Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

In this study, we investigate the role of radiomics for prediction of overall survival (OS), locoregional recurrence (LRR) and distant metastases (DM) in stage III and IV HNSCC patients treated by chemoradiotherapy. We hypothesize that radiomic analysis of (peri-)tumoral tissue may detect invasion of surrounding tissues indicating a higher chance of locoregional recurrence and distant metastasis.

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.

Premature loss of sister chromatid cohesion at metaphase is a diagnostic marker for different cohesinopathies. Here, we report that metaphase spreads of many cancer cell lines also show premature loss of sister chromatid cohesion. Cohesion loss occurs independently of mutations in cohesion factors including SA2, a cohesin subunit frequently inactivated in cancer. In untransformed cells, induction of DNA replication stress by activation of oncogenes or inhibition of DNA replication is sufficient to trigger sister chromatid cohesion loss. Importantly, cell growth under conditions of replication stress requires the cohesin remover WAPL. WAPL promotes rapid RAD51-dependent repair and restart of broken replication forks. We propose that active removal of cohesin allows cancer cells to overcome DNA replication stress. This leads to oncogene-induced cohesion loss from newly synthesized sister chromatids that may contribute to genomic instability and likely represents a targetable cancer cell vulnerability.

HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. These precancerous cells contain cancer-associated genomic changes and cause primary tumors and local relapses. Therapeutic strategies to eradicate these precancerous cells are very limited. Using functional genomic screens, we identified the therapeutic vulnerabilities of premalignant mucosal cells, which are shared with fully malignant HNSCC cells. We screened 319 previously identified tumor-lethal siRNAs on a panel of cancer and precancerous cell lines as well as primary fibroblasts. In total we identified 147 tumor-essential genes including 34 druggable candidates. Of these 34, 13 were also essential in premalignant cells. We investigated the variable molecular basis of the vulnerabilities in tumor and premalignant cell lines and found indications of collateral lethality. Wee1-like kinase (WEE1) was amongst the most promising targets for both tumor and precancerous cells. All four precancerous cell lines were highly sensitive to Wee1 inhibition by Adavosertib (AZD1775), while primary keratinocytes tolerated this inhibitor. Wee1 inhibition caused induction of DNA damage during S-phase followed by mitotic failure in (pre)cancer cells. In conclusion, we uncovered Wee1 inhibition as a promising chemopreventive strategy for precancerous cells, with comparable responses as fully transformed HNSCC cells.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, pneumothoraces and renal cell carcinomas. Here, we reveal a novel hereditary disorder in a family with skin and mucosal lesions, extensive lipomatosis and renal cell carcinomas. The proband was initially diagnosed with BHD based on the presence of fibrofolliculomas, but no pathogenic germline variant was detected in FLCN, the gene associated with BHD. By whole exome sequencing we identified a heterozygous missense variant (p.(Cys677Tyr)) in a zinc-finger encoding domain of the PRDM10 gene which co-segregated with the phenotype in the family. We show that PRDM10Cys677Tyr loses affinity for a regulatory binding motif in the FLCN promoter, abrogating cellular FLCN mRNA and protein levels. Overexpressing inducible PRDM10Cys677Tyr in renal epithelial cells altered the transcription of multiple genes, showing overlap but also differences with the effects of knocking out FLCN. We propose that PRDM10 controls an extensive gene program and acts as a critical regulator of FLCN gene transcription in human cells. The germline variant PRDM10Cys677Tyr curtails cellular folliculin expression and underlies a distinguishable syndrome characterized by extensive lipomatosis, fibrofolliculomas and renal cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody-drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy.

Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

The response rate to immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) receptor is 13%-18% for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Detailed understanding of the tumor immune microenvironment (TIME) is crucial in order to explain and improve this response rate. HNSCCs arise at various anatomical locations including the oral cavity, hypopharynx, larynx and oropharynx. Studies directly comparing immune infiltration between anatomical sites are scarce. Since the distinct locations could drive deviating microenvironments, we questioned whether the immune composition varies across these HNSCC sites.