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Background High grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known. Methods We analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs. Results The CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes ( EGFR , RPTOR , ATRIP ) exerted synergic effects on HGSOC PDO viability. Conclusions CDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers.

Background Over the past thirty years, the identification of species-specific molecular markers has significantly advanced our understanding of genetic diversity in both plants and animals. Among these, short InDels have emerged as vital genomic features, contributing more to sequence divergence than single nucleotide polymorphisms do in closely related species. This study aimed to identify specific InDels for Bos taurus , Bubalus bubalis , Capra hircus , and Ovis aries via an in silico approach and validated them in 400 individuals (100 for each species). Results We identified and characterized short, specific InDels in the sequences of the CSN1S1 , CSN1S2 , MSTN , and PRLR genes, which can be used for species identification of Capra hircus, Ovis aries, Bos taurus , and Bubalus bubalis , respectively. We developed a Tetraplex Specific PCR assay to enable efficient discrimination among these species. Conclusions This study highlights the utility of InDels as biallelic, codominant markers that are cost-effective and easy to analyse, providing valuable tools for genetic diversity analysis and species identification.

Cervical cancer (CC) is the fourth most common cause of cancer-related death in women. According to international guidelines, a standard treatment for locally advanced cervical cancer (LACC) consists of exclusive concurrent chemoradiation treatment (CRT). However, chemoradioresistance and subsequent relapse and metastasis of cancer occur in many patients, and survival for these women has generally remained poor. Therefore, strategies to overcome resistance are urgently needed. We have recently reported a radiosensitizing effect of the signal transducer and activator of transcription 1 (STAT1) in CC, associated with the control of [Poly(ADP-ribose) polymerase −1] PARP1 levels, a key factor in cell response to DNA damage induced by radiation. Here, we sought to decipher the underlying mechanism of STAT1-mediated control of PARP1, elucidating its role as a radiosensitizer in CC. Functional and molecular biology studies demonstrated that STAT1 may act at both transcriptional and posttranscriptional levels to modulate PARP1 expression in CC cells. In light of these results, we tested the effect of Olaparib in sensitizing CC cells to radiation and investigated signaling pathways involved in the activity observed. Results showed that PARP1 inhibition, at clinically achievable doses, may indeed selectively improve the sensitivity of resistant CC cells to DNA-damaging treatment. The translational relevance of our findings was supported by preliminary results in a limited patient cohort, confirming that higher PARP1 levels are significantly associated with a radioresistant phenotype. Finally, bioinformatics analysis of GEPIA and TCGA databases, demonstrated that PARP1 mRNA is higher in CC than in normal tissues and that increased PARP1 mRNA expression levels are associated with poor prognosis of LACC patients. Overall, our data open new opportunities for the development of personalized treatments in women diagnosed with CC.

Background High-grade serous ovarian cancer (HGSOC) has poor survival rates due to a combination of diagnosis at advanced stage and disease recurrence as a result of chemotherapy resistance. In BRCA1 (Breast Cancer gene 1) - or BRCA2-wild type (BRCAwt) HGSOC patients, resistance and progressive disease occur earlier and more often than in mutated BRCA. Identification of biomarkers helpful in predicting response to first-line chemotherapy is a challenge to improve BRCAwt HGSOC management. Methods To identify a gene signature that can predict response to first-line chemotherapy, pre-treatment tumor biopsies from a restricted cohort of BRCAwt HGSOC patients were profiled by RNA sequencing (RNA-Seq) technology. Patients were sub-grouped according to platinum-free interval (PFI), into sensitive (PFI > 12 months) and resistant (PFI < 6 months). The gene panel identified by RNA-seq analysis was then tested by high-throughput quantitative real-time PCR (HT RT-qPCR) in a validation cohort, and statistical/bioinformatic methods were used to identify eligible markers and to explore the relevant pathway/gene network enrichments of the identified gene set. Finally, a panel of primary HGSOC cell lines was exploited to uncover cell-autonomous mechanisms of resistance. Results RNA-seq identified a 42-gene panel discriminating sensitive and resistant BRCAwt HGSOC patients and pathway analysis pointed to the immune system as a possible driver of chemotherapy response. From the extended cohort analysis of the 42 DEGs (differentially expressed genes), a statistical approach combined with the random forest classifier model generated a ten-gene signature predictive of response to first-line chemotherapy. The ten-gene signature included: CKB (Creatine kinase B), CTNNBL1 (Catenin, beta like 1), GNG11 (G protein subunit gamma 11), IGFBP7 (Insulin-like growth factor-binding protein 7), PLCG2 (Phospholipase C, gamma 2), RNF24 (Ring finger protein 24), SLC15A3 (Solute carrier family 15 member 3), TSPAN31 (Tetraspanin 31), TTI1 (TELO2 interacting protein 1) and UQCC1 (Ubiquinol-cytochrome c reductase complex assembly factor). Cytotoxicity assays, combined with gene-expression analysis in primary HGSOC cell lines, allowed to define CTNNBL1, RNF24, and TTI1 as cell-autonomous contributors to tumor resistance. Conclusions Using machine-learning techniques we have identified a gene signature that could predict response to first-line chemotherapy in BRCAwt HGSOC patients, providing a useful tool towards personalized treatment modalities.

Background In spite of great progress in the surgical and clinical management, until now no significant improvement in overall survival of High-Grade Serous Ovarian Cancer (HGSOC) patients has been achieved. Important aspects for disease control remain unresolved, including unclear pathogenesis, high heterogeneity and relapse resistance after chemotherapy. Therefore, further research on molecular mechanisms involved in cancer progression are needed to find new targets for disease management. The Krüppel-like factors (KLFs) are a family of transcriptional regulators controlling several basic cellular processes, including proliferation, differentiation and migration. They have been shown to play a role in various cancer-relevant processes, in a context-dependent way. Methods To investigate a possible role of KLF family members as prognostic biomarkers, we carried out a bioinformatic meta-analysis of ovarian transcriptome datasets in different cohorts of late-stage HGSOC patients. In vitro cellular models of HGSOC were used for functional studies exploring the role of KLF7 in disease development and progression. Finally, molecular modelling and virtual screening were performed to identify putative KLF7 inhibitors. Results Bioinformatic analysis highlighted KLF7 as the most significant prognostic gene, among the 17 family members. Univariate and multivariate analyses identified KLF7 as an unfavourable prognostic marker for overall survival in late-stage TCGA-OV and GSE26712 HGSOC cohorts. Functional in vitro studies demonstrated that KLF7 can play a role as oncogene, driving tumour growth and dissemination. Mechanistic targets of KLF7 included genes involved in epithelial to mesenchymal transition, and in maintaining pluripotency and self-renewal characteristics of cancer stem cells. Finally, in silico analysis provided reliable information for drug-target interaction prediction. Conclusions Results from the present study provide the first evidence for an oncogenic role of KLF7 in HGSOC, suggesting it as a promising prognostic marker and therapeutic target.

Background Alemtuzumab (ALEM), an immune reconstitution therapy for multiple sclerosis (MS), has been associated with an increased risk of secondary autoimmune diseases, including Graves’ disease (GD). Graves’ orbitopathy following ALEM (GO-f-ALEM) is a rare, but relevant manifestation. GO-f-ALEM epidemiology and clinical course are still poorly characterized. Main body The first aim of this paper was to perform a systematic review of published evidence of GO-f-ALEM in patients with MS, focusing on clinical features and management. Second aim was to integrate and compare literature data with real-world pharmacovigilance reports and institutional cases. A systematic review of published literature databases (PubMed, Embase, Scopus) from inception to December 2024 was performed according to PRISMA guidelines. A total of 42 published GO-f-ALEM cases in MS patients were identified. In parallel, as additional sources of data, which were not included in the systematic review analysis, 272 cases were retrieved from the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) database, and three institutional cases were described and analysed. GO-f-ALEM occurred more frequently in middle-aged adults (mean age 41 years old), with female predominance (60%) and a high rate of smoking (60%). GO generally occurred after 2–5 years from ALEM initiation. Fluctuating thyroid function and thyrotropin receptor antibody (TRAb) levels were commonly observed, with frequent need for definitive thyroid treatment (surgery or radioactive iodine - RAI). GO presentation was heterogeneous - from mild to sight-threatening forms - and mainly manifesting with lid retraction, proptosis, and diplopia. Concurrently, pharmacovigilance data from FAERS identified almost 300 reports of eye disorders potentially consistent with GO, thus suggesting under-diagnosis or under-reporting prevalence in literature. Institutional cases confirmed the potential for sight-threatening GO with dysthyroid optic neuropathy. Conclusions Our data suggest that GO-f-ALEM is an underrecognized disorder within the spectrum of ALEM-induced autoimmunity, with peculiar features, as suggested by clinical and immunological observations and simultaneously by pharmacovigilance reports. Routine ophthalmic monitoring and standardized GO classification are essential for diagnosis and follow-up. Prospective studies are warranted to better define its epidemiology and guide optimised management strategies.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ET A R) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ET A R/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ET A R, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ET A R/β-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ET A R/β-arr1 regulates Intα5β1 functional pathway.

ObjectiveAim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the α-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs).MethodsDNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5′-CACTGATCTTAAAGCTCAGGTT-3′ (forward) and 5′-TCAGA GTAGGCCACAGAAG-3′ (reverse).ResultsSequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic “within” and variable in size “among” the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources (0.25 ng/μL).ConclusionWe developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

Purpose Diabetic macular edema (DME) is a leading cause of vision loss in patients with diabetes. In developed countries, intravitreal (IVT) anti-vascular endothelial growth factor (VEGF) injections are the standard-of-care first-line treatment for DME. However, despite the efficacy of anti-VEGF and associated improvements in prognosis, some patients show only a partial response and continue to require monthly injections. The aim of this study was to investigate the effect of switching from aflibercept 2.0 mg to faricimab (which targets both angiopoietin-2 [Ang-2] and VEGF-A) on visual function, retinal anatomy and intraretinal fluid (IRF) dynamics in patients with refractory DME. Methods A single-center, observational study of patients with aflibercept-resistant DME who switched to IVT faricimab treatment, comprising a 3-month loading phase, during which faricimab was administered monthly (total of four injections), followed by a treat-and-extend regimen. Visual acuity, anatomical parameters, and fluid dynamics were assessed from baseline to Month 6 in an interim analysis. Results Fourteen eyes from 10 patients were included. At Month 6, mean best-corrected visual acuity improved by + 2.7 Early Treatment Diabetic Retinopathy Study (ETDRS) letters (not statistically significant). Mean changes in central macular thickness and outer nuclear layer (ONL) thickness at Month 6 were not significant. However, ONL thickness was significantly reduced in multiples ETDRS macular grid subfields at Month 4. Subretinal fluid volume was negligible through Month 6, with most fluid located in the intraretinal layers (97.8–100%). Total IRF decreased by 22% at Month 4, reaching a nadir of − 37% at Month 2. There was no significant change in mean vascular density from Month 0 to Month 4. Conclusion Faricimab treatment led to modest early improvements in visual acuity and retinal anatomy overall in patients with refractory DME. The reduction in total IRF at Month 4 may be attributable to Ang-2 inhibition in these patients, who had previously not responded to anti-VEGF treatment alone. Longer-term studies are needed to evaluate the durability and long-term efficacy of faricimab for the treatment of refractory DME. Key messages What is known Despite the success of anti-vascular endothelial growth factor (VEGF) therapies in treating diabetic macular edema (DME), many patients still require monthly injections, and some only show a partial response to them. For those with persistent DME, treatment often involves switching anti-VEGF agents or using corticosteroids; while this can be effective, it is associated with adverse events such as ocular hypertension and cataracts. Faricimab, a novel anti-VEGF and anti-angiopoietin 2 monoclonal antibody, has shown superior drying capacity in the past. What is new A detailed analysis of the retinal layers showed a significant reduction in outer nuclear layer (ONL) thickness following faricimab initiation, while inner nuclear layer and outer plexiform layer thickness remained unchanged. The reduction in ONL thickness, indicated a centripetal drying pattern, was achieved by the end of the loading phase. In our study population, mean vascular density (VD) on optical coherence tomography angiography remained stable at Month 4, and, to our knowledge, these are the first data regarding the effect of faricimab on VD in diabetic patients

The tumor immune microenvironment (TIME) has largely been reported to cooperate on tumor onset and progression, as a consequence of the phenotype/functional plasticity and adaptation capabilities of tumor-infiltrating and tumor-associated immune cells. Immune cells within the tumor micro (tissue-local) and macro (peripheral blood) environment closely interact by cell-to-cell contact and/or via soluble factors, also generating a tumor-permissive soil. These dangerous liaisons have been investigated for pillars of tumor immunology, such as tumor associated macrophages and T cell subsets. Here, we reviewed and discussed the contribution of selected innate immunity effector cells, namely neutrophils and natural killer cells, as “soloists” or by their “dangerous liaisons”, in favoring tumor progression by dissecting the cellular and molecular mechanisms involved.