Biology subjects

Stewart-Ornstein, J.

Publications and source records attributed to Stewart-Ornstein, J..

5 recordsLinked to original sources

bioRxiv2022

PKC-alpha regulates the phosphorylation of KRAS that suppresses its oncogenic properties

Oncogenic KRAS-driven cancers have long been considered as "undruggable" due to limited therapeutic options. While the recent success of KRAS-G12C inhibitors argues against the "undruggability" of KRAS, this treatment only benefits a small proportion of patients with KRAS mutant cancers, leaving an urgent need for modalities to target other KRAS mutants. KRAS-calmodulin (CaM) signaling axis reportedly regulates the oncogenic properties of KRAS through its C-terminal hypervariable region. Phosphorylation of KRAS by activated protein kinase C (PKC) uncouples KRAS-CaM, resulting in growth inhibition effective against the entire spectrum of KRAS hotspot mutations. However, broadly activating PKC could mediate tumor promoting signaling nodes and cause systemic toxicity, undermining its applicability as an anti-KRAS therapy. Here, we found that prostratin induces KRAS phosphorylation, resulting in an elevated level of active CaM in the cytosol of KRAS mutant cells, and consequentially suppresses their malignancies. A whole-genome wide CRISPR/Cas9 knockout screening, further confirmed by biochemical analysis, revealed that prostratin acts through activating PKC. Functional studies confirmed PKC as the sole kinase to phosphorylate KRAS and, therefore, a KRAS suppressor. Activation of PKC induces senescence in KRAS mutant tumor cells through PTPN14, accompanied by a secretory phenotype contributing to the growth inhibition, and parallelly mediates a nuclear translocation of a CaM-dependent transcription activator, CAMTA-1, which can be a biomarker to indicate the activity of PKC-KRAS-CaM axis. Our findings reveal a previously understudied regulation of KRAS-CaM axis by PKC, which can be an actionable target for developing anti-KRAS therapeutics. One Sentence SummaryThis study deciphers a PKC-led tumor suppressive effect specific to the "undruggable" KRAS-mutant tumor cells through the phosphorylation of KRAS and a consequently altered KRAS-CaM signaling axis.

cancer biology↗

bioRxiv2021

Telomeric 8-oxoguanine drives rapid premature senescence in the absence of telomere shortening

Oxidative stress is a primary cause of cellular senescence and contributes to the pathogenesis of numerous human diseases. Oxidative damage to telomeric DNA is proposed to trigger premature senescence by accelerating telomere shortening. Here we tested this model directly using a precision tool to produce the common base lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblast and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and delayed mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions which drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.

molecular biology↗

bioRxiv2020

Acetylation-mediated phase control of the nucleolus regulates cellular acetyl-CoA responses

The metabolite acetyl-CoA serves as an essential element for a wide range of cellular functions including ATP production, lipid synthesis and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using CRISPR-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa HDAC deacetylases regulating the phase state of the nucleolus. Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

cell biology↗

bioRxiv2019

Optogenetic control reveals differential promoter interpretation of transcription factor nuclear translocation dynamics

The dynamic translocation of transcription factors (TFs) in and out of the nucleus is thought to encode information, such as the identity of a stimulus. A corollary is the idea that gene promoters can decode different dynamic TF translocation patterns. Testing this TF encoding/promoter decoding hypothesis requires tools that allow direct control of TF dynamics without the pleiotropic effects associated with general perturbations. In this work, we present CLASP (Controllable Light Activated Shuttling and Plasma membrane sequestration), a tool that enables precise, modular, and reversible control of TF localization using a combination of two optimized LOV2 optogenetic constructs. The first sequesters the cargo in the dark at the plasma membrane and releases it upon exposure to blue light, while light exposure of the second reveals a nuclear localization sequence that shuttles the released cargo to the nucleus. CLASP achieves minute-level resolution, reversible translocation of many TF cargos, large dynamic range, and tunable target gene expression. Using CLASP, we investigate the relationship between Crz1, a naturally pulsatile TF, and its cognate promoters. We establish that some Crz1 target genes respond more efficiently to pulsatile TF inputs than to continuous inputs, while others exhibit the opposite behavior. We show using computational modeling that efficient gene expression in response to short pulsing requires fast promoter activation and slow inactivation and that the opposite phenotype can ensue from a multi-stage promoter activation, where a transition in the first stage is thresholded. These data directly demonstrate differential interpretation of TF pulsing dynamics by different genes, and provide plausible models that can achieve these phenotypes.

systems biology↗

bioRxiv2017

Stereotyped p53 binding tuned by chromatin accessibility

The tumor suppressor p53 is a major regulator of the DNA damage response and has been suggested to selectively bind and activate cell type specific gene expression programs, however recent studies and meta-analyses of genomic data propose largely uniform, and condition independent, p53 binding. To systematically assess the cell type specificity of p53, we measured its association with DNA in 12 p53 wild-type cell lines, from a range of epithelial linages, in response to ionizing radiation. We found that the majority of bound sites were occupied across all cell lines, however we also identified a subset of binding sites that were specific to one or a few cell lines. Unlike the shared p53-bound genome, which was not dependent on chromatin accessibility, the association of p53 with these atypical binding sites was well explained by chromatin accessibility and could be modulated by forcing cell state changes such as the epithelial-to-mesenchymal transition. These results position p53 as having both universal and cell type specific regulatory programs that have different regulators and dependence on chromatin state.

genomics↗