6110.
Here we present data on the characterization and implementation of a set of reference materials for the standardization of homologous recombination deficiency (HRD) assessment. The safety and effectiveness profile of a drug can be improved by using it in patients where that drug is most likely to be effective. While PARP inhibitors are indicated for use in some patients with ovarian, breast, pancreatic, and prostate cancer, patients with other cancers that are deficient in homologous recombination repair (HRR) might also benefit from their use. However, determining whether a cancer is HRD-positive is neither trivial nor precise. While clinical trials have shown that patients with deleterious mutations in BRCA2 and BRCA1 are most likely to benefit from PARP inhibitors, determining that a given BRCA mutation (or set of such mutations) is in fact deleterious is frequently not straightforward. Therefore, assays are now looking for signs of HRD, such as genomic instability consistent with non-homologous end joining (NHEJ) being used for the repair of double-stranded breaks in DNA as opposed to HRR. For example, some assays look at the combination of loss of heterozygosity (LOH) and large-scale state transitions (LST) across chromosomes as well as telomeric allelic imbalance (TAI) when determining whether a cancer is HRD-positive, while other assays use alternative approaches. How those characteristics are measured, integrated, and distilled into a final determination of whether HRD is present or absent varies, which can create uncertainty around treatment options and enrollment into clinical trials. This also makes the path of follow-on companion diagnostics challenging because perfect agreement between imprecise measurements is unlikely. In order to enable more standardized reporting of HRD and to enable the assessment of HRD assays, we created a set of characterized reference materials composed of HRD negative, borderline, and positive tumor/normal matched cell lines that were analyzed using both array-based and next generation sequencing-based assays in order to characterize chromosomal changes across their genomes and obtain HRD scores. Because of differences in the results, assay imprecision should be taken into account in clinical trials that implement cutoffs in order to establish safety data for patients who are biomarker-negative but may be biomarker-positive in a future assay, which is similar to what is done for complementary diagnostics. Citation Format: Yves Konigshofer, Matthew G. Butler, Krystyna Nahlik, Dana Ruminski Lowe, Catherine Huang, Omoshile Clement, Russell K. Garlick. The challenge of standardizing the measurement of an imprecise biomarker like HRD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1024.
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