Human Papillomavirus-Induced Chromosomal Instability and Aneuploidy in Squamous Cell Cancers.

Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. CIN is defined as a continuous rate of chromosome missegregation events over the course of multiple cell divisions. CIN causes aneuploidy, a state of abnormal chromosome content differing from a multiple of the haploid. Human papillomavirus (HPV) is a well-known cause of squamous cancers of the oropharynx, cervix, and anus. The HPV E6 and E7 oncogenes have well-known roles in carcinogenesis, but additional genomic events, such as CIN and aneuploidy, are often required for tumor formation. HPV+ squamous cancers have an increased frequency of specific types of CIN, including polar chromosomes. CIN leads to chromosome gains and losses (aneuploidies) specific to HPV+ cancers, which are distinct from HPV- cancers. HPV-specific CIN and aneuploidy may have implications for prognosis and therapeutic response and may provide insight into novel therapeutic vulnerabilities. Here, we review HPV-specific types of CIN and patterns of aneuploidy in squamous cancers, as well as how this impacts patient prognosis and treatment.

p53 Gain-of-Function Mutation Induces Metastasis via BRD4-Dependent CSF-1 Expression.

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease. SIGNIFICANCE: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489.

Outcomes in ANCA-Associated Vasculitis in Scotland: Validation of the Renal Risk Score in a Complete National Cohort.

Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) causes autoimmune-mediated inflammation of small blood vessels in multiple organs, including the kidneys. The ability to accurately predict kidney outcomes would enable a more personalized therapeutic approach. Methods: We used our national renal biopsy registry to validate the ability of ANCA Renal Risk Score (ARRS) to predict end-stage kidney disease (ESKD) for individual patients. This score uses histopathological and biochemical data to stratify patients as high, medium, or low risk for developing ESKD. Results: A total of 288 patients were eligible for inclusion in the study (low risk n = 144, medium risk n = 122, high risk n = 12). Using adjusted Cox proportional hazard models with the low-risk group as reference, we show that outcome differs between the categories: high-risk hazard ratio (HR) 16.69 (2.91-95.81, P = 0.002); medium risk HR 4.14 (1.07-16.01, P = 0.039). Incremental multivariable-adjusted Cox proportional hazards models demonstrated that adding ARRS to a model adjusted for multiple clinical parameters enhanced predictive discrimination (basic model C-statistic 0.864 [95% CI 0.813-0.914], basic model plus ARRS C-statistic 0.877 [95% CI 0.823-0.931]; P <0.01). Conclusion: The ARRS better discriminates risk of ESKD in AAV and offers clinicians more prognostic information than the use of standard biochemical and clinical measures alone. This is the first time the ARRS has been validated in a national cohort. The proportion of patients with high-risk scores is lower in our cohort compared to others and should be noted as a limitation of this study.

Oncogene-like addiction to aneuploidy in human cancers.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses p53 signaling, and we show that TP53 mutations are mutually exclusive with 1q aneuploidy in human cancers. Thus, tumor cells can be dependent on specific aneuploidies, raising the possibility that these "aneuploidy addictions" could be targeted as a therapeutic strategy.

Cancer aneuploidies are shaped primarily by effects on tumour fitness.

Aneuploidies-whole-chromosome or whole-arm imbalances-are the most prevalent alteration in cancer genomes1,2. However, it is still debated whether their prevalence is due to selection or ease of generation as passenger events1,2. Here we developed a method, BISCUT, that identifies loci subject to fitness advantages or disadvantages by interrogating length distributions of telomere- or centromere-bounded copy-number events. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage specific. BISCUT identified the helicase-encoding gene WRN as a haploinsufficient tumour-suppressor gene on chromosome 8p, which is supported by several lines of evidence. We also formally quantified the role of selection and mechanical biases in driving aneuploidy, finding that rates of arm-level copy-number alterations are most highly correlated with their effects on cellular fitness1,2. These results provide insight into the driving forces behind aneuploidy and its contribution to tumorigenesis.

HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E.

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

Oncogene-like addiction to aneuploidy in human cancers.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses TP53 signaling, and we show that TP53 mutations are mutually-exclusive with 1q aneuploidy in human cancers. Thus, specific aneuploidies play essential roles in tumorigenesis, raising the possibility that targeting these "aneuploidy addictions" could represent a novel approach for cancer treatment.

Multimodal single-cell and whole-genome sequencing of small, frozen clinical specimens.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial12, we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases13, we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena.

Molecular Subtypes of Head and Neck Cancer in Patients of African Ancestry.

PURPOSE: The purpose of this study was to better understand the complex molecular biomarkers and signatures of head and neck cancer (HNC) among Black patients and identify possible molecular changes associated with HNC disparities. EXPERIMENTAL DESIGN: Molecular subtypes and genomic changes in HNC samples from patients of African and European ancestry in The Cancer Genome Atlas, Memorial Sloan Kettering Cancer Center, Broad Institute, MD Anderson Cancer Center, and John Hopkins University were identified. Molecular features (genomic, proteomic, transcriptomic) associated with race and genomic alterations associated with clinical outcomes were determined. An independent cohort of HNC tumor specimens was used to validate the primary findings using IHC. RESULTS: Black patients were found to have a younger age at diagnosis, more aggressive tumor types, higher rates of metastasis, and worse survival compared with White patients. Black patients had fewer human papillomavirus-positive tumor types and higher frequencies of laryngeal subtype tumors. Higher frequencies of TP53, MYO18B, KMT2D, and UNC13C mutations and a lower frequency of PIK3CA mutations were observed in Black patients. Tumors of Black patients showed significant enrichment of c-MYC and RET-tyrosine signaling and amplifications. A significant increase in tumor expression of c-MYC in Black patients was observed and was associated with poor survival outcomes in the independent cohort. CONCLUSIONS: Novel genomic modifications and molecular signatures may be related to environmental, social, and behavioral factors associated with racial disparities in HNC. Unique tumor mutations and biological pathways have potential clinical utility in providing more targeted and individualized screening, diagnostic, and treatment modalities to improve health outcomes.

Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer.

The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types1,2. Intratumoral host-microbiota studies have so far largely relied on bulk tissue analysis1-3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies4 and single-cell RNA sequencing5 to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host-microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling6, we show that bacterial communities populate microniches that are less vascularized, highly immuno­suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion-adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.

Elraglusib (9-ING-41), a selective small-molecule inhibitor of glycogen synthase kinase-3 beta, reduces expression of immune checkpoint molecules PD-1, TIGIT and LAG-3 and enhances CD8+ T cell cytolytic killing of melanoma cells.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with multiple roles in tumour growth, cell invasion and metastasis. We have previously established GSK-3 as an upstream regulator of PD-1 gene expression in CD8 + T cells and demonstrated that GSK-3 inhibition is as effective as anti-PD-1 mAb blockade in controlling tumour growth. Elraglusib (9-ING-41) is a specific small-molecule inhibitor of GSK-3ß with clinical activity in patients with advanced cancers, including a patient with refractory melanoma whose response provided the rationale for the current study. METHODS: The B16 melanoma mouse model was used to observe the effect of elraglusib on tumour growth either as a single agent or in combination (simultaneously and sequentially) with anti-PD-1 mAb treatment. B16 tumour cells were implanted in either the flank, brain or both locations, and Kaplan-Meier plots were used to depict survival and significance determined using log rank tests. Expression of the immune checkpoint molecules, TIGIT, LAG-3 and PD-1, was evaluated using flow cytometry alongside expression of the chemokine receptor, CXCR3. Further evaluation of PD-1 expression was determined through RT-qPCR and immunohistochemistry. RESULTS: We demonstrated that elraglusib has a suppressive effect against melanoma as a single agent and enhanced anti-PD-1 therapy. There was a synergistic effect when elraglusib was used in combination with anti-PD-1 mAb, and an even greater effect when used as sequential therapy. Suppression of tumour growth was associated with a reduced expression of immune checkpoint molecules, PD-1, TIGIT and LAG-3 with upregulation of CXCR3 expression. CONCLUSIONS: These data highlight the potential of elraglusib as an immune-modulatory agent and demonstrate the benefit of a sequential approach with immune checkpoint inhibition followed by GSK-3ß inhibition in melanoma and provide a rationale for clinical investigation of elraglusib combined with immune checkpoint inhibitory molecules, including those targeting PD-1, TIGIT and LAG-3. This has several potential implications for current immunotherapy regimes, including possibly reducing the intensity of anti-PD-1 mAb treatment needed for response in patients receiving elraglusib, especially given the benign adverse event profile of elraglusib observed to date. Based on these data, a clinical study of elraglusib, an anti-PD-1 mAb and chemotherapy is ongoing (NCT NCT05239182).

Clinical course of inflammatory bowel disease and impact on liver disease outcomes in patients with autoimmune sclerosing cholangitis.

BACKGROUND & AIMS: Autoimmune sclerosing cholangitis (ASC) is a childhood sclerosing cholangitis frequently associated with inflammatory bowel disease (IBD). We describe the IBD phenotype in ASC patients and associated liver disease outcomes. METHODS: Single center retrospective observational review of ASC patients, with a control population of pediatric IBD. Demographic and clinical parameters were obtained. Clinical endpoints were escalation of IBD therapy (biologic or colectomy) and transplant-free survival. RESULTS: In 93 ASC patients (53.8% female) and median follow up of 172 months: 70% had IBD, 25.8% underwent liver transplant. Median age at liver transplant was 21.7 years, at 131 months from ASC diagnosis. There was no association between presence of IBD and transplant-free survival, whilst those requiring second-line immunomodulators for ASC had poorer long-term liver prognosis. During follow-up 22 (33.8%) ASC-IBD required biologic or colectomy. On multivariate analysis ASC was associated with a lower risk of escalation of IBD therapy (HR 0.14, 95% CI 0.05-0.42; P=.001), including biologic therapy (HR 0.21, 95% CI 0.08-0.55, P=.002), but not colectomy on univariate analysis (HR 1.54, 95% CI 0.43-5.44, P=.51). CONCLUSIONS: IBD is common in ASC and during longterm follow up a third of ASC-IBD required escalation of IBD therapy; however ASC-IBD was lower risk compared to IBD alone. IBD does not appear to impact on transplant-free survival in patients with ASC, however second-line immunomodulators for ASC are associated with poorer IBD and liver outcomes.

SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response.

BACKGROUND: Understanding the role and potential therapeutic targeting of tumor-associated macrophages (TAMs) is crucial to developing new biomarkers and therapeutic strategies for cancer immunotherapies. The epigenetic reader SP140 has emerged as a master regulator of macrophage transcriptional programs; however, its role in the signaling of TAMs and response to immunotherapy has not been investigated. METHODS: We evaluated the correlation between SP140 expression in head and neck squamous cell carcinoma (HNSCC) TAMs and clinical outcomes. We also used complementary bioinformatics and experimental approaches to study the association of SP140 expression with tumor mutation burden, patient survival, immunogenic signature of tumors, and signaling of TAMs. SP140 overexpression or knockdown was implemented to identify the role of SP140 in downstream signaling and production of inflammatory cytokine and chemokines. Chromatin immunoprecipitation and analysis of assay of transposase accessible chromatin sequencing data were used to demonstrate the direct binding of SP140 on the promoters of STAT1. Finally, correlation of SP140 with immune cell infiltrates and response to immune-checkpoint blockade in independent cohorts of HNSCC, metastatic melanoma, and melanoma was assessed. RESULTS: We found that SP140 is highly expressed in TAMs across many cancer types, including HNSCCs. Interestingly, higher expression of SP140 in the tumors was associated with higher tumor mutation burden, improved survival, and a favorable response to immunotherapy. Tumors with high SP140 expression showed enrichment of inflammatory response and interferon-gamma (IFN-γ) pathways in both pan-cancer analysis and HNSCC-specific analysis. Mechanistically, SP140 negatively regulates transcription and phosphorylation of STAT1 and induces IFN-γ signaling. Activating SP140 in macrophages and TAMs induced the proinflammatory macrophage phenotype, increased the antitumor activity of macrophages, and increased the production of IFN-γ and antitumor cytokines and chemokines including interleukin-12 and CXCL10. SP140 expression provided higher sensitivity and specificity to predict antiprogrammed cell death protein 1 immunotherapy response compared with programmed death-ligand 1 in HNSCCs and lung cancer. In metastatic melanoma, higher levels of SP140 were associated with a durable response to immunotherapy, higher immune score estimates, high infiltrations of CD8+ T cells, and inflammatory TAMs. CONCLUSIONS: Our findings suggest that SP140 could serve as both a therapeutic target and a biomarker to identify immunotherapy responders.

A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer.

Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3' noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.

Tumour cell CD99 regulates transendothelial migration via CDC42 and actin remodelling.

Metastasis requires tumour cells to cross endothelial cell (EC) barriers using pathways similar to those used by leucocytes during inflammation. Cell surface CD99 is expressed by healthy leucocytes and ECs, and participates in inflammatory transendothelial migration (TEM). Tumour cells also express CD99, and we have analysed its role in tumour progression and cancer cell TEM. Tumour cell CD99 was required for adhesion to ECs but inhibited invasion of the endothelial barrier and migratory activity. Furthermore, CD99 depletion in tumour cells caused redistribution of the actin cytoskeleton and increased activity of the Rho GTPase CDC42, known for its role in actin remodelling and cell migration. In a xenograft model of breast cancer, tumour cell CD99 expression inhibited metastatic progression, and patient samples showed reduced expression of the CD99 gene in brain metastases compared to matched primary breast tumours. We conclude that CD99 negatively regulates CDC42 and cell migration. However, CD99 has both pro- and anti-tumour activity, and our data suggest that this results in part from its functional linkage to CDC42 and the diverse signalling pathways downstream of this Rho GTPase. This article has an associated First Person interview with the first author of the paper.

Non-redundant activity of GSK-3α and GSK-3ß in T cell-mediated tumor rejection.

Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3ß. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b-/- mice suppressed tumor growth to the same degree as Gsk3a/b-/- mice, whereas Gsk3a-/- mice behaved similarly to wild-type, revealing an important role for GSK-3ß in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and ß isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy.

Prevalence of COVID-19 in patients with autoimmune liver disease in Europe: A patient-oriented online survey.

BACKGROUND: During the current SARS-CoV-2 pandemic it is important to identify risk factors for COVID-19. Registry studies are providing growing evidence on the elevated risk of mortality from COVID-19 in patients with chronic liver disease, especially in advanced stages. Results may, however, have a selection bias towards severe cases. Limited data is available on COVID-19 in patients with autoimmune liver disease (AILD). AIM: To perform an online survey to capture the prevalence of COVID-19 and the state of medical care of patients with AILD in Europe during the pandemic. METHODS: Data was collected via an anonymous patient-oriented, online survey, which was available on the EUSurvey platform in nine European languages between 24th June 2020 and 14th October 2020. Of 1834 contributions, 51 were excluded because participants did not name an underlying AILD, and four were excluded because of duplicate data entry. RESULTS: Of 1,779 participants, 1,752 resided in 20 different countries of the European Union and the United Kingdom (UK). The five countries with the highest numbers of contributions were France (n = 450), Germany (n = 318), the Netherlands (n = 267), Spain (n = 225), and the UK (n = 183). 2.2% of participants (39/1779) had been diagnosed with COVID-19. There were no differences regarding age, sex, AILD, the status of liver cirrhosis, or status post liver transplantation between COVID-19 and non-COVID-19 cases. Of the 39 COVID-19 cases, five patients were admitted to a regular ward, one patient was admitted to ICU and required ventilation. CONCLUSION: In our Europe-wide, patient-oriented survey on COVID-19 in patients with AILD, we detected a low rate of COVID-19, comparable to the period prevalence of the general population. These results suggest that patients with AILD are not at elevated risk of COVID-19.