Biology subjects

Keith, B.

Publications and source records attributed to Keith, B..

7 recordsLinked to original sources

bioRxiv2026

HAP40 functions as a proteostasis regulator by controlling huntingtin interactions and its release into the extracellular space

Huntingtin-associated protein 40 (HAP40) forms a stable protein complex with huntingtin (HTT). Its cellular function and how HAP40 loss influences mutant HTT (mHTT) abundance and pathobiology are currently unclear. Here, using diverse cellular models and OMICs methods, we demonstrate that HAP40 is an obligate interaction partner of full-length HTT and through its binding controls the abundance of HTT-associated proteins, indicating that it functions as HTT interaction regulatory unit. Also, loss of HAP40 in mHTT-expressing striatal cells impairs autophagosome-lysosome flux, triggers massive transcriptional dysregulation, including the activation of the CLEAR network, demonstrating that it functions as proteostasis regulator that acts on quality control pathways. Finally, mHTT-expressing cells lacking HAP40 showed increased secretion of mHTT through the ER-to-Golgi route, indicating that striatal cells reduce intracellular mHTT-induced proteotoxicity through activation of secretory pathways. Together, these results establish HAP40 as a critical proteostasis regulator that through controlling HTT interactions maintains cellular homeostasis.

cell biology↗

bioRxiv2024

Chemoresistance of TP53 mutant AML requires the mevalonate byproduct, GGPP, for regulation of ROS and induction of a mitochondria stress response

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene, TP53 (TP53mut AML), is fatal with a median survival of only 6 months. RNA sequencing on purified AML patient samples show TP53mut AML has higher expression of mevalonate pathway genes. We retrospectively identified a survival benefit in TP53mut AML patients who received chemotherapy concurrently with a statin, which inhibits the mevalonate pathway. Mechanistically, TP53mut AML resistance to standard AML chemotherapy, cytarabine (AraC), correlates with increased mevalonate pathway activity and a mitochondria stress response with increased mitochondria mass and oxidative phosphorylation. Pretreatment with a statin reverses these effects and chemosensitizes TP53mut AML cell lines and primary samples in vitro and in vivo. Mitochondria-dependent chemoresistance requires the geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway and novel GGPP-dependent synthesis of glutathione to manage AraC-induced reactive oxygen species (ROS). Overall, we show that the mevalonate pathway is a novel therapeutic target in TP53mut AML. SignificanceChemotherapy-persisting TP53mut AML cells induce a mitochondria stress response that requires mevalonate byproduct, GGPP, through its novel role in glutathione synthesis and regulation of mitochondria metabolism. We provide insight into prior failures of the statin family of mevalonate pathway inhibitors in AML. We identify clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML.

cancer biology↗

bioRxiv2023

Translational mapping of spatially resolved transcriptomes in human and mouse pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. To address these limitations, we generated spatially resolved transcriptome maps of human IPF and bleomycin-induced mouse lung fibrosis. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by TGF{beta} signaling alongside factors such as p53 and ApoE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches, and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.

molecular biology↗

bioRxiv2023

Nuclear speckles regulate HIF-2α programs and correlate with patient survival in kidney cancer

Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2 transcription factor, and we demonstrate here that HIF-2 drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2-driven speckle association via deletion of its speckle targeting motifs (STMs)--defined in this study--led to defective induction of speckle-associating HIF-2 target genes without impacting non-speckle-associating HIF-2 target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2 gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation. HIGHLIGHTS- Nuclear speckles shown to reproducibly vary in cancer, predicting patient survival in ccRCC - HIF-2 drives DNA/gene-speckle contacts dependent on identified speckle targeting motifs within HIF-2 - Putative speckle targeting motifs are highly enriched among regulators of gene expression - Partitioning of transcription factor functional programs may be a major biological function of nuclear speckles

cancer biology↗

bioRxiv2022

The Cdc42 effector and non-receptor tyrosine kinase, ACK defines a distinct STAT5 transcription signature in a chronic myeloid leukaemia cell model

Activated Cdc42-associated kinase (ACK) is a Rho family effector that is widely implicated in cancer. Here, we describe new roles for ACK in transcriptional regulation mediated by its relationship with the signal transducer and activators of transcription (STAT) family. We show that ACK can interact with STAT3, STAT5A and STAT5B, and augments phosphorylation at the conserved activation tyrosine on these STAT members. ACK stimulates oncogenic STAT nuclear relocation and transcriptional activation. We also identify endogenous relationships between ACK and STAT family members in haematopoietic disease cell lines. In the K562 chronic myeloid leukaemia cell line, we confirm that ACK contributes to the pool of active, nuclear STAT5. By interrogating ACK knock out cells we describe an ACK-driven STAT5 transcriptional signature in K562s. We propose ACK as a contributor to hyperactivated STAT5 signalling in this CML cell line and reveal a new route for therapeutic intervention. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/494364v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1c4973dorg.highwire.dtl.DTLVardef@1a54ba1org.highwire.dtl.DTLVardef@1083bf9org.highwire.dtl.DTLVardef@1997ad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Human genetic disease is greatly influenced by the underlying fragility of evolutionarily ancient genes

The ability to predict disease association in human genes is enhanced by an evolutionary understanding. Importantly genes linked with heritable disease, particularly dominant disorders, tend to have undergone duplication in our early vertebrate ancestors, with a strong asymmetric relationship between disease-association within duplicate/paralog pairs. Using a novel phylogenetic approach, alongside a whole-genome comparative analysis, we show that contrary to the accepted compensatory model of disease evolution, the majority of disease-associations reside with the more evolutionary constrained gene, inferred to most closely resemble the progenitor. This indicates that the strong association between paralogs, specifically ohnologs, and dominant disorders is often a consequence of a mechanism through which pre-existing dosage sensitive/haploinsufficient genes are successfully duplicated and retained. Heritable disease is thus as much a consequence of the fragility of evolutionarily more ancient genes as compensatory mechanisms. From these findings, we demonstrate the utility of a new model with which to predict disease associated genes in the human genome.

genetics↗

bioRxiv2017

CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

Interleukin 18 (IL18) is known to induce the expression of interferon-{gamma} (IFNG), but its effects on T cell proliferation and costimulation are not completely understood. In this study, we demonstrate that ectopic expression of IL18 in CART cells caused significant T cell proliferation in vitro and in vivo, and enhanced antitumor effects in xenograft models. Moreover, IL18 mediated T cell expansion required neither tumor antigen nor CAR expression, and produced severe GVHD in NSG mice. Furthermore, recombinant IL18 costimulated IFNG secretion and proliferation of anti-CD3 beads treated T cells. Interestingly, IL18 costimulation could expand purified CD4 T cells, but not CD8 T cells. However, CD8 T cells proliferated greater than CD4 T cells in magnitude within bulk T cells, suggesting CD4 help effect was involved. Using CRISPR/Cas9 gene editing, we confirmed that IL18-driven expansion was both TCR and IL18 receptor (IL18R) dependent. Importantly, we demonstrated that TCR-deficient, IL18-expressing CD19 CART cells exhibited remarkable proliferation and persistent antitumor activity against CD19-expressing tumor cells in vivo, without eliciting any detectable GVHD symptom. Finally, we describe APACHE T cells, a novel strategy for coupling IL18 expression in CART cells to antigen stimulation, thereby limiting potential toxicity associated with persistent IL18 production. In sum, our study supports human IL18 as a T cell costimulatory cytokine for fueling CART therapy.

synthetic biology↗