Biology subjects

McCormick, K.

Publications and source records attributed to McCormick, K..

9 recordsLinked to original sources

bioRxiv2023

Stress-induced Rab11a-exosomes induce AREG-mediated cetuximab resistance in colorectal cancer

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the Epidermal Growth Factor Receptor (EGFR) ligand, Amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/572341v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18f47bborg.highwire.dtl.DTLVardef@1ae627corg.highwire.dtl.DTLVardef@1c3cb23org.highwire.dtl.DTLVardef@14da12e_HPS_FORMAT_FIGEXP M_FIG C_FIG This study highlights a clinically relevant mechanism in which stress-induced Rab11a-exosomes carrying the EGFR ligand, Amphiregulin (AREG) transfer drug resistance between genetically distinct colorectal cancer cells. Resistance to cetuximab, an anti-EGFR therapy, can be passed via Rab11a-exosomes from drug-resistant KRAS-mutant cells to previously drug-responsive KRAS-wild type cells. Unlike soluble AREG, Rab11a-exosome-associated AREG competes with cetuximab to activate EGFR signalling and promote EGFR-dependent outcomes, such as growth. This mechanism may support the co-operative evolution of clonal heterogeneity during tumour progression.

cancer biology↗

bioRxiv2022

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. TF SHIV.C.CH505 is an extensively characterized virus shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides {Delta}375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure. Author SummaryThe power of the nonhuman primate model of HIV to predict outcomes in people living with HIV (PLWH) depends on authentic virus-host interactions. In pursuit of viruses that generate infection that mirrors the effects of HIV-1 in PLWH, we developed a minimally adapted version of a commonly used virus, SHIV.C.CH505, which has better fitness than the parental virus while retaining important biological properties. First, we studied virus sequences from SHIV.C.CH505-infected rhesus macaques to identify a signature of mutations common to animals with higher viral loads. We then tested viruses containing the various mutations in the lab and in animals to determine the most fit version and to identify the contribution of each mutation. Ultimately, we identified a minimally adapted version of SHIV.C.CH505 with just 5 amino acid substitutions that enhances virus replication and preserves CH505 envelope properties, including sensitivity to clinically relevant broadly neutralizing antibodies. This new virus, called SHIV.C.CH505.v2 replicates well in macaques over time and persists through antiretroviral therapy. SHIV.C.CH505.v2 could be an important component of nonhuman primate studies of HIV prevention, therapy, and cure.

microbiology↗

bioRxiv2021

A Gene Replacement Humanization Platform for Rapid Functional Testing of Clinical Variants in Epilepsy-associated STXBP1.

PurposeFunctional evidence is a pillar of variant interpretation according to ACMG guidelines. Functional evidence can be obtained in a variety of models and assay systems, including patient-derived tissues and iPSCs, in vitro cellular assays, and in vivo assays. Here we evaluate the reliability and practicality of variant interpretation in the small animal model, C. elegans, through a series of experiments evaluating the function of syntaxin binding protein, STXBP1, a well-known causative gene for Early infantile epileptic encephalopathy 1 (EIEE1). MethodsUsing CRISPR, we replaced the coding sequence for unc-18 with the coding sequence for the human ortholog STXBP1. Next, we used CRISPR to introduce precise point mutations in the human STXBP1 coding sequence, reflecting three clinical categories (benign, pathogenic, and variants of uncertain significance (VUS)). We quantified 26 features of the resulting worms movement to train Random Forest (RF) and Support Vector Machines (SVM) machine learning classifiers on known pathogenic and benign variants. We characterized the classifiers, and then used the behavioral data from the VUS-expressing animals to predict the categorization of the VUS. ResultsWhereas knock-out worms without unc-18 are severely impaired in motor function, worms expressing STXBP1 in its place have restored motor function. We produced worms with STXBP1 variants previously classified by ACMG criteria, including 25 benign variants, 32 pathogenic, and 24 variants of uncertain significance (VUS). Using either SVM or RF classifiers, we were able to obtain a sensitivity of 0.84-0.97 on known benign and pathogenic strains. By comparing multiple ML classification methods, we were able to classify 9 of the VUS as functionally abnormal, suggesting that these VUS are likely to be pathogenic. ConclusionsWe demonstrate that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1, one of the most common causes of genetic epilepsies and neurodevelopmental disorders.

genetics↗

bioRxiv2020

Antigen-driven clonal selection shapes the persistence of HIV-1 infected CD4+ T cells in vivo

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here we show that it is possible to link antigen responsiveness, full proviral sequence, integration site, and T cell receptor {beta}-chain (TCR{beta}) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated Cytomegalovirus (CMV)- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCR{beta} repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCR{beta}-integration site analysis showed that infection could occur early or late in the course of a clones response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.

microbiology↗

bioRxiv2020

Anti-androgens induce Rab11a-exosome secretion in prostate cancer by suppressing amino acid-sensitive PAT4-mTORC1 signalling

Advanced prostate cancer is typically treated with anti-androgens to reduce cancer growth, but patients almost inevitably develop treatment resistance and castration-resistant disease. Recently, extracellular vesicles known as exosomes, which are secreted from the endosomal compartments in which they are formed, have been implicated in drug resistance mechanisms. Here we investigate whether growth regulation by the amino acid-dependent kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1), and associated extracellular vesicle secretion might be involved in the adaptive responses to anti-androgens. We show that expression and intracellular localisation of the glutamine-sensing PAT4 (SLC36A4) amino acid transporter is increased in malignant versus benign prostatic tissue, mirroring earlier in vivo fly studies suggesting that these transporters are more effective at promoting growth from internal versus cell surface membranes. Furthermore, androgens induce PAT4 expression in prostate cancer cell lines and PAT4 is required for a proportion of androgen-stimulated mTORC1 activation and growth. Consistent with previous studies in other cancer cell lines, we find that glutamine depletion, PAT4 knockdown and mTORC1 inhibition all independently increase the production of a specific exosome subtype, Rab11a-exosomes, which has recently been implicated in pro-tumorigenic signalling responses to mTORC1 inhibition. Furthermore, we show that these exosomes are also induced by anti-androgens. We hypothesise that the uptake of Rab11a-exosomes by cells with higher PAT4 levels could provide a growth-promoting boost, enabling them to out-compete others with lower PAT4 expression, resulting in tumours that are more resistant to nutrient-deprivation and anti-androgen treatment.

cancer biology↗

bioRxiv2019

Glutamine deprivation regulates the origin and function of cancer cell exosomes

Exosomes are secreted extracellular vesicles (EVs) carrying diverse cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of these exosomes from cancer cells is increased by reducing Akt/mechanistic Target of Rapamycin (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. The resulting vesicles promote tumour cell proliferation and turnover, and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against Amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release of Rab11a-exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

cancer biology↗

bioRxiv2019

Acoustic and visual stimulus parameters underlying sound-symbolic crossmodal correspondences

It is often assumed that a fundamental property of language is the arbitrariness of the relationship between sound and meaning. Sound symbolism, which refers to non-arbitrary mapping between the sound of a word and its meaning, contradicts this assumption. Sensitivity to sound symbolism has been studied through crossmodal correspondences (CCs) between auditory pseudowords (e.g. loh-moh) and visual shapes (e.g. a blob). We used representational similarity analysis to examine the relationships between physical stimulus parameters and perceptual ratings that varied on dimensions of roundedness and pointedness, for a range of auditory pseudowords and visual shapes. We found that perceptual ratings of these stimuli relate to certain physical features of both the visual and auditory domains. Representational dissimilarity matrices (RDMs) of parameters that capture the spatial profile of the visual shapes, such as the simple matching coefficient and Jaccard distance, were significantly correlated with those of the visual ratings. RDMs of certain acoustic parameters of the pseudowords, such as the temporal fast Fourier transform (FFT) and spectral tilt, that reflect spectral composition, as well as shimmer and speech envelope that reflect aspects of amplitude variation over time, were significantly correlated with those of the auditory perceptual ratings. RDMs of the temporal FFT (acoustic) and the simple matching coefficient (visual) were significantly correlated. These findings suggest that sound-symbolic CCs are related to basic properties of auditory and visual stimuli, and thus provide insights into the fundamental nature of sound symbolism and how this might evoke specific impressions of physical meaning in natural language.

neuroscience↗

bioRxiv2018

Neural Basis Of The Sound-Symbolic Crossmodal Correspondence Between Auditory Pseudowords And Visual Shapes

Sound symbolism refers to the association between the sounds of words and their meanings, often studied using the crossmodal correspondence between auditory pseudowords, e.g. takete or maluma, and pointed or rounded visual shapes, respectively. In a functional magnetic resonance imaging study, participants were presented with pseudoword-shape pairs that were sound-symbolically congruent or incongruent. We found no significant congruency effects in the blood oxygenation level-dependent (BOLD) signal when participants were attending to visual shapes. During attention to auditory pseudowords, however, we observed greater BOLD activity for incongruent compared to congruent audiovisual pairs bilaterally in the intraparietal sulcus and supramarginal gyrus, and in the left middle frontal gyrus. We compared this activity to independent functional contrasts designed to test competing explanations of sound symbolism, but found no evidence for mediation via language, and only limited evidence for accounts based on multisensory integration and a general magnitude system. Instead, we suggest that the observed incongruency effects are likely to reflect phonological processing and/or multisensory attention. These findings advance our understanding of sound-to-meaning mapping in the brain.

neuroscience↗

bioRxiv2018

Sera from individuals with narrowly focused influenza virus antibodies rapidly select viral escape mutations in ovo

Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo. We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Remarkably, sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals that produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.\n\nImportanceInfluenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.

microbiology↗