Approximately 50% of prostate cancer (PCa) patients harbor fusions involving the TMPRSS2 and ERG genes. Despite this, tailored therapies targeting the fused gene, tERG, remain undeveloped. Our study analyzed biopsy samples from two clinical trials assessing the efficacy of androgen receptor (AR) signaling inhibitors (ARSIs). The results revealed that tERG promotes resistance to ARSIs and is associated with elevated levels of the glucocorticoid receptor (GR). Subsequent assays showed that GR directly interacts with tERG, alleviates allosteric autoinhibition, and prevents chemotherapy-induced tERG degradation. In PCa models, either inhibiting GR or lowering cortisol levels suppressed tumor growth in tERG-positive models, but not in tERG-negative models. In addition, patient-derived fusion-positive xenografts displayed enhanced sensitivity to combined GR and AR inhibitors. Collectively, these findings highlight TMPRSS2-ERG as a new biomarker and propose that simultaneous inhibition of GR and AR may specifically benefit tERG-positive patients. However, GR stimulatory corticosteroid therapies may not be advisable for this patient subgroup.

Abstract Chemotherapy response in breast cancer is highly heterogeneous and influenced by tumor-intrinsic drivers of drug sensitivity, including cancer stem cell abundance. We previously reported that the scaffold protein p140Cap limits breast cancer stem cell traits and delays tumor progression. Here, we investigated the role of p140Cap in shaping sensitivity to chemotherapy in HER2-positive and triple-negative breast cancer. In preclinical and patient-derived models, p140Cap enhances chemotherapy response by increasing intracellular doxorubicin retention, DNA damage and subsequent apoptosis. Mechanistically, p140Cap constrained a doxorubicin-negative side population enriched for stem-like properties and elevated ABCC1 expression via inhibition of β-Catenin signaling. Constitutively active β-Catenin expression reversed this phenotype, whereas pharmacological inhibition of the Wnt/β-Catenin pathway with IWR-1 or LGK-974 sensitized p140Cap-deficient tumors to chemotherapy. Clinically, analyses of breast cancer cohorts and patient-derived xenograft models identify p140Cap as predictive biomarker of chemotherapy response, proposing p140Cap-guided patient stratification, dose optimization and rational combination therapies.

BACKGROUND: Bladder cancer (BLC) remains a clinically challenging malignancy due to its pronounced inter- and intra-patient heterogeneity, which contributes to therapeutic resistance and poor clinical outcomes. Capturing and modeling this complexity is essential for the development of effective, personalized therapeutic strategies. METHODS: To investigate molecularly and functionally BLC heterogeneity, this study employed three-dimensional patient-derived organoids (PDOs) and ex vivo tissue slice culture as advanced preclinical models. PDOs were established from a patient’s cohort enrolled at Regina Elena National Cancer Institute in Rome using spatially distinct tumor samples from central (TC) and peripheral (TP) tumor regions to preserve intratumoral heterogeneity. Genomic and transcriptomic fidelity between PDOs and their parental tumors was assessed through multi-omics analyses. Functional assays were conducted to evaluate therapeutic responses. A second patient cohort from the University of Bern, was used to further characterize cellular and microenvironmental features of BLC samples combining ex vivo tissue culture and multiparametric-Flow Cytometry (FACS) to address treatment-induced cancer cell plasticity and epithelial-marker expression dynamics. RESULTS: PDOs recapitulated the genomic and transcriptomic landscapes of the original tumors. Early passage PDOs retained components of the tumor microenvironment, including immune cell subsets, suggesting their relevance for ex vivo modeling of tumor-immune interactions. Functional assays revealed spatial heterogeneous responses to both chemotherapy and EGFR/FGFR-targeted therapies. A corresponding reduction of the EGFR-high basal-like population was observed in ex vivo tissue cultures. In contrast, treatment with a PD-1 immune checkpoint inhibitor showed consistent responses across PDOs regions but correlated with the degree of immune infiltration observed in the parental tumors. CONCLUSION: This integrated dual-cohort approach demonstrates that both BLC PDOs and ex vivo tissue cultures offer a versatile and faithful platform for dissecting BLC heterogeneity and advancing functional precision medicine recapitulating patient-specific immune-tumor interactions observed in native tissues.

Background: We have previously demonstrated that ovarian tumor (Otu) domain-containing ubiquitin aldehyde-binding protein 2 (Otub2), a deubiquitinating enzyme, exerts anti-apoptotic effects in primary human islets. The present study aims to further elucidate the molecular mechanisms underlying the role of Otub2 as a regulator of insulin secretion and β-cell function. Methods: Otub2 overexpression or silencing was employed to study its effects on cultured MIN6 cells and dispersed human islets. To evaluate its in vivo effects, Otub2 knockout (KO) mice were employed, as well as a pancreata-specific Otub2 overexpression model. RNA sequencing was performed on pancreatic tissue from Otub2-KO and control mice to study its effects on gene expression patterns. Co-immunoprecipitation followed by mass spectrometry identified Otub2-interacting proteins. Results: Overexpression of Otub2 inhibited NF-κB activity and enhanced glucose-stimulated insulin secretion (GSIS) in cultured MIN6 cells and primary human islets. Otub2 KO mice exhibited impaired glucose tolerance and upregulation of NF-κB target genes. Conversely, selective in vivo overexpression of Otub2 in pancreata of C57BL wild-type mice resulted in significantly lower (~30%) blood glucose levels, post glucose injection, compared to control mice. Transcriptomic analysis of KO pancreata revealed downregulation of K+ transporter-related genes and upregulation of oxidative phosphorylation genes, consistent with defective insulin secretion. Mass spectrometry identified the voltage-gated potassium channel subunit Kv9.3 as a major Otub2 binding partner, along with paternally expressed 3 (Peg3) and calcium/calmodulin dependent protein kinase II delta (Camk2d) proteins known to promote NF-κB signaling and β-cell apoptosis. Conclusions: Otub2 is a critical regulator of β-cell function, acting through modulation of NF-κB signaling and K+ channel-associated complexes. By deubiquitinating components such as Peg3 and Camk2d, Otub2 may protect β-cells from cytokine-induced apoptosis and sustain insulin secretory capacity. These findings position Otub2 as a potential therapeutic target for preserving β-cell function in diabetes.

The polynucleotide kinase/phosphatase (PNKP) is a DNA repair enzyme possessing bifunctional DNA 3'-phosphatase and DNA 5'-kinase activities. It plays an important role in the rejoining of single- and double-strand DNA breaks and is considered as a potential therapeutic target for different cancer types. Here we show that PNKP is highly expressed in triple negative breast cancer (TNBC) and associated with poor prognosis and chemoresistance. Targeting of PNKP enhanced ferroptosis in TNBC, which was associated with increased labile iron pool and ROS and concomitantly decreased in intracellular glutathione, SCD1 and GPX4 levels. Transcriptomic profiling and mechanistic data indicate that PNKP targeting robustly enhances the lysosomal and the autophagic machinery by activating STING and concurrently inhibiting STAT3, thereby increasing ferritinophagy, intracellular iron level and modulating the expression of key ferroptosis regulators. Importantly, PNKP and STAT3 are rapidly phosphorylated, colocalize, and interact upon ferroptosis induction or doxorubicin treatment, the first line treatment for TNBC patients. Targeting PNKP together with doxorubicin synergistically inhibited the growth of TNBC in an animal model and of TNBC-patients derived organoids. These results offer a promising therapeutic combination for TNBC and highlight the clinical potential of PNKP targeting and ferroptotic death for TNBC therapy.

EMT converts epithelial (E) phenotypes to invasive mesenchymal (M) states. However, analyses of circulating tumor cells (CTCs) indicated that biphenotypic (E+M) CTCs better correlate with metastasis. Similarly, investigations of murine tumors undergoing EMT concluded that early E+M states posses the highest metastatic potential. To explore this, we selected in animals with breast cancer CTCs having progressively increasing intravasation abilities. This revealed that downregulation of arrestin Arrdc4 associates with CTC aggressiveness. In xenografts, depleting Arrdc4 accelerated tumor progression, whereas overexpression hindered progression in immunocompetent, but not in immunocompromised mice. Mechanistically, high Arrdc44 suppresses glucose uptake and enhances gasdermin E, triggering pyroptosis a type of pro-inflammatory cell death. Consistently, Arrdc4's lowest levels characterize the most metastatic biphenotypic states. In patients, both epigenetic and chromosomal aberrations downregulate ARRDC4 and predict poor prognosis. In summary, the uncovered mechanism portrays pyroptosis of biphenotypic EMT cells as a rheostat of CTCs, which may resolve the controversy on the role played by EMT in metastasis. ### Competing Interest Statement The authors have declared no competing interest.

Cellular plasticity mediates tissue development as well as cancer growth and progression. In breast cancer, a shift to a more epithelial phenotype (epithelialization) underlies a state of reversible cell growth arrest called tumor dormancy, which enables drug resistance, tumor recurrence, and metastasis. Here, we explored the mechanisms driving epithelialization and dormancy in aggressive mesenchymal-like breast cancer cells in three-dimensional cultures. Overexpressing either of the epithelial lineage-associated transcription factors OVOL1 or OVOL2 suppressed cell proliferation and migration and promoted transition to an epithelial morphology. The expression of OVOL1 (and of OVOL2 to a lesser extent) was regulated by steroid hormones and growth factors and was more abundant in tumors than in normal mammary cells. An uncharacterized and indirect target of OVOL1/2, C1ORF116 , exhibited genetic and epigenetic aberrations in breast tumors, and its expression correlated with poor prognosis in patients. We further found that C1ORF116 was an autophagy receptor that directed the degradation of antioxidant proteins, including thioredoxin. Through C1ORF116 and unidentified mediators, OVOL1 expression dysregulated both redox homeostasis (in association with increased ROS, decreased glutathione, and redistribution of the transcription factor NRF2) and DNA damage and repair (in association with increased DNA oxidation and double-strand breaks and an altered interplay among the kinases p38-MAPK, ATM, and others). Because these effects, as they accumulate in cells, can promote metastasis and dormancy escape, the findings suggest that OVOLs not only promote dormancy entry and maintenance in breast cancer but also may ultimately drive dormancy exit and tumor recurrence.

Endometrial cancer (EC) is one of the most common gynecologic malignancies amongst women worldwide. Its incidence and mortality rates have been increasing in the last decade. In the present work, we built a patient EC-derived organoid (PDOs) platform that faithfully recapitulated tumor phenotype, genomic alterations, and expression profiles of matched-primary cancer tissues. Interestingly, we found that the response of EC-derived PDOs to both standard therapy and a wide range of targeted drugs accordingly to their specific druggable genetic alterations was congruent with that of the originating patients. We also isolated and genomically characterized matched-PDO stromal cells, specifically cancer-associated fibroblasts (CAFs). Unlike PDOs matched CAFs were poorly responsive and underwent to pro-inflammatory senescence upon treatment with standard therapy. Collectively, our findings established a EC-PDOs preclinical platform which allows assessing the therapeutic response of tumor and surrounding tumor microenvironment cellular landscape.

Here, we present a computational protocol for predicting repressors of cancer cell death by combining vulnerability and transcriptomic responses to cell death inducers. We describe steps for calculating a set of predictors for each gene and aggregating them into a single metric, ranking the genes according to their predictive power. We then detail procedures for selecting candidate genes for experimental validation based on this ranking and other considerations. This protocol enables the identification of several experimentally validated cell death repressors. For complete details on the use and execution of this protocol, please refer to Vinik et al.1.

Approximately 50% of prostate cancer (PCa) patients harbor fusions involving the TMPRSS2 and ERG genes. Despite this, tailored therapies targeting the fused gene, tERG , remain undeveloped. Our study analyzed biopsy samples from two clinical trials assessing the efficacies of androgen receptor (AR) signaling inhibitors (ARSIs). The results revealed that tERG promotes resistance to ARSIs and is associated with elevated levels of the glucocorticoid receptor (GR). Subsequent assays showed that GR directly interacts with tERG, alleviates allosteric autoinhibition and prevents chemotherapy-induced tERG degradation. In PCa models, either inhibiting GR or lowering cortisol levels suppressed tumor growth in tERG-positive models, but not in fusion-negative models. In addition, patient-derived fusion-positive xenografts displayed enhanced sensitivity to combined GR and AR inhibitors. Collectively, these findings highlight TMPRSS2-ERG as a new biomarker and propose that simultaneous inhibition of GR and AR may specifically benefit tERG -positine patients. However, GR stimulatory corticosteroid therapies may not be advisable for this patient subgroup.

Epithelial lineage differentiation is pivotal to mammary gland development and it can pause metastasis of breast cancer (BC) by inducing tumor dormancy. To simulate this, we expressed epithelial genes in mesenchymal BC cells. Inducible expression of the epithelial OVOL genes in metastatic BC cells suppressed proliferation and migration. We found that C1ORF116, an OVOL's target, is susceptible to genetic and epigenetic aberrations in BC. It is regulated by steroids and functions as a putative autophagy receptor that inhibits antioxidants like thioredoxin. Accordingly, boosting epithelialization lowered glutathione, elevated reactive oxygen species and increased both DNA oxidation and double strand breaks. Epithelialization also associated with redistribution of NRF2 and an altered interplay among p38, ATM, and the other kinases regulating the DNA damage response. Hence, hormonal regulation of OVOLs and chronic stress might permit epithelial differentiation and retard exit from dormancy, while altering redox homeostasis and permitting DNA damage accumulation, which may awaken dormant tumors. ### Competing Interest Statement The authors have declared no competing interest.

Ferroptosis and apoptosis are key cell-death pathways implicated in several human diseases including cancer. Ferroptosis is driven by iron-dependent lipid peroxidation and currently has no characteristic biomarkers or gene signatures. Here a continuous phenotypic gradient between ferroptosis and apoptosis coupled to transcriptomic and metabolomic landscapes is established. The gradual ferroptosis-to-apoptosis transcriptomic landscape is used to generate a unique, unbiased transcriptomic predictor, the Gradient Gene Set (GGS), which classified ferroptosis and apoptosis with high accuracy. Further GGS optimization using multiple ferroptotic and apoptotic datasets revealed highly specific ferroptosis biomarkers, which are robustly validated in vitro and in vivo. A subset of the GGS is associated with poor prognosis in breast cancer patients and PDXs and contains different ferroptosis repressors. Depletion of one representative, PDGFA-assaociated protein 1(PDAP1), is found to suppress basal-like breast tumor growth in a mouse model. Omics and mechanistic studies revealed that ferroptosis is associated with enhanced lysosomal function, glutaminolysis, and the tricarboxylic acid (TCA) cycle, while its transition into apoptosis is attributed to enhanced endoplasmic reticulum(ER)-stress and phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC) metabolic shift. Collectively, this study highlights molecular mechanisms underlying ferroptosis execution, identified a highly predictive ferroptosis gene signature with prognostic value, ferroptosis versus apoptosis biomarkers, and ferroptosis repressors for breast cancer therapy.

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient’s outcome, and reliable biomarkers for effective anticancer therapies.

Apoptosis and ferroptosis are two regulated cell death (RCD) pathways implicated in different human diseases and considered as promising strategies to eliminate cancer cells. These two pathways are characterized by distinct morphological and biochemical properties, induce cell death through different mechanisms, but share common regulators in different cancer types. Although apoptosis and ferroptosis have been extensively studied over the last few years, their transcriptomic responses have not yet been systematically compared due to remarkable variability in the transcriptomic data. Here we provide a brief snapshot of the transcriptomic landscapes of the apoptosis and ferroptosis responses in cancer, discuss their divergent and convergent properties, and implications to cancer therapy.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted in chromosome 10) is genetically deleted or downregulated in many cancer types. Loss of PTEN protein expression is frequently found in lung cancer while genetic alterations are less abundant. PTEN expression is regulated at multiple genetic and epigenetic levels and even partial reduction of its expression increases cancer occurrence. We show that YAP and TAZ cooperate with EZH2, and MYC to transcriptionally repress onco-suppressor genes, including PTEN, in non-small cell lung cancer (NSCLC) cells. YAP/TAZ-EZH2-MYC transcriptional regulators form a nuclear complex that represses PTEN transcription, while their combinatorial targeting restores PTEN expression, attenuates NSCLC cell growth, and prevents compensatory responses induced by single treatments. Datasets analysis of NSCLC patients revealed that PTEN expression is negatively correlated to YAP/TAZ, EZH2 and MYC and that low expression of PTEN is predictive of poor prognosis, especially at earlier stages of the disease. These findings highlight the repressive role of the YAP/TAZ-EZH2-MYC axis on tumor-suppressor genes and offer a potential therapeutic strategy for lung cancer patients with low PTEN levels.

Identification of promising targets for cancer therapy is a global effort in precision medicine. Here, we describe a computational pipeline integrating transcriptomic and vulnerability responses to cell-death inducing drugs, to predict cell-death suppressors as candidate targets for cancer therapy. The prediction is based on two modules; the transcriptomic similarity module to identify genes whose targeting results in similar transcriptomic responses of the death-inducing drugs, and the correlation module to identify candidate genes whose expression correlates to the vulnerability of cancer cells to the same death-inducers. The combined predictors of these two modules were integrated into a single metric. As a proof-of-concept, we selected ferroptosis inducers as death-inducing drugs in triple negative breast cancer. The pipeline reliably predicted candidate genes as ferroptosis suppressors, as validated by computational methods and cellular assays. The described pipeline might be used to identify repressors of various cell-death pathways as potential therapeutic targets for different cancer types.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

We have previously shown that otubain 2 (OTUB2), a deubiquitinating enzyme, inhibits caspase-3/7 activity in primary human islets; promotes insulin secretion and inhibits cytokine-induced nuclear factor-κB (NFκB) activity. In the present work we show that overexpression of Otub2 in MIN6 cells inhibits NFκB activity and the expression of its target genes MCP-1 and iNOS. Consequently, both the basal and the cytokine-induced apoptosis of cultured MIN6 cells and dispersed human islets were inhibited. Overexpression of Otub2 in MIN6 cells increase the mRNA levels of NKx6.1 and Glut2 and concomitantly increased glucose-stimulated insulin secretion (GSIS) (by 2-3-fold). The beneficial effects of Otub2 on β-cell function was demonstrated by the phenotype of Otub2-/+ and Otub2-/- mice, which manifested impaired glucose tolerance and increased expression of NFkB target genes (e.g. IP-10, MCP-1 and IL-1β). RNAseq analysis of pancreata derived from OTUB2 KO mice revealed reduced expression of genes that down regulate K+ transporters (e.g. Ank2, Cacna1a and Kcnab1) combined with an increase in oxidative phosphorylation related genes. Given that closure of K+ channels is crucial for insulin secretion, these results could account, at least in part, for the impaired GSIS in the OTUB2 KO mice. Indeed, mass-spectrometry analysis of proteins co-immunoprecipitated with Otub2 revealed the voltage-gated potassium channel subunit Kv9.3 as a major Otub2 binding-partner. Additional binding partners included the Peg3 and Camk2d proteins, which promote NFκB signaling and β-cell death. Hence, by deubiquitinating proteins in complexes that contain Peg3 and Camk2d, Otub2 might inhibit propagation of NFκB signaling and β-cell apoptosis. Collectively our findings implicate Otub2 as a key regulator of β-cell function, mainly affecting NFκB signaling and the K+ channels that regulate insulin secretion. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND AND PURPOSE: A malformed corpus callosum carries a risk for abnormal neurodevelopment. The advent of high-frequency transducers offers the opportunity to assess corpus callosum development in early pregnancy. The aim of the study was to construct a reference chart of the fetal corpus callosum length on ultrasound between 13 and 19 weeks of gestation and to prospectively examine growth patterns in pathologic cases. MATERIALS AND METHODS: We performed a prospective cross-sectional study between 2020 and 2022 in well-dated, low-risk, singleton pregnancies between 13 and 19 weeks of gestation. A standardized image was obtained in the midsagittal plane. Imaging criteria were used as a confirmation of the early corpus callosum. Measurements were taken by 4 trained sonographers. Intra- and interobserver variability was assessed. Corpus callosum length in centiles were calculated for each gestational week. RESULTS: One hundred eighty-seven fetuses were included in the study. All cases met inclusion criteria. At 13 weeks of gestation, the margins of the early corpus callosum were sufficiently clear to be measured in 80% (20/25) of fetuses. A cubic polynomial regression model best described the correlation between corpus length and gestational age. The correlation coefficient (r2) was 0.929 (P < .001). Intra- and interobserver variability had high interclass correlation coefficients (>0.99). Presented is the earliest published case of agenesis of corpus callosum and a case of dysgenetic corpus callosum in Rubinstein-Taybi syndrome. CONCLUSIONS: Provided is a nomogram of the early fetal corpus callosum. Applying imaging criteria helped to identify a case of complete agenesis of the corpus callosum as early as 14 weeks.