Biology subjects

Kelly, T. K.

Publications and source records attributed to Kelly, T. K..

6 recordsLinked to original sources

bioRxiv2026

Pharmacological targeting of EED is an effective therapeutic strategy in cellular models of incurable neuroendocrine prostate cancer

BackgroundNeuroendocrine Prostate Cancer (NEPC) is an incurable malignancy, originating from the trans-differentiation of prostate adenocarcinoma (PRAD). Compared to PRAD, NEPC shows over-activation of Polycomb Repressive complex-1(PRC1) and-2 (PRC2), which are multiprotein epigenetic writers that drive cancer progression via tumour suppressor gene silencing. Tazemetostat is a PRC2 inhibitor approved for the treatment of sarcomas and lymphomas. ORIC-944 is a novel EED (Embryonic Ectoderm Development) inhibitor, which is being tested in clinical trials. EED is an attractive target as it functions as a key component of both PRC1 and PRC2. Objective and MethodsWe compared the anticancer effects of tazemetostat and ORIC-944 in NEPC and PRAD cells. Cells were exposed to various concentrations of the two compounds to measure effects on cell viability (IC50) and apoptosis (flow cytometry). PRC2 inhibition was confirmed by measuring histone H3 Lys 27 trimethylation (H3K27me3) via ELISA and Western Blot. RNA Sequencing and pathway analysis was conducted to study modes of actions of tazemetostat vs ORIC-944. ResultsUnlike tazemetostat, ORIC-944 causes dose-dependent growth inhibition in both NEPC and PRAD cells. In this context, EED targeting achieves IC50 values that are comparable to those of compounds used for the clinical treatment of advanced prostate cancer. Moreover, ORIC-944 (but not tazemetostat) causes significant apoptosis in NEPC cells. Both tazemetostat and ORIC-944 reduce H3K27me3. Mechanistically, both compounds reactivate the expression of known PRC2 targets, such as genes that control neural differentiation. However, the EED inhibitor also reactivates PRC1 targets, including pro-apoptotic and anti-proliferating genes (e.g. metallothionines). This evidence suggests that EED inhibition is a promising therapeutic strategy for NEPC.

cancer biology↗

bioRxiv2026

Cytokine-induced Chromatin Accessibility in Whole Blood Neutrophils Links to Sepsis Transcriptional 1 States

BackgroundNeutrophils play an important role in the immune system and sense environmental perturbations including pathogens. Upon pathogen detection, neutrophils extrude their chromatin, forming neutrophil extracellular traps (NETs) trapping and removing pathogens. Previous studies have shown that controlled chromatin decondensation occurs during NET formation, reflecting NET inducing pathways, and the cellular environment. While NET inducing stimuli like phorbol 12-myristate 13-acetate (PMA) is commonly used to study NET formation, it bypassing regulatory mechanisms, limiting insights. MethodsWe used the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) to profile chromatin accessibility in neutrophils stimulated in whole blood with PMA and physiologically relevant inflammatory factors (NFs), including TNF-, GM-CSF, fMLP, C5a, and IL-1{beta}, alone and in combination. Chromatin responses were compared across conditions and integrated with publicly available transcriptomic sepsis cohorts. ResultsWe found that NF stimulation induced stimulus specific chromatin accessibility programs distinct from PMA. Individual NFs increased specific transcription factor (TF) motif enrichments in a stimulus dependent manner, with GM-CSF increasing STATs, TNF- increasing NF-{kappa}B, C5a/fMLP increasing AP-1, and Combined with a cooperative response including CEBP. Integration with sepsis transcriptomic datasets revealed that promoter accessibility changes within NF stimulations correspond to transcriptional states associated with sepsis disease severity, highlighting the upstream regulatory programs linked to clinical outcomes. ConclusionsThese findings demonstrate that NF stimulation in whole blood reveals chromatin accessibility programs in neutrophils that correlate with disease severity in sepsis. This approach provides a framework for linking cytokine driven neutrophil regulation to heterogenous inflammatory states in sepsis and other NET-associated diseases.

immunology↗

bioRxiv2024

Understanding Complex Chromatin Dynamics of Primary Human Neutrophils During PMA Induced NETosis

BackgroundPrimary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases such as sepsis, systemic lupus erythematosus, rheumatoid arthritis, and others. MethodsThe study presented here investigates chromatin dynamics during NETosis by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response. ResultsWe find that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 minutes, with many continuing across the 90 minutes assessed in this study. Regulatory regions gaining accessibility are associated with activity of pathways that have been implicated in NOX-dependent NET formation. ConclusionsOur findings enhance the understanding of the chromatin changes underlying NETosis and also identify potential early-acting targets for modulating this process in inflammatory diseases.

immunology↗

bioRxiv2024

Long-read sequencing reveals aberrant fragmentation patterns and origins of circulating DNA in cancer

Circulating cell-free DNA (cfDNA) carries fragmentation patterns that serve as biomarkers of cancer, but standard sequencing approaches miss large portions of the fragment length spectrum. In addition to altered fragmentation patterns, cancer patients often have elevated levels of cfDNA, but the underlying mechanisms are not well understood. To address both questions, we analyzed cancer cases with elevated cfDNA levels using Oxford Nanopore (ONT) sequencing. Long-read ONT sequencing captures the full spectrum of cfDNA fragment lengths and enables cell type inference based on DNA methylation markers. One cohort included cases from several cancer types with elevated cfDNA levels, and a second consisted of patients from a single neuroendocrine cancer study. In each cohort, cases with the highest cfDNA levels showed either hypofragmentation (excess fragments of 1-4 kb) or hyperfragmentation (excess fragments <145 bp). Hypofragmentation reflected blood cell DNA released during delayed sample processing, bearing DNASE1L3-associated hallmarks, while in one cohort we also observed ultra-long fragments (>7.5 kb) lacking these hallmarks and consistent with plasma lysis. By contrast, hyperfragmented samples often had elevated levels of both cancer- and blood-derived DNA, indicating an inflammatory or other system process rather than cancer-specific origin. These findings clarify the distinction between biological and artifactual fragmentation, expand our understanding of cfDNA biology, and highlight long-read sequencing as a powerful tool for biomarker discovery.

cancer biology↗

bioRxiv2023

Novel rapid high-throughput method of NETosis Induction and Inhibition with physiological triggers and inhibitors

Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil Extracellular Trap (NET) formation in response to pathogens is increasingly recognized as an essential rapid innate immune response, but when dysregulated contributes to pathogenesis of sepsis and immunothrombotic disease. Current models of NETosis are limited, routinely employing non-physiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines, do not reflect the complex biology underlying neutrophil activation and NETosis, that occurs in whole-blood. Here we describe a novel, high-throughput ex-vivo whole blood induced NETosis model using combinatorial pooling of native NETosis inducing factors in a more biologically relevant Synthetic-Sepsis model. We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite inter-donor variability, similar sets of pro-inflammatory molecules induced consistent responses across donors. We found at least three biological triggers were necessary to induce NETosis in our system including either TNF- or LT-. To our knowledge, we report the first human ex-vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially, providing novel strategies for disease intervention and treatment. EssentialsO_LINETosis is a vital immune response, but dysregulation leads to disastrous health outcomes. C_LIO_LICurrent NETosis models dont reflect the complex endogenous signaling that occurs in whole blood C_LIO_LINetosis induction stimuli differs between isolated neutrophils and whole blood. C_LIO_LIA minimum of three physiological factors are required to induce NETosis in whole blood. C_LI

immunology↗

bioRxiv2023

Monitoring plasma nucleosome concentrations to measure of disease response and progression in dogs with hematopoietic malignancies.

BackgroundHematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. MethodsCirculating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. ResultsPlasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. ConclusionsPlasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

cancer biology↗