Biology subjects

Brown, M.

Publications and source records attributed to Brown, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Inhibition of CD226 Co-Stimulation Suppresses Diabetes Development in the NOD Mouse by Augmenting Tregs and Diminishing Effector T Cell Function

Aims/hypothesisImmunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. A growing number of T cell-directed therapeutics have demonstrated partial therapeutic efficacy, with anti-CD3 (-CD3) representing the only regulatory agency-approved drug capable of slowing disease progression through a mechanism involving the induction of partial T cell exhaustion. There is an outstanding need to augment the durability and effectiveness of T cell targeting by directly restraining proinflammatory T helper type 1 (Th1) and type 1 cytotoxic CD8+ T cell (Tc1) subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse model using a blocking monoclonal antibody targeting the type 1 diabetes-risk associated T cell co-stimulatory receptor, CD226. MethodsFemale NOD mice were treated with anti-CD226 between 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action. ResultsCompared to isotype-treated controls, anti-CD226 treated NOD mice showed reduced insulitis severity at 12 weeks and decreased disease incidence at 30 weeks. Flow cytometric analysis performed five weeks post-treatment demonstrated reduced proliferation of CD4+ and CD8+ effector memory T cells in spleens of anti-CD226 treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression and STAT5 phosphorylation following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+ T cell responders in vitro. Anti-CD226 treated mice exhibited reduced frequencies of islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP)-reactive CD8+ T cells in the pancreas, using both ex vivo tetramer staining and single-cell T cell receptor sequencing (scTCR-seq) approaches. 51Cr-release assays demonstrated reduced cell-mediated lysis of beta-cells by anti-CD226-treated autoreactive cytotoxic T lymphocytes. Conclusions/interpretationCD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIThe co-stimulatory receptor CD226 is upregulated upon activation and is highly expressed on NK cell subsets, myeloid cells, and effector T cells. C_LIO_LIA single nucleotide polymorphism in CD226 (rs763361; C>T) results in a Gly307Ser missense mutation linked to genetic susceptibility for type 1 diabetes. C_LIO_LIGlobal knockout of Cd226 and conditional Cd226 knockout in FoxP3+ Tregs reduced insulitis severity and diabetes incidence in NOD mice, indicating a crucial role for CD226 in disease pathogenesis. C_LI What is the key question?O_LICan CD226 blockade reduce T cell cytotoxicity and improve Treg function to diminish diabetes incidence in NOD mice? C_LI What are the new findings?O_LIAnti-CD226 treatment reduced insulitis, decreased disease incidence, and inhibited splenic CD4+ and CD8+ effector memory T cell proliferation. C_LIO_LIPancreatic Tregs from anti-CD226 treated mice exhibited increased CD25 expression; splenic Tregs displayed augmented STAT5 phosphorylation and suppressive capacity in vitro. C_LIO_LIAnti-CD226 treatment reduced IGRP-specific pancreatic CD8+ T cell frequencies, and reduced autoreactive CD8+ T cell-mediated lysis of beta-cells in vitro. C_LI How might this impact on clinical practice in the foreseeable future?O_LICD226 blockade could reduce autoreactive T cell cytotoxicity, enhance Treg function, and slow disease progression in high-risk or recent-onset type 1 diabetes cases. C_LI

immunology↗

bioRxiv2024

Regulation of brain development by the Minibrain/Rala signaling network

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized early role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in brain development. Significance statementThe kinase Minibrain(Mnb)/DYRK1A regulates the development of the brain and other tissues across many organisms. Here we show the critical importance of Mnb within the developing neuroepithelium. Advancing our understanding of Mnb function, we identified novel protein interactors of Mnb, Reps and Rlip, which function together with Mnb to regulate growth in Drosophila melanogaster. We also identify and characterize a role for the small GTPase Rala in Mnb-regulated growth and nervous system development. This work reveals an early role of Mnb in brain development and identifies a new Mnb/Reps/Rlip/Rala signaling axis.

developmental biology↗

bioRxiv2024

Cholesterol biosynthesis inhibition synergizes with AKT inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer deaths due to its molecular heterogeneity, high recurrence rate and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. We performed a genome-wide CRISPR/Cas9 screen with PI3K and AKT inhibitors to find targetable synthetic lethalities in TNBC. We identified cholesterol homeostasis as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin. This was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. Our work motivates combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC.

cancer biology↗

bioRxiv2024

Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

cancer biology↗

bioRxiv2024

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation

Enteroendocrine cells (EECs), which secrete serotonin (enterochromaffin cells, EC) or a dominant peptide hormone, serve vital physiologic functions. As with any adult human lineage, the basis for terminal cell diversity remains obscure. We replicated human EEC differentiation in vitro, mapped transcriptional and chromatin dynamics that culminate in discrete cell types, and studied abundant EEC precursors expressing selected transcription factors (TFs) and gene programs. Before expressing the pre-terminal factor NEUROD1, non-replicating precursors oscillated between epigenetically similar but transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor substantially accelerated EEC differentiation and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and hormone-producing cell features. Expressed late in EEC differentiation, the latter TFs mainly bind cis-elements that are accessible in undifferentiated stem cells and tailor the subsequent expression of TF combinations that specify EEC types. Thus, TF oscillations retard EEC maturation to enable accurate EEC diversification.

molecular biology↗

bioRxiv2023

Teaching transposon classification as a means to crowd source the curation of repeat annotation - a tardigrade perspective

The advancement of sequencing technologies results in the rapid release of hundreds of new genome assemblies a year providing unprecedented resources for the study of genome evolution. Within this context, the significance of in-depth analyses of repetitive elements, transposable elements (TEs) in particular, is increasingly recognized in understanding genome evolution. Despite the plethora of available bioinformatic tools for identifying and annotating TEs, the phylogenetic distance of the target species from a curated and classified database of repetitive element sequences constrains any automated annotation effort. Manual curation of raw repeat libraries is deemed essential due to the frequent incompleteness of automatically generated consensus sequences. However, manual curation and classification are time-consuming processes that offer limited short-term academic rewards and are typically confined to a few research groups where methods are taught through hands-on experience. Crowd sourcing efforts could offer a significant opportunity to bridge the gap between learning the methods of curation effectively and empowering the scientific community with high-quality, reusable repeat libraries. Here, we present an example of such crowd sourcing effort developed through both in-person and online courses built around a collaborative peer-reviewed teaching process that can be used as teaching reference guide for similar projects. The collaborative manual curation of TEs from two tardigrade species, for which there were no TE libraries available, resulted in the successful characterization of hundreds of new and diverse TEs: A hidden treasure awaits discovery within non-model organisms.

evolutionary biology↗

bioRxiv2023

Breast cancer cells can recognize and respond to different levels of progestins to achieve different phenotypic outputs

The steroid hormone progesterone, acting through its nuclear progesterone receptor (PR), has complex physiologic activities with different levels of hormones manifesting distinct and sometimes opposing phenotypic responses in target tissues. However, most of what is currently known about the transcriptional activity of PR comes from studies performed using progestins at levels that are in the high physiologic range ([≥]10nM), relevant only in the luteal phase of the reproductive cycle and pregnancy in humans. These studies do not consider the non-linearity of responses to progestins that exist in physiology and are not informative as to the mechanisms by which low levels of progestins, as occurs during menopause, exert their biological activities. Thus, we undertook to define the mechanisms which enable cells to recognize and respond to different levels of progestins. Using a PR expressing cell model of luminal breast cancer (T47D cells) we demonstrated that low concentration progestins (0.1-0.3nM) drive proliferation while high dose progestins ([≥]10nM) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 and SGK1, which are both required to initiate a signaling cascade that leads to increased phospho-Rb and E2F1 transcriptional activity. CCND1 cooperates with CDK4/6 to phosphorylate Rb, while SGK1 phosphorylates p21, thereby excluding it from the nucleus and inhibiting its anti-proliferative function. Expression of CCND1 and SGK1 mRNAs are primary responses to low dose progestin treatment. However, these responses occur at very low levels of receptor occupancy and in the absence of receptor phosphorylation events that have been shown to be required for nuclear translocation and transcriptional activity. These findings challenge the assumption of linearity in response to progestin dose. Further, they suggest that concentrations of progestins found in post-menopausal women (0.1-0.3nM) have the potential to exert proliferative responses in PR expressing cancers.

cancer biology↗

bioRxiv2023

Loss of PAX6 alters the excitatory/inhibitory neuronal ratio in human cerebral organoids

The transcription factor PAX6 is a crucial regulator of multiple aspects of embryonic forebrain development. It has well-established roles in the regulation of excitatory and inhibitory neuron development in the embryonic cortex in mice but PAX6s roles during human forebrain development are less well understood. Using human cerebral organoids, we investigated PAX6s roles in human neurodevelopment. Homozygous PAX6 mutant (PAX6-/-) organoids were larger than controls and contained increased inhibitory cell types. Excitatory neurons were still generated in PAX6-/- organoids but they were less mature, and a subset showed dysregulated expression of inhibitory identity genes compared to PAX6+/+ controls. The inhibitory cells found in PAX6-/- organoids physically segregated from excitatory neurons and presented a distinct transcriptomic profile when compared to in vivo cortical inhibitory neurons. PAX6-/- organoids showed a dysregulated cellular response to PTN-PTPRZ1 signalling, which contributed to the observed increase in inhibitory neurons and the consequent altered excitatory to inhibitory neuronal ratio. Summary StatementHuman cerebral organoids lacking PAX6 expression show an altered excitatory to inhibitory neuronal ratio, concomitant with altered responses to intercellular PTN-PTPRZ1 signalling.

developmental biology↗

bioRxiv2023

scATAnno: Automated Cell Type Annotation for single-cell ATAC-seq Data

Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.

bioinformatics↗

bioRxiv2023

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which can interbreed with Mus musculus but lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

cell biology↗

bioRxiv2023

Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function

The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.

genomics↗

bioRxiv2023

Analysis of evolutionary dynamics and clonal architecture in prostate cancer

The extent to which clinical and genomic characteristics associate with prostate cancer clonal architecture, tumor evolution, and therapeutic response remains unclear. Here, we reconstructed the clonal architecture and evolutionary trajectories of 845 prostate cancer tumors with harmonized clinical and molecular data. We observed that tumors from patients who self-reported as Black had more linear and monoclonal architectures, despite these men having higher rates of biochemical recurrence. This finding contrasts with prior observations relating polyclonal architecture to adverse clinical outcomes. Additionally, we utilized a novel approach to mutational signature analysis that leverages clonal architecture to uncover additional cases of homologous recombination and mismatch repair deficiency in primary and metastatic tumors and link the origin of mutational signatures to specific subclones. Broadly, prostate cancer clonal architecture analysis reveals novel biological insights that may be immediately clinically actionable and provide multiple opportunities for subsequent investigation. Statement of significanceTumors from patients who self-reported as Black demonstrate linear and monoclonal evolutionary trajectories yet experience higher rates of biochemical recurrence. In addition, analysis of clonal and subclonal mutational signatures identifies additional tumors with potentially actionable alterations such as deficiencies in mismatch repair and homologous recombination.

cancer biology↗

bioRxiv2023

Operant training for highly palatable food alters translating mRNA in nucleus accumbens D2 neurons and reveals a modulatory role of Neurochondrin

BACKGROUNDHighly palatable food triggers behavioral alterations reminiscent of those induced by addictive drugs. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the effects of highly palatable food on feeding behavior are poorly understood. METHODSWe studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral effects and dendritic spine modifications in the NAc. We compared the translating mRNA in NAc neurons identified by the type of dopamine receptors they express, depending on the type of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn (Neurochondrin). RESULTSOperant conditioning for highly palatable food increases motivation for food even in well-fed mice. In control mice, free access to regular or highly palatable food results in increased weight as compared to regular food only. Highly palatable food increases spine density in the NAc. In animals trained for highly palatable food, translating mRNAs are modified in NAc dopamine D2-receptor-expressing neurons, mostly corresponding to striatal projection neurons, but not in those expressing D1-receptors. Knock-out of Ncdn, an abundant down-regulated gene, opposes the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting down-regulation may be a compensatory mechanism. CONCLUSIONSOur results emphasize the importance of mRNA alterations D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

neuroscience↗

bioRxiv2023

The search behavior of terrestrial mammals

Animals moving through landscapes need to strike a balance between finding sufficient resources to grow and reproduce while minimizing encounters with predators 1,2. Because encounter rates are determined by the average distance over which directed motion persists 1,3-5, this trade-off should be apparent in individuals movement. Using GPS data from 1,396 individuals across 62 species of terrestrial mammals, we show how predators maintained directed motion ~7 times longer than for similarly-sized prey, revealing how prey species must trade off search efficiency against predator encounter rates. Individual search strategies were also modulated by resource abundance, with prey species forced to risk higher predator encounter rates when resources were scarce. These findings highlight the interplay between encounter rates and resource availability in shaping broad patterns mammalian movement strategies.

ecology↗

bioRxiv2022

A novel mechanistic framework for precise sequence replacement using reverse transcriptase and diverse CRISPR-Cas systems

CRISPR/Cas systems coupled with reverse transcriptase (RT), such as the recently described Prime editing, allow for site-specific replacement of DNA sequences. Despite widespread testing of Prime editing, it is currently only compatible with type II CRISPR/Cas proteins such as Streptococcus pyogenes and Staphylococcus aureus Cas9. Enabling RT compatibility with other CRISPR/Cas domains, such as type V enzymes with orthogonal protospacer adjacent motif specificities and smaller protein size would expand the range of edits that can be made in therapeutic and industrial applications. We achieve this with a novel mode of DNA editing at CRISPR-targeted sites that reverse transcribes the edit into the target strand DNA (e.g., the complement of the PAM-containing strand), rather than the non-target strand DNA, as in Prime editing. We term this technology RNA encoded DNA replacement of alleles with CRISPR (hereafter, REDRAW). We show that REDRAW extends the utility of RT-mediated editing beyond type II to include multiple type V CRISPR domains. REDRAW features a broad (8-10 bases) targeting window, at which all types of substitutions, insertions and deletions are possible. REDRAW combines the advantages of type V CRISPR domains with the extensive range of genetic variation enabled by RT-mediated, templated sequence replacement strategies.

molecular biology↗

bioRxiv2022

The BRCA1 isoform, BRCA1-IRIS, operates independently of the full-length BRCA1 in the Fanconi anemia pathway

The tumor suppressor BRCA1 encodes multiple protein products including the canonical BRCA1-p220 (p220), which plays important roles in repair of diverse types of DNA damage. However, contributions of other BRCA1-encoded protein isoforms to DNA damage repair are less clear. Here, we report that the BRCA1-IRIS (IRIS) isoform has critical functions in the Fanconi anemia (FA) pathway and in repair of DNA interstrand crosslinks (ICLs). Loss of IRIS expression sensitizes cells to ICLs and impairs ICL repair. ICL formation stimulates association of IRIS with both FANCD2 and the FA core complex, which promotes FANCD2 recruitment to damage sites. The unique, BRCA1 intron 11-encoded C-terminal tail of IRIS is required for complex formation with FANCD2 and for ICL-inducible FANCD2 mono-ubiquitylation. Collectively, our findings reveal that IRIS plays an essential role, upstream of the p220-directed HR, in the FA pathway through a previously unrecognized mechanism that depends on the IRIS-FANCA-FANCD2 interaction. HighlightsO_LIBRCA1 splicing isoform BRCA1-IRIS is required for interstrand crosslink (ICL) repair. C_LIO_LIBRCA1-IRIS interacts with FANCD2 and promotes its recruitment to sites of ICL damage. C_LIO_LIBRCA1-IRIS, but not BRCA1-p220, promotes ICL-inducible FANCD2 mono-ubiquitylation. C_LIO_LIThe unique C-terminal tail of BRCA1-IRIS is essential for its function in ICL repair. C_LI

molecular biology↗

bioRxiv2022

Targeting the BAG-1 family of co-chaperones in lethal prostate cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR drives transcriptional activity in CRPC but is intrinsically disordered and remains a challenging therapeutic target. Therefore, inhibiting critical co-chaperones, such as BAG-1L, is an attractive alternative strategy. We performed druggability analyses demonstrating the BAG domain to be a challenging drug target. Thio-2, a tool compound, has been reported to bind the BAG domain of BAG-1L and inhibit BAG-1L-mediated AR transactivation. However, despite these data, the mechanism of action of Thio-2 is poorly understood and the BAG domain which is present in all BAG-1 isoforms has not been validated as a therapeutic target. Herein, we demonstrate growth inhibiting activity of Thio-2 in CRPC cell lines and patient derived models with decreased AR genomic binding and AR signaling independent of BAG-1 isoform function. Furthermore, genomic abrogation of BAG-1 isoforms did not recapitulate the described Thio-2 phenotype, and NMR studies suggest that Thio-2 may bind the AR NTD, uncovering a potential alternative mechanism of action, although in the context of low compound solubility. Furthermore, BAG-1 isoform knockout mice are viable and fertile, in contrast to previous studies, and when crossed with prostate cancer mouse models, BAG-1 deletion does not significantly impact prostate cancer development and growth. Overall, these data demonstrate that Thio-2 inhibits AR signaling and growth in CRPC independent of BAG-1 isoforms, and unlike previous studies of the activated AR, therapeutic targeting of the BAG domain requires further validation before being considered a therapeutic strategy for the treatment of CRPC.

molecular biology↗

bioRxiv2022

IRF7 impacts on prostate cancer cell survival in response to radiation

Understanding the impact of radiotherapy on the evolution of treatment resistant prostate cancer is critical for selecting effective treatment combinations. Whilst activation of Type 1 interferon signalling is a hallmark of how cells respond to viral infection, in cancer cells, multiple stresses are known to activate this same response. In this study we have evaluated for the first time the changes in the interferon response induced by culturing prostate cancer cells under sphere- forming conditions and following irradiation. We report a conserved upregulated transcript profile for both conditions that is strongly associated with therapeutic resistance and cell survival in vitro and in vivo. The profile includes and is regulated by the Type 1 interferon master regulator IRF7 which, when depleted, delays tumour re-growth following irradiation. We immuno-stained two independent prostate cohorts for IRF7 and found that increased expression, particularly in cases with low PTEN expression, correlated with poor prognosis. To more comprehensively characterise the impact of IRF7 and radiation on cells, RNA-Seq was performed on IRF7 knockdown cells at different radiation doses. We identified a number of biological processes that were IRF7-dependent, including the formation of stem-like cell populations and also therapeutic vulnerabilities. For example, irradiation sensitised surviving cells to either a combination of an IKK{varepsilon}/TBK1 and a MEK inhibitor or treatment with an inhibitor of IDO1, an IRF7- dependent gene. Translationally our work suggests that IRF7 expression can be used to stratify patients who may not benefit from receiving radiotherapy alone but rather may benefit from treatment combinations. In two cohorts treated with radical intent, strong IRF7 staining was associated with disease-specific death implicating this pathway as a convergence point for therapeutic resistance in prostate and potentially other cancer types.

cancer biology↗