The popularity of microRNA expression analyses is reflected by the existence of thousands of sRNA-seq studies in which matched total RNA-seq data are often unavailable. The lack of paired sequencing experiments limits the analysis of microRNA-gene regulatory networks. Here, we explore whether protein-coding gene expression can be quantified directly from transcript fragments present in sRNA-seq experiments. We analyze studies containing matched total RNA and small RNA from four human tissues and recover transcript fragments from the sRNA-seq data sets. We find that the expression levels of protein-coding gene transcripts derived from sRNA-seq data sets are comparable to those from total RNA-seq experiments (R 2 ranging from 0.33 to 0.76). Analyses across multiple tissues and species show similar correlations, indicating that the approach is applicable across organisms. We confirm that transcript half-life and the expression of housekeeping or highly abundant genes do not bias the results. Analysis of the expression of both microRNAs and coding genes from the same sRNA-seq experiments demonstrates that known microRNA-target interactions are, as expected, inversely correlated with the expression profiles of these microRNA-mRNA pairs. For a dual mRNA/miRNA profile, we recommend sequencing the ≥25 nucleotide fraction at 5 million or more reads. To confirm the utility of this approach, we apply our method to breast cancer sRNA-seq data sets lacking total RNA-seq data and achieve 75% recall and 64% accuracy comparing inferred coding gene expression with qPCR-validated targets. Our findings demonstrate that quantifying mRNA fragments from sRNA-seq experiments provides a reliable approach to investigate microRNA-mRNA interactions when total RNA-seq is unavailable.

Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the TP53 tumour suppressor gene, which encodes the multifunctional transcription factor p53. p53 is the most commonly mutated protein in human cancer, with the majority occurring within the DNA-binding domain, often disrupting transcriptional activity and resulting in a loss-of-function. Here, we characterise the novel p53N263Tfs*7 truncated mutant, identified as a germline mutation from a patient with LFS who developed breast cancer. Functional assays revealed a partial loss-of-function across key cellular processes, including proliferation, cell death, cell motility, and transcriptional transactivation. This mutant lacked a dominant-negative effect, distinguishing it from common DNA-binding domain missense mutations. Our findings demonstrate that oncogenesis in LFS can be driven by partial impairment of functional p53, rather than dominant-negative or gain-of-function mutations alone. This underscores the clinical significance of recognising subtle TP53 variants for the refined molecular classification and clinical prediction of cancer risk in TP53 mutation carriers.

Eighty-five percent of the human proteome has at least one interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Analysis of antibody variable domains and characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells indicated charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found an optimisable charge discrepancy between variable heavy framework and CDR sites. When applied to reduce the search space and rank the products of AI-led inverse folding this creates a single highly soluble, abundant and stable intrabody with parent antibody epitope recognition. Over six hundred intrabody sequences are presented targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomer specificity. Interactions were validated for p53, α-synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. Here we show reliable repurposing of the sequenced antibody interactome inside the cell. O’Shea and colleagues establish that optimisation of charge and stability is sufficient to enable any single-chain variable fragment intrabody to function within the cell. The authors use AI-led inverse folding to optimise intrabody characteristics, and they present hundreds of intrabody sequences targeting sixty cytoplasmic proteins.

Immune checkpoint therapies aim to restore anti-tumour immunity by blocking inhibitory signals that suppress T-cell activation. The most effective current strategies use humanised antibodies targeting PD1 or its ligand PDL1. However, due to their large size, antibodies often exhibit limited tissue diffusion, resulting in poor penetration into solid tumours. To address this challenge, we developed a novel checkpoint inhibitor approach that uses mesenchymal stromal cells (MSCs) to deliver a high-affinity, soluble PD1 receptor (sPD1HAC). We found that sPD1HAC produced as an IgG1-Fc fusion protein provided functional expression in MSCs. The sPD1HAC-IgG1-Fc fusion protein showed strong and specific binding to PDL1 and could outcompete recombinant PD1 and anti-PDL1 antibodies, including the clinically approved durvalumab. Although the sPD1HAC variant was designed to block human PD1-PDL1 signalling, it also bound murine PDL1 and blocked the binding of mouse-specific PD1 antibodies as well as recombinant murine PD1 protein. Accordingly, we found significant anti-tumour activity of intravenously administered MSCs.PD1 HAC in an aggressive B16-F10 cancer model. This cell-based immune checkpoint approach offers a potential therapeutic option for targeting stroma-rich, treatment-resistant tumours.

Breast cancer (BCa) is the most frequently diagnosed malignancy in women worldwide, with approximately 70

In biology proximity is paramount and eighty-five percent of the human proteome has at least one documented interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Sequence analysis of 106 antibody variable domains alongside the cytoplasmic human proteome shows charge and isoelectric point are characteristics ill adapted to intracellular monodispersity. Characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells confirmed charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found variable heavy domain framework sites to be generally positively charged, and promote insolubility, but be amenable to optimisation. This is applied in combination to reduce the search space and refine the products of AI-led inverse folding to create highly soluble, abundant and thermally stable intrabodies that maintain parent antibody epitope recognition. Over six hundred intrabody sequences are described targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomeric state specificity. Interactions were validated for p53, α-synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. This approach removes obstacles hindering intracellular repurposing of the vast sequenced antibody interactome with applications relevant to many human disease states. ### Competing Interest Statement N.R.C. is Chief Science Officer of ProMIS Neurosciences, and a Professor Emeritus at the University of British Columbia.

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.

Abstract Breast cancer is the most frequently diagnosed cancer in women worldwide. Oestrogen Receptor-α (ERα), is expressed in two thirds of human breast cancers, and the majority of patients are sensitive to endocrine therapy, i.e. anti-oestrogens or aromatase inhibitors that aim to block oestrogen signalling. In addition to ERα, the other members of the steroid receptor family (androgen, AR; progesterone, PR; glucocorticoid, GR; mineralocorticoid receptors, MR) also appear to play an important role in tumour development and recurrence, but the role of this signalling in cancer progression has not been fully characterised. Therefore, this study aims to address the role of the steroid receptor family members in breast cancer and to assess the effect of downstream signalling upon prognosis. Investigation of receptor signalling cross-talk using reporter, proliferation and migration assays, demonstrated that ERα and the other steroid receptors inhibit each other’s activity in endocrine responsive breast cancer cell lines, MCF7 and T47D. Furthermore, treatment with antioestrogen reversed the suppressive effects of ER𝛼;;; on the activity of the androgen, glucocorticoid progesterone receptors. To characterize receptor crosstalk on a global scale, RNA-seq and ChIP-seq datasets were analysed. Analysis of the ChIP-seq data demonstrated that the PR, GR and AR reprogramming of ERα results in the loss of ERα canonical binding sites and the gain of novel response elements/binding sites. Motif enrichment analysis of the novel ERα binding sites suggested that the altered binding in response to AR, GR and PR signalling, promotes the recruitment of ERα to androgen, glucocorticoid and progesterone response elements, respectively. Gene ontology analysis also revealed that this change in ERα binding sites is correlated with alterations in genes linked to cell proliferation and growth. Moreover, transcriptome studies showed that cotreatment of cells with estradiol and glucocorticoid or mineralocorticoid resulted in the upregulation of apoptotic signalling pathways. These findings may provide an explanation for the observed downregulation of GR and MR expression in ERα positive breast cancer. Patient datasets were used to investigate the prognostic significance of the steroid receptor target genes of interest, more specifically, those deregulated as a result of cross-talk. Considering overall survival and disease specific survival, univariate and multivariate Cox models with Lasso Regression were used to identify prognostic gene signatures using RNA_seq datasets. Kaplan-Meier (KM) and ROC analyses were applied to evaluate risk models. Established genetic markers associated with MR, AR, GR and PR was used to stratify BC patients into low and high risk groups. Patients survival was found to be positively correlated with these signatures, demonstrates that these gene sets have prognostic value. Further studies are required to validate these preliminary results to understand the role of steroid receptor crosstalk in breast cancer formation, progression, and survival. Citation Format: Aygun Azadova, Greg Brooke, Antonio Marco. Steroid receptor cross-talk in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB335.

Abstract Background Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. Results We have generated high-resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with ~ 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. The tumour tissues were characterised by a 5–10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. This effect was correlated with gene activity, differential DNA methylation and changes in local occupancy of linker histone variants H1.4 and H1X. Conclusions Our study offers a novel resource of high-resolution nucleosome maps in breast cancer patients and reports for the first time the effect of systematic decrease of NRL in paired tumour versus normal breast tissues from the same patient. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient diagnostics, stratification and monitoring.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly against prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and replication in both TMPRSS2- and non-expressing immortalised cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E-and OC43- infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72h post-infection. The virus-mediated effect to AR signaling was further confirmed with a consensus androgen response element (ARE)-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen signaling, offering insights for potential antiviral interventions.

Hydrogen peroxide (H2O2) and lipid hydroperoxides (LOOH) are initiators and transducers of inter- and intra-cellular signaling in response to diverse environmental, pathological and developmental cues. The accumulation of both H2O2 and LOOH is often temporally and spatially coincident in tissues, but it is unknown if this coincidence extends to subcellular compartments. If distinct accumulation of different peroxides occurs at this smaller spatial scale, then it would be an important factor in signaling specificity. Fusion of the redox-sensitive (ro)GFP2 to the Saccharomyces cerevisiae (yeast) OXIDANT RECEPTOR PEROXIDASE1 (ORP1), also known as GLUTATHIONE PEROXIDASE3 (GPX3), created a now widely used biosensor that is assumed to detect H2O2 in vivo. This is despite monomeric GPX enzymes, such as ORP1/GPX3, possessing wide peroxide substrate specificities. Consequently, we confirmed in vitro that roGFP2-ORP1 is not only oxidized by H2O2, but also by phospholipid fatty acid peroxides generated in lecithin-derived liposomes by lipoxygenase-catalyzed peroxidation. This led us to doubt that roGFP2-ORP1 in vivo is specific for H2O2. To address this issue of peroxide specificity, we constructed a modified biosensor called roGFP2-synORP1. This version has greatly diminished reactivity towards phospholipid fatty acid peroxides but retains high sensitivity for H2O2. These two roGFP2-based biosensors, targeted to chloroplasts, cytosol and the nucleus, were quantitatively imaged in parallel in Nicotiana benthamiana abaxial epidermal cells experiencing high light- and herbicide-induced photo-oxidative stress. From differential patterns of oxidation of these probes, we inferred that the chloroplasts accumulated both peroxide types. In contrast, LOOH and H2O2 accumulated exclusively in the cytosol and nucleus respectively. Therefore, this suggests that the signalling networks initiated by different peroxides will have a distinct spatial component.### Competing Interest StatementThe authors have declared no competing interest.

Oncogenic activation of the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways are frequent events in Prostate Cancer (PCa) that have been correlated to tumour formation, disease progression and therapeutic resistance. At the intersection of these two pathways lies the p90 ribosomal S6 kinase (RSKs) family, which regulates many proteins involved in cell survival, growth and proliferation. As such, deregulated RSKs activity has been associated with multiple cancer types, including PCa. However, the full extent of the RSKs involvement in prostate tumorigenesis remains to be determined. Here we have shown that RSKs levels are increased in PCa samples and cell lines. The RSKs were found to enhance Androgen Receptor (AR) activity, the key oncogenic driver in PCa. Indeed, all RSKs were found to interact in close proximity to the AR. However, RSK2/4, but not RSK1/3, showed changes in cell localisation following AR nuclear translocation. Consistently, silencing of RSK2/4, but not RSK1/3, inhibited PCa proliferation in an androgen-dependent and independent manner, respectively, and induced different signaling events downstream of the AR. The data suggests that RSK2 and RSK4 activity is required for PCa cell proliferation, but they are likely regulating growth via different mechanisms.### Competing Interest StatementThe authors have declared no competing interest.

Supplementary Figures 1-6, Table 1 from FUS/TLS Is a Novel Mediator of Androgen-Dependent Cell-Cycle Progression and Prostate Cancer Growth

Background Combining the power of magnetic guidance and the biological activities of stem cells transformed into biohybrid microrobots holds great promise for the treatment of several diseases including cancer. Results We found that human MSCs can be readily loaded with magnetic particles and that the resulting biohybrid microrobots could be guided by a rotating magnetic field. Rotating magnetic fields have the potential to be applied in the human setting and steer therapeutic stem cells to the desired sites of action in the body. We could demonstrate that the required loading of magnetic particles into stem cells is compatible with their biological activities. We examined this issue with a particular focus on the expression and functionality of therapeutic genes inside of human MSC-based biohybrid microrobots. The loading with magnetic particles did not cause a loss of viability or apoptosis in the human MSCs nor did it impact on the therapeutic gene expression from the cells. Furthermore, the therapeutic effect of the gene products was not affected, and the cells also did not lose their migration potential. Conclusion These results demonstrate that the fabrication of guidable MSC-based biohybrid microrobots is compatible with their biological and therapeutic functions. Thus, MSC-based biohybrid microrobots represent a novel way of delivering gene therapies to tumours as well as in the context of other diseases.

DNAzymes are short pieces of DNA with catalytic activity, capable of cleaving RNA. DNAzymes have multiple applications as biosensors and in therapeutics. The high specificity and low toxicity of these molecules make them particularly suitable as therapeutics, and clinical trials have shown that they are effective in patients. However, the development of DNAzymes has been limited due to the lack of specific tools to identify efficient molecules, and users often resort to time-consuming/costly large-scale screens. Here, we propose a computational methodology to identify 10-23 DNAzymes that can be used to triage thousands of potential molecules, specific to a target RNA, to identify those that are predicted to be efficient. The method is based on a logistic regression and can be trained to incorporate additional DNAzyme efficiency data, improving its performance with time. We first trained the method with published data, and then we validated, and further refined it, by testing additional newly synthesized DNAzymes in the laboratory. We found that although binding free energy between the DNAzyme and its RNA target is the primary determinant of efficiency, other factors such as internal structure of the DNAzyme also have an important effect. A program implementing the proposed method is publicly available.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), is estimated to have caused over 6.5 million deaths worldwide. The emergence of fast-evolving SARS-CoV-2 variants of concern alongside increased transmissibility and/or virulence, as well as immune and vaccine escape capabilities, highlight the urgent need for more effective antivirals to combat the disease in the long run along with regularly updated vaccine boosters. One of the early risk factors identified during the COVID-19 pandemic was that men are more likely to become infected by the virus, more likely to develop severe disease and exhibit a higher likelihood of hospitalisation and mortality rates compared to women. An association exists between SARS-CoV-2 infectiveness and disease severity with sex steroid hormones and, in particular, androgens. Several studies underlined the importance of the androgen-mediated regulation of the host protease TMPRSS2 and the cell entry protein ACE2, as well as the key role of these factors in the entry of the virus into target cells. In this context, modulating androgen signalling is a promising strategy to block viral infection, and antiandrogens could be used as a preventative measure at the pre- or early hospitalisation stage of COVID-19 disease. Different antiandrogens, including commercial drugs used to treat metastatic castration-sensitive prostate cancer and other conditions, have been tested as antivirals with varying success. In this review, we summarise the most recent updates concerning the use of antiandrogens as prophylactic and therapeutic options for COVID-19.

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide-a well-tolerated drug widely used in advanced prostate cancer-reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

Abstract COVID-19 symptoms and mortality are largely due to its devastating effects in the lungs. The disease is caused by the SARS (Severe Acute Respiratory Syndrome)-CoV-2 coronavirus, which requires host cell proteins such as ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) for infection of lung epithelia. The expression and function of the steroid hormone receptor family is important in many aspects that impact on COVID-19 effects in the lung – notably lung development and function, the immune system, and expression of TMPRSS2 and ACE2. This review provides a brief summary of current knowledge on the roles of the steroid hormone receptors [androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR) and oestrogen receptor (ER)] in the lung, their effects on host cell proteins that facilitate SARS-CoV-2 uptake, and provides a snapshot of current clinical trials investigating the use of steroid receptor (SR) ligands to treat COVID-19.

The COVID-19 pandemic, caused by the novel human coronavirus SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. To determine therapeutic potential, we assessed uptake of SARS-CoV-2 Spike protein pseudotyped lentivirus and live SARS-CoV-2 into human lung cells and saw a significant reduction in viral entry and infection upon treatment with the antiandrogens enzalutamide and bicalutamide. In support of this new experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.