Biology subjects

Watson, Z. L.

Publications and source records attributed to Watson, Z. L..

11 recordsLinked to original sources

bioRxiv2024

Ablation of hematopoietic stem cell derived adipocytes reduces tumor burden in syngeneic mouse models of high-grade serous carcinoma

Hematopoietic stem cell-derived adipocytes (HSCDAs) are an adipose subtype derived from myeloid precursors that are distinct from conventional mesenchymal adipocytes (CMAs). We hypothesized that HSCDAs promote high grade serous carcinoma (HGSC), the most common form of ovarian cancer. Despite similar rates of differentiation, primary human HSCDAs from female donors showed marked transcriptional differences from CMAs, including downregulation of cell cycle and upregulation of lipid metabolic pathways. HSCDAs secreted greater amounts of inflammatory cytokines than CMAs. We also conducted two independent tumor studies using ID8 and SO syngeneic HGSC murine models in immunocompetent mice that were either HSCDA Proficient (HSCDA-Pro; can make both adipocyte subtypes) or Deficient (HSCDA-Def; can only make CMAs). Tumor burden trended lower in HSCDA-Def mice in both models. Relative to HSCDA-Pro mice, omental ID8 tumors from HSCDA-Def mice downregulated transcription of multiple metabolic pathways that were enriched in human HSCDA cells in vitro, suggesting that ablation of HSCDAs altered the tumor metabolic environment. Compared to HSCDA-Pro mice, tumors from HSCDA-Def mice had lower densities of dendritic cells (DC) and natural killer (NK) cells, as well as fewer DCs, NKs, and B-cells in proximity to tumor cells. Our data suggest that HSCDAs alter the peritoneal immune and metabolic environment to support HGSC progression. ONE SENTENCE SUMMARYHematopoietic stem cell derived adipocytes may alter the peritoneal metabolic and immune environment to establish a metastatic niche and support ovarian cancer progression.

cancer biology↗

bioRxiv2023

Combination CDC-like kinase inhibition (CLK)/Dual-specificity tyrosine-regulated kinase (DYRK) and taxane therapy in CTNNB1-mutated endometrial cancer

SM08502 (cirtuvivint) is a novel pan CDC-like kinase (CLK) and Dual specificity tyrosine kinase (DYRK) inhibitor that targets mRNA splicing and is optimized for Wnt pathway inhibition. Previous evaluation of single agent CLK/DYRK inhibition (SM04690) demonstrated inhibition of tumor progression and {beta}-catenin/TCF transcriptional activity in CTNNB1-mutant endometrial cancer (EC). In-vitro analysis of SM08502 similarly decreases Wnt transcriptional activity and cellular proliferation while increasing cellular apoptosis. SM08502 is an active single-agent therapy with IC50s in the nanomolar range for all EC cell lines evaluated. Combination of SM08502 with paclitaxel has synergistic effect in vitro, as demonstrated by Combination Index <1, and inhibits tumor progression in four endometrial cancer models (HEC265, Ishikawa, Ishikawa-S33Y, and SNGM). In our in vivo mouse models, Ishikawa demonstrated significantly lower tumor volumes of combination vs SM08502 alone (Repeated Measures one-way ANOVA, p = 0.04), but not vs paclitaxel alone. HEC265, SNGM, and Ishikawa-S33Y tumors all had significantly lower tumor volumes with combination SM08502 and paclitaxel compared to single-agent paclitaxel (Repeated Measures one-way ANOVA, p = 0.01, 0.004, and 0.0008, respectively) or single-agent SM08502 (Repeated Measures one-way ANOVA, p = 0.002, 0.005, and 0.01, respectively) alone. Mechanistically, treatment with SM08502 increases alternative splicing (AS) events compared to treatment with paclitaxel. AS regulation is an important post-transcriptional mechanism associated with the oncogenic process in many cancers, including EC. Results from these studies have led to a Phase I evaluation of this combination in recurrent EC.

cancer biology↗

bioRxiv2023

Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models

Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

cancer biology↗

bioRxiv2022

Rare Ribosomal RNA Sequences from Archaea Stabilize the Bacterial Ribosome

Ribosomes serve as the universally conserved translators of the genetic code into proteins and must support life across temperatures ranging from below freezing to above the boiling point of water. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Peptide bond formation by the ribosome requires correct positioning of the 3 s-end of the aminoacylated tRNA (aa-tRNA) substrate, which is aided by an RNA hairpin in the ribosomal RNA (rRNA) of the large subunit, termed the A loop. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not exert a dominant negative effect on cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation in the peptidyl transferase center of the archaeal ribosome that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

biophysics↗

bioRxiv2021

BRPF3 knockdown or inhibition moderately reverses olaparib resistance in high grade serous ovarian carcinoma, but depletion of H3K14 acetylation has no effect

BackgroundPARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) and epigenetic readers have known functions in DNA repair and replication. Our objectives are to examine their expression and activities in the context of PARPi-resistant HGSOC, and to determine if targeting H3K14ac or associated proteins has therapeutic potential. ResultsUsing mass spectrometry profiling of histone modifications, we observed increased H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By RT-qPCR and RNA-Seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 severely depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, which is known to interact in a complex with HBO1, did reduce PARPi resistance. ConclusionsThis study demonstrates that severe depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that bromodomain functions of HAT proteins, such as PCAF, or accessory proteins, such as BRPF3, may play a more direct role compared to direct histone acetyltransferase functions in PARPi response.

cancer biology↗

bioRxiv2020

Structure of the Bacterial Ribosome at 2 A Resolution

Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 [A], based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 [A], based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

biophysics↗

bioRxiv2020

Eliciting OTUD3/RIPK-Dependent Necroptosis to Prevent Epithelial Ovarian Cancer

BackgroundThere is an urgent need for early prevention strategies against high grade serous ovarian carcinoma (HGSOC), the deadliest gynecologic malignancy. Transformed p53-null fallopian tube epithelium (FTE) cells are precursors of HGSOC that may be eliminated by inducing necroptosis, a programmed form of inflammatory cell death. Induction of necroptosis is dependent upon activation of receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1/3). TNF and progesterone (P4) effectively promote necroptosis. In this study, we explore the activation of necroptosis as an approach to inhibit HGSOC progression. MethodsUsing gene ontology sets as a reference, we analyzed publicly available datasets of HGSOC to correlate the expression of necroptosis effectors to clinical outcomes. Using in vitro models of HGSOC we evaluated the effect of TNF, P4, and -eleostearic acid on necroptosis. In parallel, the necroptosis inhibitor Necrostatin-1 was used to confirm necroptosis-specific cell death. ResultsExpression of the P4 receptor (PGR) was sharply reduced in a HGSOC cohort compared to normal, nonmalignant FTE. However, several genes involved in necroptosis signaling were elevated in HGSOC, including TNF and RIPK1. Increased expression of PGR, the necroptosis effectors TNF and RIPK1/3, as well as ovarian tumor domain-containing deubiquitinase 3 (OTUD3) were associated with higher overall survival in 484 HGSOC cases. HGSOC cells activated necroptosis in response to P4, TNF, and -eleostearic acid treatment, while P4 or TNF treatment of HGSOC cells increased TNF, RIPK1, and OTUD3 expression. OTUD3 is a putative tumor suppressor that stabilizes PTEN and is hypothesized to be functionally similar to the necroptosis inducer, OTUD7B. shRNA knockdown of OTUD3 resulted in decreased PTEN protein and RIPK1 protein. ConclusionsWe conclude that necroptosis activation may be a viable prevention strategy that leads to the elimination of transformed FTE "founder" cells and prevents HGSOC tumorigenesis. Our data indicate that HGSOC cells activate necroptosis in response to P4, TNF, and -eleostearic acid, suggesting that established HGSOC cells may also be eliminated by activating necroptosis.

cancer biology↗

bioRxiv2019

Defects in the assembly of ribosomes selected for β-amino acid incorporation

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and {beta}-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo {beta}-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

biochemistry↗

bioRxiv2019

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

molecular biology↗

bioRxiv2018

Targeting Wnt Signaling To Overcome PARP Inhibitor Resistance

Epithelial ovarian cancer (EOC) has one of the highest deaths to incidence ratios. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype because of the lack of secondary therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, more recent studies have observed a significant clinical benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance to PARPi. Therefore, there is an urgent clinical need to better understand PARPi resistance, and to introduce novel combinatorial therapies to overcome PARPi resistance and extend HGSOC disease-free intervals. Utilizing a two BRCA2-mutated and one BRCA-wildtype HGSOC cell lines that are olaparib sensitive, we established resistant cells. Transcriptome analysis of the matched olaparib-sensitive versus resistant cells did not detect BRCA2 reversion mutations, but revealed activation of Wnt/TCF signaling pathway, as TCF transcriptional activity was significantly increased in PARPi-resistant cells. In parallel, forced activation of Wnt signaling in PARPi-sensitive cells via WNT3A stimulation reduced response to PARPi. In a recurrent-HGSOC PARPi insensitive patient-derived xenograft model there was an increase in a Wnt/TCF transcriptional target. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to inhibit Wnt signaling. We observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden and number of tumor nodules. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARPi and Wnt inhibitors.

cancer biology↗

bioRxiv2016

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 [A] or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 [A], while the resolution of the map reached 4.0 [A] for the same number of particles, when the estimated resolution of streptavidin crystal was 4 [A] or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 [A], a marked improvement over the value of 3.9 [A] obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

biophysics↗