Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

BACKGROUND: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. RESULTS: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. CONCLUSIONS: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

ABTRACT: Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. Conclusions: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Evaluation and Management of Deficiency of Adenosine Deaminase 2: An International Consensus Statement.

Importance: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited disease characterized by systemic vasculitis, early-onset stroke, bone marrow failure, and/or immunodeficiency affecting both children and adults. DADA2 is among the more common monogenic autoinflammatory diseases, with an estimate of more than 35 000 cases worldwide, but currently, there are no guidelines for diagnostic evaluation or management. Objective: To review the available evidence and develop multidisciplinary consensus statements for the evaluation and management of DADA2. Evidence Review: The DADA2 Consensus Committee developed research questions based on data collected from the International Meetings on DADA2 organized by the DADA2 Foundation in 2016, 2018, and 2020. A comprehensive literature review was performed for articles published prior to 2022. Thirty-two consensus statements were generated using a modified Delphi process, and evidence was graded using the Oxford Center for Evidence-Based Medicine Levels of Evidence. Findings: The DADA2 Consensus Committee, comprising 3 patient representatives and 35 international experts from 18 countries, developed consensus statements for (1) diagnostic testing, (2) screening, (3) clinical and laboratory evaluation, and (4) management of DADA2 based on disease phenotype. Additional consensus statements related to the evaluation and treatment of individuals with DADA2 who are presymptomatic and carriers were generated. Areas with insufficient evidence were identified, and questions for future research were outlined. Conclusions and Relevance: DADA2 is a potentially fatal disease that requires early diagnosis and treatment. By summarizing key evidence and expert opinions, these consensus statements provide a framework to facilitate diagnostic evaluation and management of DADA2.

Direct long-acting antibodies: updating the language of RSV prevention to reflect the evolution of mAbs.

Introduction: The language of medicine is constantly evolving, typically to better describe a new understanding of disease, adjust to changing social sensibilities, or simply to reflect a new drug class or category. We address the need for an updated language around monoclonal antibodies, or "mAbs" - a widely used medical term, but one which is now too general to accurately reflect the range of mAb pharmaceuticals, their effects, and the intended patients. Methods: The question of "what should we call a monoclonal antibody immunisation against respiratory syncytial virus (RSV) to ensure accurate understanding of the product?" was the basis for a virtual advisory panel in May 2022. The panel was convened by Sanofi with the intention of reviewing appropriate language in terminology in the context of mAb-based prophylaxis for RSV. The panel comprised several global experts on RSV and vaccination, a trained linguist specialising in doctor-patient interactions and medical language, and several experts in marketing and communications. Results: We suggest the term "Direct Long-acting Antibody" (DLA) for a specific sub-class of mAbs for use in prevention of RSV disease in infants. This terminology should differentiate from other mAbs, which are generally not used as therapies in infants. Discussion and Conclusions: This change will more accurately convey the specific mode of action of a mAb in infants, and how it could impact the prevention of communicable diseases: this class of mAbs is not an active treatment, but rather will offer direct and rapid protection lasting at least 5 months.

The breast is yet to come: current and future utility of circulating tumour DNA in breast cancer.

Advances in genomic strategies and the development of targeted therapies have enabled precision medicine to revolutionise the field of oncology. Precision medicine uses patient-specific genetic and molecular information, traditionally obtained from tumour biopsy samples, to classify tumours and treat them accordingly. However, biopsy samples often fail to provide complete tumour profiling, and the technique is expensive and, of course, relatively invasive. Advances in genomic techniques have led to improvements in the isolation and detection of circulating tumour DNA (ctDNA), a component of a peripheral blood draw/liquid biopsy. Liquid biopsy offers a minimally invasive method to gather genetic information that is representative of a global snapshot of both primary and metastatic sites and can thereby provide invaluable information for potential targeted therapies and methods for tumour surveillance. However, a lack of prospective clinical trials showing direct patient benefit has limited the implementation of liquid biopsies in standard clinical applications. Here, we review the potential of ctDNA obtained by liquid biopsy to revolutionise personalised medicine and discuss current applications of ctDNA both at the benchtop and bedside.

Hierarchical tumor heterogeneity mediated by cell contact between distinct genetic subclones.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.

Cyclin-dependent Kinase 1 and Aurora Kinase choreograph mitotic storage and redistribution of a growth factor receptor.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.

Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements.

BACKGROUND: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene expression or developmental patterning. The rules governing this process of developmental systems drift, including the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly delineated. RESULTS: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory elements we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyngeal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. CONCLUSIONS: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory elements. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be governed by distinct structural constraints that will be difficult to predict based on network architecture.

Validation of anti-glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells.

Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba+/+ and gba-/- immortalized neurons and primary human wild-type and type 2 GD fibroblasts.

Exploring genetic modifiers of Gaucher disease: The next horizon.

Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from mutations in the gene GBA1 that lead to a deficiency in the enzyme glucocerebrosidase. Accumulation of the enzyme's substrates, glucosylceramide and glucosylsphingosine, results in symptoms ranging from skeletal and visceral involvement to neurological manifestations. Nonetheless, there is significant variability in clinical presentations amongst patients, with limited correlation between genotype and phenotype. Contributing to this clinical variation are genetic modifiers that influence the phenotypic outcome of the disorder. In this review, we explore the role of genetic modifiers in Mendelian disorders and describe methods to facilitate their discovery. In addition, we provide examples of candidate modifiers of Gaucher disease, explore their relevance in the development of potential therapeutics, and discuss the impact of GBA1 and modifying mutations on other more common diseases like Parkinson disease. Identifying these important modulators of Gaucher phenotype may ultimately unravel the complex relationship between genotype and phenotype and lead to improved counseling and treatments.

Stress-Induced Hyperglycemia in White-Throated and White-Crowned Sparrows: A New Technique for Rapid Glucose Measurement in the Field.

Organisms experience stressors, and the physiological response to these stressors is highly conserved. Acute stress activates both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, increasing epinephrine, norepinephrine, and glucocorticoids, collectively promoting glucose mobilization. While this is well characterized in mammals, the hyperglycemic response to stress in avian and nonavian reptiles has received less attention. A number of factors, ranging from time of day to blood loss, are reported to influence the extent to which acute stress leads to hyperglycemia in birds. Here we characterized the glycemic response to acute handling stress in two species of free-living sparrows: white-throated sparrows (WTSPs: Zonotrichia albicollis) in St. Mary's County, Maryland, and white-crowned sparrows (WCSPs: Zonotrichia leucophrys) in Tioga Pass Meadow, California. We validated a novel technique for rapid field measurement of glucose using a human blood glucose meter, FreeStyle Lite. As expected, acute handling stress elevated blood glucose at both 15 and 30 min postcapture as compared to baseline for both WTSPs and WCSPs. In addition, handling for 30 min without bleeding had the same hyperglycemic effect as handling with serial bleeds in WCSPs. Finally, body condition that was measured as abdominal fat score predicted stress-induced blood glucose in WTSPs but not in WCSPs. Our results are consistent with the mammalian literature on acute stress and energy mobilization, and we introduce a new field technique for avian field biologists.

Establishment of lateral organ asymmetries in the invertebrate chordate, Ciona intestinalis.

BACKGROUND: The evolutionary emergence and diversification of the chordates appear to involve dramatic changes in organ morphogenesis along the left/right axis. However, the ancestral chordate mechanism for establishing lateral asymmetry remains ambiguous. Additionally, links between the initial establishment of lateral asymmetry and subsequent asymmetries in organ morphogenesis are poorly characterized. RESULTS: To explore asymmetric organ morphogenesis during chordate evolution, we have begun to characterize left/right patterning of the heart and endodermal organs in an invertebrate chordate, Ciona intestinalis. Here, we show that Ciona has a laterally asymmetric, right-sided heart. Our data indicate that cardiac lateral asymmetry requires H+/K+ ion flux, but is independent of Nodal signaling. Our pharmacological inhibitor studies show that ion flux is required for polarization of epidermal cilia and neurula rotation and suggest that ion flux functions synergistically with chorion contact to drive cardiac laterality. Live imaging analysis revealed that larval heart progenitor cells undergo a lateral shift without displaying any migratory behaviors. Furthermore, we find that this passive shift corresponds with the emergence of lateral asymmetry in the endoderm, which is also ion flux dependent. CONCLUSIONS: Our data suggest that ion flux promotes laterally asymmetric morphogenesis of the larval endoderm rudiment leading to a passive, Nodal-independent shift in the position of associated heart progenitor cells. These findings help to refine hypotheses regarding ancestral chordate left/right patterning mechanisms and how they have diverged within invertebrate and vertebrate chordate lineages.

Fibronectin contributes to notochord intercalation in the invertebrate chordate, Ciona intestinalis.

BACKGROUND: Genomic analysis has upended chordate phylogeny, placing the tunicates as the sister group to the vertebrates. This taxonomic rearrangement raises questions about the emergence of a tunicate/vertebrate ancestor. RESULTS: Characterization of developmental genes uniquely shared by tunicates and vertebrates is one promising approach for deciphering developmental shifts underlying acquisition of novel, ancestral traits. The matrix glycoprotein Fibronectin (FN) has long been considered a vertebrate-specific gene, playing a major instructive role in vertebrate embryonic development. However, the recent computational prediction of an orthologous "vertebrate-like" Fn gene in the genome of a tunicate, Ciona savignyi, challenges this viewpoint suggesting that Fn may have arisen in the shared tunicate/vertebrate ancestor. Here we verify the presence of a tunicate Fn ortholog. Transgenic reporter analysis was used to characterize a Ciona Fn enhancer driving expression in the notochord. Targeted knockdown in the notochord lineage indicates that FN is required for proper convergent extension. CONCLUSIONS: These findings suggest that acquisition of Fn was associated with altered notochord morphogenesis in the vertebrate/tunicate ancestor.

Mitotic Membrane Turnover Coordinates Differential Induction of the Heart Progenitor Lineage.

In response to microenvironmental cues, embryonic cells form adhesive signaling compartments that influence survival and patterning. Dividing cells detach from the surrounding matrix and initiate extensive membrane remodeling, but the in vivo impact of mitosis on adhesion-dependent signaling remains poorly characterized. We investigate in vivo signaling dynamics using the invertebrate chordate, Ciona intestinalis. In Ciona, matrix adhesion polarizes fibroblast growth factor (FGF)-dependent heart progenitor induction. Here, we show that adhesion inhibits mitotic FGF receptor internalization, leading to receptor enrichment along adherent membranes. Targeted disruption of matrix adhesion promotes uniform FGF receptor internalization and degradation while enhanced adhesion suppresses degradation. Chimeric analysis indicates that integrin ß chain-specific impacts on induction are dictated by distinct internalization motifs. We also found that matrix adhesion impacts receptor enrichment through Caveolin-rich membrane domains. These results redefine the relationship between cell division and adhesive signaling, revealing how mitotic membrane turnover orchestrates adhesion-dependent signal polarization.

The challenge of offering long-acting antipsychotic therapies: a preliminary discourse analysis of psychiatrist recommendations for injectable therapy to patients with schizophrenia.

OBJECTIVE: To characterize patterns of communication in the offer of long-acting injectable (LAI) antipsychotic medication made by psychiatrists to patients with schizophrenia by (1) examining the style and content of their interaction and (2) determining how these may have driven the ultimate response to recommendations for LAI therapy. METHOD: This was an observational study conducted at 10 community mental health centers in 3 waves from July 2010 to May 2011. The final dataset for discourse analysis was 33 recorded conversations in which a psychiatrist offered an injectable antipsychotic to a patient with schizophrenia. These visits were transcribed and analyzed by a team of linguists and social scientists. RESULTS: Our primary finding is that, based on analyses of their language during the interview, psychiatrists presented LAI therapy in a negative light. Supporting this, 11 of 33 recommendations (33%) were accepted during the discussion, whereas in the postvisit interview, 27 of 28 patients (96%) who seemed to decline the initial recommendation said they actually would be willing to try LAI treatment. CONCLUSIONS: These data support a preliminary hypothesis that the relatively low use of injectable antipsychotic therapies in the United States relative to other parts of the world is not fully attributable to patient rejection of the injectable modality. Rather, psychiatrists' ambivalence regarding the value of LAIs may play a significant role in the perceived difficulty with patient acceptance of this recommendation.

Heart genetics in a small package, exploiting the condensed genome of Ciona intestinalis.

Defects in the initial establishment of cardiogenic cell fate are likely to contribute to pervasive human congenital cardiac abnormalities. However, the molecular underpinnings of nascent cardiac fate induction have proven difficult to decipher. In this review we explore the participation of extracellular, cellular and nuclear factors in comprehensive specification networks. At each level, we elaborate on insights gained through the study of cardiogenesis in the invertebrate chordate Ciona intestinalis and propose productive lines of future research. In-depth discussion of pre-cardiac induction is intended to serve as a paradigm, illustrating the potential use of Ciona to elucidate comprehensive networks underlying additional aspects of chordate cardiogenesis, including directed migration and subspecification of cardiac and pharyngeal lineages.

Matrix adhesion polarizes heart progenitor induction in the invertebrate chordate Ciona intestinalis.

Cell-matrix adhesion strongly influences developmental signaling. Resulting impacts on cell migration and tissue morphogenesis are well characterized. However, the in vivo impact of adhesion on fate induction remains ambiguous. Here, we employ the invertebrate chordate Ciona intestinalis to delineate an essential in vivo role for matrix adhesion in heart progenitor induction. In Ciona pre-cardiac founder cells, invasion of the underlying epidermis promotes localized induction of the heart progenitor lineage. We found that these epidermal invasions are associated with matrix adhesion along the pre-cardiac cell/epidermal boundary. Through targeted manipulations of RAP GTPase activity, we were able to manipulate pre-cardiac cell-matrix adhesion. Targeted disruption of pre-cardiac cell-matrix adhesion blocked heart progenitor induction. Conversely, increased matrix adhesion generated expanded induction. We were also able to selectively restore cell-matrix adhesion and heart progenitor induction through targeted expression of Ci-Integrin ß2. These results indicate that matrix adhesion functions as a necessary and sufficient extrinsic cue for regional heart progenitor induction. Furthermore, time-lapse imaging suggests that cytokinesis acts as an intrinsic temporal regulator of heart progenitor adhesion and induction. Our findings highlight a potentially conserved role for matrix adhesion in early steps of vertebrate heart progenitor specification.

Initial deployment of the cardiogenic gene regulatory network in the basal chordate, Ciona intestinalis.

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification.

Cytoskeletal polarity mediates localized induction of the heart progenitor lineage.

Cells must make appropriate fate decisions within a complex and dynamic environment. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart progenitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients.