Biology subjects

Sykes, D. B.

Publications and source records attributed to Sykes, D. B..

8 recordsLinked to original sources

bioRxiv2022

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that modulating aspects of metabolism could alter cell state along differentiation trajectories. Here we find that nucleotide depletion and DNA replication stress are common drivers of cell state progression across a variety of normal and transformed hematopoietic systems. DNA replication stress-induced cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress leads to increased activity at primed regulatory loci and expression of lineage-appropriate maturation genes while progenitor TF activity is still present. Altering the baseline cell state by manipulating the cohort of transcription factors expressed redirects the effect of replication stress towards induction of a different set of lineage-specific genes. The ability of replication stress to selectively activate primed maturation programs across different cellular contexts suggests a general mechanism by which metabolism can promote lineage-appropriate and potentially therapeutically relevant cell state transitions.

cancer biology↗

bioRxiv2022

A new transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we take advantage of fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors revealed a distinct transcriptional signature that was predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrated vascular remodeling within the endothelial cells and a proliferative signature within the fibroblasts that was associated with poor survival. An in silico cell-to-cell interaction analysis highlighted the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.

cancer biology↗

bioRxiv2022

Brequinar and Dipyridamole in Combination Exhibits Synergistic Antiviral Activity Against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of currently available vaccines and monoclonal antibody (mAb)-based treatment options for COVID-19. The limited number of authorized small-molecule direct-acting antivirals present challenges with pill burden, the necessity for intravenous administration or potential drug interactions. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

microbiology↗

bioRxiv2022

Integrated single-cell and spatial transcriptomic analyses unravel the heterogeneity of the prostate tumor microenvironment

The treatment of primary prostate cancer delicately balances an active surveillance approach for low-risk disease with multimodal treatment including surgery, radiation therapy, and hormonal therapy for high-risk disease. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we sought to comprehensively describe the tumor microenvironment of localized prostate cancer contrasting this with adjacent normal samples and healthy controls. We performed single-cell RNA sequencing and high-resolution spatial transcriptomic analysis. This revealed tumor context dependent changes in gene expression. Our data point towards an immune suppressive tumor microenvironment associated with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We inferred cell-to-cell relationships at an unprecedented scale for ligand-receptor interactions within undissociated tissue sections. Our work provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions. HighlightsO_LICharacterization of prostate cancer by combined scRNA-seq and spatial transcriptomic analysis C_LIO_LIPrimary prostate cancer establishes a suppressive immune microenvironment C_LIO_LIThe prostate tumor microenvironment exhibits a high angiogenic gene expression pattern C_LIO_LIA new computational analysis pipeline to deconvolute context-specific differential gene expression C_LI

cancer biology↗

bioRxiv2021

DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a pan-cancer, multi-omic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. By combining metabolomics, CRISPR screen and gene expression data from more than 700 solid tumor cell lines, we identified DHODH, a critical enzyme in pyrimidine synthesis, as a potential novel treatment target in neuroblastoma. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of high-risk neuroblastoma tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In neuroblastoma cell lines, DHODH gene dependency was found to correlate with MYCN dependency, rendering these cell lines highly sensitive to DHODH inhibition in vitro. In xenograft and transgenic neuroblastoma mouse models, tumor growth was dramatically reduced, and survival extended following treatment with the DHODH inhibitor brequinar. A combination of brequinar and temozolomide cured the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy with curative potential. Overall, DHODH inhibition combined with temozolomde has clear therapeutic potential in neuroblastoma and we propose this combination as a candidate for clinical testing.

cancer biology↗

bioRxiv2021

tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche

Extracellular vesicles transfer complex biologic material between cells, whose role in in-vivo organismal physiology is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. Stimulating osteoblastic cells with parathyroid hormone or activating its receptor enhanced extracellular vesicle transfer, myeloid recovery post radiation and improved animal survival from Candida sepsis. The extracellular vesicles contained tiRNAs known to modulate protein translation. 5-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and when transferred to granulocyte macrophage progenitors, increased protein translation, cell proliferation and myeloid differentiation. Therefore, EV-mediated tiRNA transfer provides a stress modulated signaling axis distinct from conventional cytokine-driven stress responses. One sentence summaryStress regulated tiRNA transfer alters hematopoiesis

cell biology↗

bioRxiv2021

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

Signaling cascades that convert the recognition of pathogens to efficient inflammatory responses by immune cells, specifically neutrophils, are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al., 2020, Shaban et al., 2020, Boras et al., 2017). SKAP2 is also required for the host defense against K. pneumoniae and{Delta} yopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al., 2020, Nguyen et al., 2020). Another class of pattern recognition receptors (PRR) is the C-type lectin receptors (CLR), such as Dectin-1, Dectin-2 and Mincle, that are critical to trigger innate immune responses. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 is crucial for maximal ROS response to purified CLR agonists and to the fungal pathogens Candida glabrata and C. albicans, as well as for robust killing of C. glabrata. Skap2-/- murine neutrophils failed to generate ROS and exhibited reduced cellular adhesion in response to trehalose-6,6-dibehenate (TDB), furfurman, and curdlan, Mincle, Dectin-2, and Dectin-1 agonists, respectively. TDB, furfurman, and curdlan stimulation also led to SKAP2-independent integrin conformational changes, showing that inside-out signaling by these CLRs to integrin occurs in the absence of SKAP2. Pyk2 phosphorylation was significantly reduced after infection with C. glabrata in Skap2-/- neutrophils, while Syk phosphorylation was unaffected by the loss of SKAP2. These data strengthen the importance of SKAP2 in the activation of neutrophil ROS production by PRRs to include CLRs and extend the role of SKAP2 in host defense beyond antibacterial immunity to include Candida species.

immunology↗

bioRxiv2021

Cyclophilin A regulates protein phase separation and mitigates haematopoietic stem cell aging

Loss of protein quality is a driving force of aging1. The accumulation of misfolded proteins represents a vulnerability for long-lived cells, such as haematopoietic stem cells. How these cells, which have the ability to reconstitute all haematopoietic lineages throughout life2, maintain their regenerative potential and avert the effects of aging is poorly understood. Here, we determined the protein content in haematopoietic stem and progenitor cells to identify prevalent chaperones that support proteome integrity. We identified Peptidyl-Prolyl Isomerase A (PPIA or Cyclophilin A) as the dominant cytosolic foldase in this cell population. Loss of PPIA accelerated aging in the mouse stem cell compartment. In an effort to define targets of PPIA, we found that RNA- and DNA-binding proteins are common substrates of this chaperone. These proteins are enriched in intrinsically disordered regions (IDRs), which can catalyse protein condensation3. Isomerized target prolines are almost exclusively located within IDRs. We discovered that over 20% of PPIA client proteins are known to participate in liquid-liquid phase separation, enabling the formation of supramolecular membrane-less organelles. Using the poly-A binding protein PABPC1 as an example, we demonstrate that PPIA promotes phase separation of ribonucleoprotein particles, thereby increasing cellular stress resistance. Haematopoietic stem cell aging is associated with a decreased expression of PPIA and reduced synthesis of intrinsically disordered proteins. Our findings link the ubiquitously expressed chaperone PPIA to phase transition and identify macromolecular condensation as a potential determinant of the aging process in haematopoietic stem cells.

cell biology↗