Biology subjects

Fisher, D.

Publications and source records attributed to Fisher, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Highly diverged non-vertebrate Ki-67 proteins with conserved biophysical and functional properties

The Ki-67 protein is a widely used marker of mammalian cell proliferation that plays critical roles in heterochromatin organisation and mitotic perichromosomal layer formation. Thus, its apparent restriction to vertebrates is puzzling. However, sequences with limited homology to subdomains of Ki-67 are present in genomes from some non-vertebrate species. Here, using structural modelling, biophysical and functional analysis, we identify Ki-67-like proteins across diverse non-vertebrate eukaryotes, including tunicates, sponges, molluscs, insects and fungi. Despite extremely low sequence homology, the disordered domains of Ki-67 homologues have generally conserved biophysical and molecular properties. This is further reflected by comparison of cellular localisation, dynamics and effects on chromatin organisation of human, Ciona and Drosophila Ki-67 homologues. These results reinforce the emerging paradigm that functions may be encoded by general biophysical features in highly diverged intrinsically disordered domains. Our approach provides a blueprint for comparative biology of disordered proteins and a foundation for future studies exploring functions of Ki-67 homologues across eukaryotes.

Cell Biology↗

bioRxiv2026

The 3D Ultrastructure of C. elegans Gut Granules

We identify an endoderm-restricted organelle in published volume electron microscopy datasets of C. elegans embryos. The organelle consists of a tubular ring surrounding a membrane-bound compartment harboring a prominent dense particle and exhibits a basal polarity and size concordant with canonical gut granules. This finding offers ultrastructural detail to recent evidence that gut granules are bi-lobed organelles with two distinct compartments.

cell biology↗

bioRxiv2025

Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development.

Protein kinase inhibitors are a key class of targeted cancer therapies, but finding compounds that show on-target efficacy with minimal toxicity remains challenging. CDK8 and CDK19 are paralogous kinases that regulate Mediator complex to promote transcriptional responses to extracellular signals and have also been reported to facilitate genome replication. Preclinical studies show that CDK8/19 inhibitors may have therapeutic benefit in several cancers, but whether effective inhibition of Mediator kinases is cytotoxic is debated. Here, we present a multi-modal approach for discovery of specific kinase inhibitors, and find that most CDK8/19 inhibitors show off-target toxicity and/or insufficient on-target engagement in cells. We describe several novel high affinity CDK8/19 inhibitors, report that loss of CDK8/19 activity is not cytotoxic but reduces cell proliferation without affecting DNA replication, and provide evidence that most inhibitors can bind in type I or type II modes. Our approach is applicable to any kinase inhibitor development program.

biochemistry↗

bioRxiv2025

YAP1 drives aggressive and therapy resistant state in melanoma through reprogramming the chromatin and regulating immune evasive programs

Despite promising initial results in targeting the RAF-MEK-ERK cascade, resistance to BRAF/MEK inhibitors remains a critical challenge in nearly 50% of melanoma patients. Our study demonstrates that robust YAP1 activation in metastatic melanoma correlates with poor survival and drives transcriptional programs linked to therapeutic resistance. Mechanistically, YAP1 predominantly remodels the chromatin landscape in resistant tumors by partnering with BRD4 and TEAD, creating a permissive transcriptional state that sustains oncogenic signaling. Clinical validation in biopsies from resistant melanoma confirms elevated expression of YAP1 target genes. Furthermore, pharmacological inhibition of BRD4 or TEAD reduces YAP1-driven transcription and reactivates antitumor immunity programs. TEAD specific inhibitors (and not verteporfin which is a highly non-specific inhibitor) synergize with immune checkpoint blockade in in vivo model system by promoting increased CD8 T cell infiltration and prolonged survival in the melanoma mouse model. Collectively, these findings reveal a chromatin-centric vulnerability in BRAF/MEK inhibitor-resistant melanoma and propose TEAD specific inhibitors as a promising dual strategy to overcome resistance and reinvigorate the immune response, offering a novel therapeutic avenue for patients. Resistance to BRAF and MEK inhibitors remains a major obstacle in the treatment of melanoma. Here, we show that elevated YAP1 activity in metastatic melanoma is associated with poor patient survival and drives transcriptional programs that promote therapeutic resistance. Mechanistically, YAP1 cooperates with BRD4 and TEAD to reprogram the chromatin landscape, establishing an oncogenic transcriptional state that sustains resistance. Analysis of patient-derived melanoma biopsies confirms increased expression of YAP1 target genes in resistant tumors. Targeting this epigenetic circuitry using pharmacological inhibitors of BRD4 or TEAD effectively suppresses YAP1-driven transcriptional output and restores antitumor immune activity, as evidenced by enhanced CD8 T cell infiltration and prolonged survival in resistant melanoma models. These effects are further supported by transcriptomic and chromatin accessibility analyses, which reveal reduced expression of immune-suppressive and proliferation-associated gene networks upon TEAD inhibition. Collectively, our findings identify a chromatin-centric mechanism underlying resistance to MAPK-targeted therapy and nominate TEAD inhibition as a promising dual-action strategy to both overcome resistance and re-engage antitumor immunity. This work offers a compelling therapeutic avenue for patients with drug-resistant melanoma.

cancer biology↗

bioRxiv2025

Metabolic rewiring of cancer cells induces metastasis via ERK5 but triggers recognition by NK cells

Metastasis is largely controlled by Natural Killer (NK) cell-mediated immune surveillance. To colonize new environments, cancer cells undergo epithelial to mesenchymal transition (EMT), which allows them to detach and migrate. EMT is fueled by fatty acid oxidation (FAO), which partially replaces glycolytic-based tumor metabolism. Whether metabolic rewiring affects the targeting of cancer cells by NK cells remains unknown. Here, we show that forcing solid cancer cells to perform FAO by inhibiting pyruvate dehydrogenase kinase 1 with dichloroacetate (DCA) activates extracellular signal-regulated kinase-5 (ERK5), triggering EMT and tumor cell migration and invasion. Concomitantly, FAO induces the expression of ligands that mediate NK cell recognition. Consequently, NK cells better infiltrated DCA-treated 3D tumor spheroids, where they exerted their cytotoxic effects. DCA-treated cells showed increased migration in a zebrafish model, whereas metastasis from mammary cancer cells grafted into immune-deficient mice was enhanced by DCA. These migrating/metastatic cells are preferentially killed by NK cells, which strongly limit their invasive potential. Hence, FAO promotes both metastasis and NK-mediated tumor surveillance, highlighting the Achilles heel of metastatic cells, which may offer new therapeutic opportunities. TeaserMetastasis recognition and killing by immune cells, such as NK cells, requires a metabolic shift that relies on lipid metabolism.

cancer biology↗

bioRxiv2022

Non-standard viral genome-derived RNA activates TLR3 and type I IFN signaling to induce cDC1-dependent CD8+ T-cell responses during vaccination in mice

There is a critical need to develop vaccine adjuvants that induce robust immune responses able to protect against intracellular pathogens, including viruses. Previously, we described the virus-derived adjuvant, defective viral genome-derived oligonucleotide (DDO), as a strong inducer of type 1 immune responses, including protective Th1 CD4+ T-cells and effector CD8+ T-cells in mice. Here we unravel the early innate response required for this type 1 immunity induction. Upon DDO subcutaneous injection, type 1 conventional dendritic cells (cDC1s) accumulate rapidly in the draining lymph node in a type I interferon (IFN)-dependent manner. cDC1 accumulation in the lymph node is required for antigen-specific CD8+ T-cell responses. Notably, in contrast to poly I:C, DDO administration resulted in type I IFN expression at the injection site, but not in the draining lymph node. Additionally, DDO induced an inflammatory cytokine profile distinct from that induced by poly I:C. Therefore, DDO represent a powerful new adjuvant to be used during vaccination against intracellular pathogens. IMPORTANCEThere is a paucity of vaccine adjuvants able to trigger effective and safe protective responses to many intracellular pathogens. Defining the minimal requirements to achieve type 1 immunity, which includes antigen specific CD8+ T cells capable of eliminating infected cells, is essential for the development of adjuvants that lead to optimal protective immune responses during vaccination against intracellular pathogens. We use a virus-derived immunostimulatory molecule, defective viral genome-derived oligonucleotide (DDO), to provide insights into how type 1 immune responses are triggered during vaccination using an inactivated influenza vaccine model. Understanding the mechanism of action of vaccine adjuvants not only aids in the advancement of vaccine development, but also in understanding specific immune pathways required for efficient induction of adaptive immune responses to infections.

immunology↗

bioRxiv2022

Chromosome-scale assembly of the lablab genome - A model for inclusive orphan crop genomics

Orphan crops (also described as underutilised and neglected crops) hold the key to diversified and climate-resilient food systems. After decades of neglect, the genome sequencing of orphan crops is gathering pace, providing the foundations for their accelerated domestication and improvement. Recent attention has however turned to the gross under-representation of researchers in Africa in the genome sequencing efforts of their indigenous orphan crops. Here we report a radically inclusive approach to orphan crop genomics using the case of Lablab purpureus (L.) Sweet (syn. Dolichos lablab, or hyacinth bean) - a legume native to Africa and cultivated throughout the tropics for food and forage. Our Africa-led South-North plant genome collaboration produced a high-quality chromosomescale assembly of the lablab genome - the first chromosome-scale plant genome assembly locally sequenced in Africa. We also re-sequenced cultivated and wild accessions of lablab from Africa confirming two domestication events and examined the genetic diversity in lablab germplasm conserved in Africa. Our approach provides a valuable resource for lablab improvement and also presents a model that could be explored by other researchers sequencing indigenous crops particularly from Low and middle income countries (LMIC).

genomics↗

bioRxiv2022

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also promotes transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell differentiation is unknown. Here, we find that CDK8 is dispensable for RNA polII CTD phosphorylation, regulation of gene expression, normal intestinal homeostasis and efficient tumourigenesis in mice. Furthermore, CDK8 is largely redundant with CDK19 in the control of gene expression. Yet, while their combined deletion in intestinal organoids reduces long-term proliferative capacity, it is not lethal and allows differentiation. Nevertheless, in double mutant organoids, the cystic fibrosis transmembrane conductance regulator (CFTR) pathway is transcriptionally and functionally downregulated, leading to mucus accumulation and increased secretion by goblet cells. This phenotype can be recapitulated by pharmacological inhibition of CDK8/19 kinase activity. Thus, the Mediator kinases are not essential for cell proliferation and differentiation, but they cooperate to regulate tissue-specific transcriptional programmes.

cell biology↗

bioRxiv2021

Data-driven discovery of targets for bipotent anticancer drugs identifies Estrogen Related Receptor Alpha

Drugs that kill tumors through multiple mechanisms have potential for broad clinical benefits, with a reduced propensity to resistance. We developed BipotentR, a computational approach to find cancer-cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway. Using tumor metabolism as proof-of-principle, BipotentR identified 38 candidate immune-metabolic regulators by combining epigenomes with bulk and single-cell tumor transcriptomes from patients. Inhibition of top candidate ESRRA (Estrogen Related Receptor Alpha) killed tumors by direct effects on energy metabolism and two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization (ii) antigen-presentation stimulation, recruiting CD8+T cells into tumors. ESRRA is activated in immune-suppressive and immunotherapy-resistant tumors of many types, suggesting broad clinical relevance. We also applied BipotentR to angiogenesis and growth-suppressor pathways, demonstrating a widely applicable approach to identify drug targets that act simultaneously through multiple mechanisms. BipotentR is publicly available at http://bipotentr.dfci.harvard.edu/. One-Sentence SummaryBipotentR identifies targets for bipotent anticancer drugs, as shown by the energy and immune effects of ESRRA inhibition.

cancer biology↗

bioRxiv2021

Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

cell biology↗

bioRxiv2021

Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

Senescence is an irreversible proliferation withdrawal that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 is not well understood; it requires p53 and RB family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the CDK inhibitor p21 and Chk1 controls D-type cyclin-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting Cyclin D1-CDK2/CDK4. The resulting G2 exit, which precedes appearance of senescence markers, is associated with a mitotic bypass, Chk1 inhibition and DNA damage foci reduction. In p53/RB-proficient cancer cells, compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely Cyclin E1 re-expression and genome reduplication. Chk1 depletion promotes cell cycle exit by inducing p21 binding to Cyclin D1 and Cyclin E1-CDK complexes and down-regulating CDK6, whereas Chk2 knockdown promotes RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus controlling DNA damage-induced senescence onset in G2.

cell biology↗

bioRxiv2021

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

Chronic myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed bulk transcriptome profiling accompanied by single cell RNA-sequencing on CD34+ stem/progenitor cells from serial patient samples obtained at the chronic MPN and sAML phases, and identified aberrantly increased expression of dual-specificity phosphatase 6 (DUSP6) underlying disease transformation. Genetic and pharmacologic targeting of DUSP6 led to inhibition of S6 and JAK/STAT signaling, resulting in potent suppression of cell proliferation, while also reducing inflammatory cytokine production in primary samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells. Mechanistically, DUSP6 perturbation dampened S6 signaling via inhibition of RSK1, which we identified as a second indispensable candidate associated with poor clinical outcome. Lastly, DUSP6 inhibition potently suppressed disease development across Jak2 V617F and MPL W515L MPN mouse models, and sAML patient-derived xenografts. These findings underscore DUSP6 in driving disease transformation and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies.

cancer biology↗

bioRxiv2020

Heritable Sexual Attraction under Parental Control

It is unknown whether transient transgenerational epigenetic responses to environmental challenges affect the process of evolution, which typically unfolds over many generations. Here we show that in C. elegans, inherited small RNAs shape the hard-wired genome and control genetic variation by regulating the decision of whether to self-fertilize or outcross. We found that under stressful temperatures younger hermaphrodites secrete a male-attracting pheromone. Attractiveness transmits transgenerationally to unstressed progeny via heritable small RNAs and the Argonaute Heritable-RNAi-Deficient-1. We identified an endogenous small interfering RNA pathway, enriched in endo-siRNAs which target sperm genes, that transgenerationally regulates sexual attraction, male prevalence, and outcrossing rates. Multigenerational mating competitions and mathematical simulations revealed that over generations, animals that inherit attractiveness mate more, and their alleles spread in the population. We propose that sperm serves as a "stress sensor" which, via small RNA inheritance, can promote outcrossing in challenging environments, when increasing genetic variation is advantageous.

molecular biology↗

bioRxiv2020

Seeding of outbreaks of COVID-19 by contaminated fresh and frozen food

The authors have withdrawn this manuscript because some aspects of the published work were completed prior to regulatory approval. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

microbiology↗

bioRxiv2020

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Reverse transcription, an essential event in the HIV-1 lifecycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral core. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically-favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import and reverse transcription.

biophysics↗

bioRxiv2020

Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of {beta}-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

microbiology↗

bioRxiv2019

CDK8 and CDK19 kinases have non-redundant oncogenic functions in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common cancer with high mortality. The limited therapeutic options for advanced disease include treatment with Sorafenib, a multi-kinase inhibitor whose targets include the Mediator kinase CDK8. Since CDK8 has reported oncogenic activity in Wnt-dependent colorectal cancer, we investigated whether it is also involved in HCC. We find that CDK8 and its paralogue CDK19 are significantly overexpressed in HCC patients, where high levels correlate with poor prognosis. Liver-specific genetic deletion of CDK8 in mice is well supported and protects against chemical carcinogenesis. Deletion of either CDK8 or CDK19 in hepatic precursors had little effect on gene expression in exponential cell growth but prevented oncogene-induced transformation. This phenotype was reversed by concomitant deletion of TP53. These data support important and non-redundant roles for mediator kinases in liver carcinogenesis, where they genetically interact with the TP53 tumor suppressor.

cancer biology↗

bioRxiv2019

Dynamic perineuronal net expression during learned maternal behavior is cortical hemisphere-specific and dependent on methyl-CpG-binding protein 2

Cortical neuronal circuits along the sensorimotor pathways are shaped by experience during critical periods of heightened plasticity in early postnatal development. After closure of critical periods, measured histologically by the formation and maintenance of extracellular matrix structures called perineuronal nets (PNNs), the adult mouse brain exhibits restricted plasticity and maturity. Mature PNNs are typically considered to be stable structures that restrict synaptic plasticity on cortical parvalbumin+ GABAergic neurons. Changes in environment (i.e. novel behavioral training) or social contexts (i.e. motherhood) are known to elicit synaptic plasticity in relevant neural circuitry. However, little is known about concomitant changes in the PNNs surrounding the cortical parvalbumin+ GABAergic neurons. Here, we show novel changes in PNN density in the primary somatosensory cortex (SS1) of adult female mice after maternal experience, using systematic microscopy analysis of a whole brain region. On average, PNNs were increased in the right barrel field and decreased in the left forelimb regions. Individual mice had left hemisphere dominance in PNN density. Using adult female mice deficient in methyl-CpG-binding protein 2 (MECP2), an epigenetic regulator involved in regulating experience-dependent plasticity, we found that MECP2 is critical for this precise and dynamic expression of PNN. Adult naive Mecp2-heterozygous females (Het) had increased PNN density in specific subregions in both hemispheres before maternal experience. The laterality in PNN expression seen in naive Het was lost after maternal experience, suggesting possible intact mechanisms for plasticity. Together, our results identify subregion and hemisphere-specific alterations in PNN expression in adult females, suggesting extracellular matrix plasticity as a possible neurobiological mechanism for adult behaviors in rodents.

neuroscience↗