BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity

Loss of BRCA2 affects genome stability and is deleterious for cellular survival. Using a genome-wide genetic screen in near-haploid KBM-7 cells, we show that tumor necrosis factor-alpha (TNF alpha) signaling is a determinant of cell survival upon BRCA2 inactivation. Specifically, inactivation of the TNF receptor (TNFR1) or its downstream effector SAM68 rescues cell death induced by BRCA2 inactivation. BRCA2 inactivation leads to proinflammatory cytokine production, including TNF alpha, and increases sensitivity to TNF alpha. Enhanced TNF alpha sensitivity is not restricted to BRCA2 inactivation, as BRCA1 or FANCD2 inactivation, or hydroxyurea treatment also sensitizes cells to TNF alpha. Mechanistically, BRCA2 inactivation leads to cGAS-positive micronuclei and results in a cell-intrinsic interferon response, as assessed by quantitative mass-spectrometry and gene expression profiling, and requires ASK1 and JNK signaling. Combined, our data reveals that micronuclei induced by loss of BRCA2 instigate a cGAS/STING-mediated interferon response, which encompasses rewired TNF alpha signaling and enhances TNF alpha sensitivity

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Purpose: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. Methods: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Results: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Conclusion: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells

Mutations in homologous recombination (HR) genes, including BRCA1 and BRCA2, compromise DNA repair and lead to genomic instability (GI). GI is lethal to normal cells but is a characteristic of many cancers. Apparently, these cancers are somehow re-wired to survive high levels of GI. Identification of the genetic alterations that allow viability of genomically instable tumor cells may uncover novel therapeutic targets. To elucidate how these tumor cells are rewired, we analyzed publically available mRNA expression data of 16,172 human cancer samples. Functional genomic mRNA profiling (FGmRNA-profiling) was applied on these samples to infer levels of GI and to capture the downstream effects of somatic copy number alterations on gene expression. A genome-wide association analysis was subsequently performed to assess the correlation between FGmRNA signals of individual genes with the degree of GI. From the top 250 genes with strong positive correlation with GI, 11 genes were prioritized based on a co-functionality network in which genes are co-regulated and share similar predicted biological function. The 11 genes that were identified in this cluster were: BIRC5, UBE2C, CENPA, CDCA3, DEK, SKP2, TPX2, KIF2C, RAD21, MYBL2 and WDR67. To validate these findings in genetically-defined models, we engineered a panel of 5 triple negative breast cancer (TNBC) cell lines with doxycycline-inducible shRNAs targeting BRCA2. BRCA2 depletion resulted in a failure of RAD51 foci to localize to DNA double strand breaks which generated isogenic cell line pairs proficient and deficient of HR repair. First, we depleted each of the identified 11 genes using RNA interference in BT-549 cells and observed that depletion of TPX2, a microtubule-associated protein, led to largest differential levels of cell death when comparing the BRCA2-deficient with the BRCA2-proficient context (86.6% vs 32.9% cell death in BRCA2-depleted vs controlled depleted cells respectively). Subsequently, we could replicate this decreased survival with TPX2 depletion in a BRCA2-deficient context in an additional 2 out of 4 other TNBC cell lines. Furthermore, we investigated whether BRCA2-depleted cells were also more sensitive to depletion of Aurora kinase A, a substrate of TPX2. For this purpose, mouse mammary tumor cell lines, derived from Tp53-/- or Brca2-/-;Tp53-/- mice, or a Brca2-reconstituted version thereof were treated with an Aurora A inhibitor, Alisertib. Again, we found that the BRCA2-deficient cell line was more sensitive to Aurora A inhibition than the two BRCA2-proficient cell lines. In conclusion, FGmRNA-profiling of mRNA expression data of human cancer samples identified TPX2 as an essential gene for survival of BRCA2-deficient breast cancer cells, when compared to BRCA2-proficient cells. Thus, targeting the TPX2/AURKA axis could potentially act as a novel therapeutic target in the treatment of genomically instable cancers

Data-Driven prioritisation of antibody-drug conjugate targets in head and neck squamous cell carcinoma

Background: For patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) palliative treatment options that improve overall survival are limited. The prognosis in this group remains poor and there is an unmet need for new therapeutic options. An emerging class of therapeutics, targeting tumor-specific antigens, are antibodies bound to a cytotoxic agent, known as antibody-drug conjugates (ADCs). The aim of this study was to prioritize ADC targets in HNSCC. Methods: With a systematic search, we identified 55 different ADC targets currently targeted by registered ADCs and ADCs under clinical evaluation. For these 55 ADC targets, protein overexpression was predicted in a dataset containing 344 HNSCC mRNA expression profiles by using a method called functional genomic mRNA profiling. The ADC target with the highest predicted overexpression was validated by performing immunohistochemistry (IHC) on an independent tissue microarray containing 414 HNSCC tumors. Results: The predicted top 5 overexpressed ADC targets in HNSCC were: glycoprotein nmb (GPNMB), SLIT and NTRK-like family member 6, epidermal growth factor receptor, CD74 and CD44. IHC validation showed combined cytoplasmic and membranous GPNMB protein expression in 92.0% of the cases. Strong expression was seen in 65.9% of the cases. In addition, 86.5% and 67.7% of cases showed >= 5% and > 25% GPNMB positive tumor cells, respectively. Conclusions: This study provides a data-driven prioritization of ADCs targets that will facilitate clinicians and drug developers in deciding which ADC should be taken for further clinical evaluation in HNSCC. This might help to improve disease outcome of HNSCC patients

TPX2/Aurora kinase A signaling as a potential therapeutic target in genomically unstable cancer cells

Genomic instability is a hallmark feature of cancer cells, and can be caused by defective DNA repair, for instance due to inactivation of BRCA2. Paradoxically, loss of Brca2 in mice results in embryonic lethality, whereas cancer cells can tolerate BRCA2 loss. This holds true for multiple DNA repair genes, and suggests that cancer cells are molecularly "rewired" to cope with defective DNA repair and the resulting high levels of genomic instability. In this study, we aim to identify genes that genomically unstable cancer cells rely on for their survival. Using functional genomic mRNA (FGmRNA) profiling, 16,172 cancer samples were previously ranked based on their degree of genomic instability. We analyzed the top 250 genes that showed a positive correlation between FGmRNA levels and the degree of genomic instability, in a co-functionality network. Within this co-functionality network, a strong cluster of 11 cell cycle-related genes was identified, including TPX2. We then assessed the dependency on these 11 genes in the context of survival of genomically unstable cancer cells, induced by BRCA2 inactivation. Depletion of TPX2 or its associated kinase Aurora-A preferentially reduced cell viability in a panel of BRCA2-deficient cancer cells. In line with these findings, BRCA2-depleted and BRCA2-mutant human cell lines, or tumor cell lines derived from Brca2(-/-); p53(-/-) mice showed increased sensitivity to the Aurora-A kinase inhibitor alisertib, with delayed mitotic progression and frequent mitotic failure. Our findings reveal that BRCA2-deficient cancer cells show enhanced sensitivity to inactivation of TPX2 or its partner Aurora-A, which points at an actionable dependency of genomically unstable cancers