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The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8   +  FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5   +  OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response. It is unclear whether fallopian tube epithelium or ovarian surface epithelium is the cell-of-origin of high grade serous ovarian carcinoma (HGSOC). Here the authors report a dualistic origin for HGSOC using genetically engineered mouse models and observe differential chemotherapy sensitivity depending on the cell-of-origin.

Large-scale human genetic data 1 – 3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor ( VHL ) 4 – 6 . VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization 6 , 7 . We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 ( CCND1 ) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8 ). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants. The lineage transcription factor PAX8 is shown to play a pivotal part in determining cancer risk in clear cell renal cell carcinoma, providing insights into how genetic mutations lead to specific types of cancer.

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves’ disease patient sera, demonstrating that organoids can model human autoimmune disease.

Most uterine cervical high-risk human papillomavirus (HPV) infections are transient, with only a small fraction developing into cervical cancer. Family aggregation studies and heritability estimates suggest a significant inherited genetic component. Candidate gene studies and previous genome-wide association studies (GWASs) report associations between the HLA region and cervical cancer. Adopting a genome-wide approach, we aimed to compare genetic variation in women with invasive cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 with that in healthy controls. We did a GWAS in a cohort of unrelated European individuals using data from UK Biobank, a population-based cohort including 273 377 women aged 40–69 years at recruitment between March 13, 2006, and Oct 1, 2010. We used an additive univariate logistic regression model to analyse genetic variants associated with invasive cervical cancer or CIN3. We sought replication of candidate associations in FinnGen, a large independent dataset of 128 123 individuals. We also did a two-sample mendelian randomisation approach to explore the role of risk factors in the genetic risk of cervical cancer. We included 4769 CIN3 and invasive cervical cancer case samples and 145 545 control samples in the GWAS. Of 9 600 464 assayed and imputed single-nucleotide polymorphisms (SNPs), six independent variants were associated with CIN3 and invasive cervical cancer. These included novel loci rs10175462 (PAX8; odds ratio [OR] 0·87, 95% CI 0·84–0·91; p=1·07 × 10−9) and rs27069 (CLPTM1L; 0·88, 0·84–0·92; p=2·51 × 10−9), and previously reported signals at rs9272050 (HLA-DQA1; 1·27, 1·21–1·32; p=2·51 × 10−28), rs6938453 (MICA; 0·79, 0·75–0·83; p=1·97 × 10−17), rs55986091 (HLA-DQB1; 0·66, 0·60–0·72; p=6·42 × 10−28), and rs9266183 (HLA-B; 0·73, 0·64–0·83; p=1·53 × 10−6). Three SNPs were replicated in the independent Finnish dataset of 1648 invasive cervical cancer cases: PAX8 (rs10175462; p=0·015), CLPTM1L (rs27069; p=2·54 × 10−7), and HLA-DQA1 (rs9272050; p=7·90 × 10−8). Mendelian randomisation further supported the complementary role of smoking (OR 2·46, 95% CI 1·64–3·69), older age at first pregnancy (0·80, 0·68–0·95), and number of sexual partners (1·95, 1·44–2·63) in the risk of developing cervical cancer. Our results provide new evidence for the genetic susceptibility to cervical cancer, specifically the PAX8, CLPTM1L, and HLA genes, suggesting disruption in apoptotic and immune function pathways. Future studies integrating host and viral, genetic, and epigenetic variation, could further elucidate complex host–viral interactions. NIHR Imperial BRC Wellcome 4i Clinician Scientist Training Programme.

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer. Lineage-restricted transcription factor PAX8 is oncogenic in ovarian cancer cells. Here the authors show that PAX8 interacts and recruits a splice variant of the MECOM locus PRDM3 to control the gene expression module involved in adhesion and extracellular matrix, and consequently promotes ovarian tumorigenesis.

Ovarian cancer is a malignant tumor originating from the ovary, characterized by its high mortality rate and propensity for recurrence. In some patients, especially those with recurrent cancer, conventional treatments such as surgical resection or standard chemotherapy yield suboptimal results. Consequently, there is an urgent need for novel anti-cancer therapeutic strategies. Ferroptosis is a distinct form of cell death separate from apoptosis. Ferroptosis inducers have demonstrated promising potential in the treatment of ovarian cancer, with evidence indicating their ability to enhance ovarian cancer cell sensitivity to cisplatin. However, resistance of cancer cells to ferroptosis still remains an inevitable challenge. Here, we analyzed genome-scale CRISPR-Cas9 loss-of function screens and identified PAX8 as a ferroptosis resistance protein in ovarian cancer. We identified PAX8 as a susceptibility gene in GPX4-dependent ovarian cancer. Depletion of PAX8 rendered GPX4-dependent ovarian cancer cells significantly more sensitive to GPX4 inhibitors. Additionally, we found that PAX8 inhibited ferroptosis in ovarian cancer cells. Combined treatment with a PAX8 inhibitor and RSL3 suppressed ovarian cancer cell growth, induced ferroptosis, and was validated in a xenograft mouse model. Further exploration of the molecular mechanisms underlying PAX8 inhibition of ferroptosis mutations revealed upregulation of glutamate-cysteine ligase catalytic subunit (GCLC) expression. GCLC mediated the ferroptosis resistance induced by PAX8 in ovarian cancer. In conclusion, our study underscores the pivotal role of PAX8 as a therapeutic target in GPX4-dependent ovarian cancer. The combination of PAX8 inhibitors such as losartan and captopril with ferroptosis inducers represents a promising new approach for ovarian cancer therapy.

MiT/TFE gene fusions like SFPQ-TFE3 drive both epithelial (translocation RCC) and mesenchymal (PEComas) neoplasms. However, no mouse models for SFPQ-TFE3 -related tumors exist and the underlying mechanisms of lineage plasticity remain unclear. Here, we demonstrate that constitutive murine renal expression of SFPQ-TFE3 disrupts kidney development with early neonatal renal failure and death, while post-natal induction induces infiltrative epithelioid tumors, that morphologically and transcriptionally resemble human PEComas, with strong activation of mTORC1 signaling via increased V-ATPase expression. Remarkably, SFPQ-TFE3 expression is sufficient to induce lineage plasticity, with down-regulation of the PAX2/PAX8 nephric lineage factors and tubular epithelial markers, and up-regulation of PEComa differentiation markers in transgenic mice, cell lines and human tRCC. mTOR inhibition downregulates SFPQ-TFE3 expression and rescues PAX8 expression and transcriptional activity in vitro. These data provide evidence of an epithelial cell-of-origin for TFE3 -driven PEComas, highlighting a reciprocal role for SFPQ-TFE3 and mTOR in driving lineage plasticity in the kidney. TFE3-fusions are known to drive both epithelial and mesenchymal renal tumors. Here, the authors generate a transgenic mouse model of renal tumorigenesis expressing the human SFPQ-TFE3 fusion, showing that the fusion regulates mTORC1 activity and induces lineage plasticity.

Müllerian tissue-specific oncogenes, prototyped by PAX8, underlie ovarian tumorigenesis and represent unique molecular vulnerabilities. Further delineating such lineage-dependency factors and associated therapeutic implications would provide valuable insights into ovarian cancer biology and treatment. In this study, we identified SOX17 as a new lineage-survival master transcription factor, which shared co-expression pattern with PAX8 in epithelial ovarian carcinoma. Genetic disruption of SOX17 or PAX8 analogously inhibited neoplastic cell viability and downregulated a spectrum of lineage-related transcripts. Mechanistically, we showed that SOX17 physically interacted with PAX8 in cultured cell lines and clinical tumor specimens. The two nuclear proteins bound to overlapping genomic regions and regulated a common set of downstream genes, including those involved in cell cycle and tissue morphogenesis. In addition, we revealed that small-molecule inhibitors of transcriptional cyclin-dependent kinases (CDKs) effectively reduced SOX17 and PAX8 expression. ZSQ1722, a novel orally bioavailable CDK12/13 covalent antagonist, exerted potent anti-tumor activity in xenograft models. These findings shed light on an actionable lineage-survival transcriptional complex in ovarian cancer, and facilitated drug discovery by generating a serial of candidate compounds to pharmacologically target this difficult-to-treat malignancy.

Disrupted circadian rhythms and reduced sleep duration are associated with several human diseases, particularly obesity and type 2 diabetes, but until recently, little was known about the genetic factors influencing these heritable traits. We performed genome-wide association studies of self-reported chronotype (morning/evening person) and self-reported sleep duration in 128,266 white British individuals from the UK Biobank study. Sixteen variants were associated with chronotype (P<5x10-8), including variants near the known circadian rhythm genes RGS16 (1.21 odds of morningness, 95% CI [1.15, 1.27], P = 3x10-12) and PER2 (1.09 odds of morningness, 95% CI [1.06, 1.12], P = 4x10-10). The PER2 signal has previously been associated with iris function. We sought replication using self-reported data from 89,283 23andMe participants; thirteen of the chronotype signals remained associated at P<5x10-8 on meta-analysis and eleven of these reached P<0.05 in the same direction in the 23andMe study. We also replicated 9 additional variants identified when the 23andMe study was used as a discovery GWAS of chronotype (all P<0.05 and meta-analysis P<5x10-8). For sleep duration, we replicated one known signal in PAX8 (2.6 minutes per allele, 95% CI [1.9, 3.2], P = 5.7x10-16) and identified and replicated two novel associations at VRK2 (2.0 minutes per allele, 95% CI [1.3, 2.7], P = 1.2x10-9; and 1.6 minutes per allele, 95% CI [1.1, 2.2], P = 7.6x10-9). Although we found genetic correlation between chronotype and BMI (rG = 0.056, P = 0.05); undersleeping and BMI (rG = 0.147, P = 1x10-5) and oversleeping and BMI (rG = 0.097, P = 0.04), Mendelian Randomisation analyses, with limited power, provided no consistent evidence of causal associations between BMI or type 2 diabetes and chronotype or sleep duration. Our study brings the total number of loci associated with chronotype to 22 and with sleep duration to three, and provides new insights into the biology of sleep and circadian rhythms in humans.

Abstract Context PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain. Objective We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain. Methods Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1. Results Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 aa residues) was essential for the PAX8-NKX2-1 interaction. Conclusions Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction.