Biology subjects

Tackett, A. J.

Publications and source records attributed to Tackett, A. J..

10 recordsLinked to original sources

bioRxiv2025

YY1 relieves p300 autoinhibition to promote histone acetylation in advanced prostate cancer, enhancing the oncogenic signaling of Androgen Receptor Splice Variant 7

Genetic and epigenetic aberrations often act in concert to establish oncogenic transcriptomic programs in aggressive cancers. For example, the development of castration-resistant prostate cancer (CRPC), an advanced prostate cancer form, is closely associated with over-expression and/or hyper-activation of transcription factors (TFs) such as Androgen Receptor (AR) and Yin Yang 1 (YY1), as well as p300, a prominent histone acetyltransferase. How exactly these cancer-related lesions are coordinated to generate a malignant cell state remains elusive. Here, we demonstrate that YY1, which is frequently over-expressed in advanced prostate cancers, allosterically stimulates the acetyltransferase activity of p300 in cis, leading to the globally elevated acetylation of histone H3 lysine 18 and 27 (H3K18ac and H3K27ac). Mechanistically, YY1s N-terminal activation domain (AD) directly interacts with p300s TAZ2 domain, relieving the autoinhibition of p300 to facilitate substrate acetylation. Our integrated genome-wide mapping and transcriptomic studies reveal significant co-localization of genomic binding sites of YY1, androgen receptor splice variant 7 (AR-V7, a constitutively active form of AR) and p300 in CRPC cells, where the YY1-mediated p300 activation and resultant histone acetylation increases promote the oncogenic gene-expression programs downstream of YY1 and AR/AR-V7. Both in vitro and in vivo functional assays demonstrate a critical requirement of the above signaling for the advanced disease progression and drug resistance seen in CRPC. Altogether, this study uncovers that YY1 acts to alleviate p300s autoinhibition at target genes co-bound by oncogenic TFs (YY1 and/or AR/AR-V7) in CRPC, thereby sustaining tumorigenicity. Additionally, the blockade of YY1-mediated gene activation resensitizes CRPC to treatment to the clinic anti-AR agent (enzalutamide), which provides a rationale for overcoming the therapeutic resistance often seen in advanced prostate cancers.

cancer biology↗

bioRxiv2025

Monensin and Its Analogs Exhibit Activity Against Breast Cancer Stem-Like Cells in an Organoid Model

Monensin (MON) is a polyether ionophore antibiotic of natural origin and is an FDA-approved drug for veterinary use. Recent studies have highlighted its potential anti-cancer activity in various in vitro and in vivo models. In this study, we evaluated the anti-breast cancer activity of MON and 37 synthetic analog compounds using cell monolayer and organoid models. Through a mini-ring cell viability assay, several compounds were identified that were more potent and selective against breast cancer cells compared to non-cancerous cells, surpassing the activity of parent MON. MON and these compounds induced significant DNA fragmentation, reduced cell migration, and downregulated SOX2 expression. Furthermore, MON and the most potent analog, compound 12, reduced the percentage of CD44+/CD24-/low stem-like cells and diminished cell self-renewal properties. Proteomics analyses revealed that several pathways, including extracellular matrix organization, were significantly dysregulated by MON and compound 12 in breast cancer cells. Among these, TIMP2, a protein associated with the suppression of tumor growth and metastasis, was identified as one of the most prominently upregulated proteins by MON and compound 12 in MDA-MB-231 cells. This finding was also validated in other breast cancer and melanoma cell lines. To simulate breast cancer metastasis to the brain, a human Hybrid Organoid System: Tumor in Brain Organoid (HOSTBO) model was developed. MON and compound 12 significantly reduced Ki-67 expression within the HOSTBOs, and compound 12 significantly downregulated SOX2 expression. Collectively, MON and compound 12 significantly reduced the proliferation of breast cancer stem-like cells in the organoid models, inhibited their migration, and dysregulated markers associated with stemness, demonstrating their potential as anti-metastatic agents and warranting further clinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/663311v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@388862org.highwire.dtl.DTLVardef@182072dorg.highwire.dtl.DTLVardef@1a55072org.highwire.dtl.DTLVardef@447150_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Dynamic global acetylation remodeling during the yeast heat shock response

All organisms experience stress and must rapidly respond to changing conditions. Thus, cells have evolved sophisticated rapid-response mechanisms such as post-translational protein modification to rapidly and reversibly modulate protein activity. One such post-translational modification is reversible lysine acetylation, where proteomic studies have identified thousands of acetylated proteins across diverse organisms. While the sheer size of the acetylome is striking, the function of acetylation for the vast majority of proteins remains largely obscure. Here, we show that global acetylation plays a previously unappreciated role in the heat shock response of Saccharomyces cerevisiae. We find that dysregulated acetylation renders cells heat sensitive, and moreover, that the acetylome is globally remodeled during heat shock over time. Using quantitative acetyl-proteomics, we identified [~]400 high-confidence acetyl marks across [~]200 proteins that significantly change in acetylation when cells are shifted to elevated temperature. Proteins with significant changes in lysine acetylation during heat shock strongly overlap with genes induced or repressed by stress. Thus, we hypothesize that protein acetylation augments the heat shock response by activating induced proteins and inactivating repressed proteins. Intriguingly, we find nearly 40 proteins with at least two acetyl marks that significantly change in the opposite directions. These proteins are strongly enriched for chaperones and ribosomal proteins, suggesting that these two key processes are coordinately regulated by protein acetylation during heat shock. Moreover, we hypothesize that the same type of activating and inactivating marks that exist on histones may be a general feature of proteins regulated by acetylation. Overall, this work has identified a new layer of post-translational regulation that likely augments the classic heat shock response.

genomics↗

bioRxiv2023

Increased response to immune checkpoint inhibitors with dietary methionine restriction

Dietary methionine restriction, defined as reduction of methionine intake by around 80%, reproducibly decreases tumor growth and synergizes with cancer therapies. Here, we combined dietary methionine restriction with immune checkpoint inhibitors in a model of colon adenocarcinoma. In vitro, we observed that methionine restriction increased the expression of MHC-I and PD-L1 in both mouse and human colorectal cancer cells. We also saw an increase in the gene expression of STING, a known inducer of type I interferon signaling. Inhibition of the cGAS-STING pathway, pharmacologically or with siRNA, blunted the increase in MHC-I and PD-L1 surface and gene expression following methionine restriction. PD-L1 expression was also This indicated that the cGAS-STING pathway in particular, and interferon in general, is playing a role in the immune response to methionine restriction. We then combined dietary methionine restriction with immune checkpoint inhibitors targeted against CTLA-4 and PD-1 in a MC38 colorectal cancer tumor model in C57BL/6 mice. The combination treatment was five times more effective at reducing tumor size than immune checkpoint inhibition alone in males. We noted sex differences in the response to dietary methionine restriction for the MC38 tumor model in C57BL/6 mice. Finally, we observed an increase in PD-L1 protein expression in MC38 tumors from animals who were fed a methionine-restricted diet. Furthermore, the distribution of CD8 staining changed from mostly peripheric in the controls, to intratumoral in the methionine-restricted tumors. MHC-I, which has a high basal expression in MC38 cells, was highly expressed in all tumors. These results indicate that methionine restriction improves the response to immune checkpoint inhibitors in mice, and that this improvement is associated with the cGAS-STING pathway and interferon signaling.

cancer biology↗

bioRxiv2023

Characterization of methionine dependence in melanoma cells

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of s-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

molecular biology↗

bioRxiv2022

Proteogenomics analysis to identify acquired resistance-specific alterations in melanoma PDXs on MAPKi therapy

Therapeutic approaches to treat melanoma include small molecule drugs that target activating protein mutations in pro-growth signaling pathways like the MAPK pathway. While beneficial to the approximately 50% of patients with activating BRAFV600 mutation, mono- and combination therapy with MAPK inhibitors is ultimately associated with acquired resistance. To better characterize the mechanisms of MAPK inhibitor resistance in melanoma, we utilize patient-derived xenografts and apply proteogenomic approaches leveraging genomic, transcriptomic, and proteomic technologies that permit the identification of resistance-specific alterations and therapeutic vulnerabilities. A specific challenge for proteogenomic applications comes at the level of data curation to enable multi-omics data integration. Here, we present a proteogenomic approach that uses custom curated databases to identify unique resistance-specific alternations in melanoma PDX models of acquired MAPK inhibitor resistance. We demonstrate this approach with a NRASQ61L melanoma PDX model from which resistant tumors were developed following treatment with a MEK inhibitor. Our multi-omics strategy addresses current challenges in bioinformatics by leveraging development of custom curated proteogenomics databases derived from individual resistant melanoma that evolves following MEK inhibitor treatment and is scalable to comprehensively characterize acquired MAPK inhibitor resistance across patient-specific models and genomic subtypes of melanoma.

bioinformatics↗

bioRxiv2020

Methionine stress induces ferroptosis in methionine dependent cancer cells

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood, except for the observation that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We found that replacing methionine with homocysteine generally induced hypomethylation in gene promoters. We isolated nuclear proteins and submitted it for tandem mass tag (TMT) proteomics. This analysis revealed that several proteins involved in the mitochondrial integrated stress response (ISR) were upregulated in response to the replacement of methionine to homocysteine in both cell lines, but to a much greater degree in the methionine dependent cell line. Consistent with the ISR signature, a proteomic analysis of a subcellular fraction enriched for mitochondrial content revealed a strong enrichment for proteins involved in oxidative phosphorylation. Analysis of cellular bioenergetics confirmed that homocysteine induces a decrease in ATP production from oxidative phosphorylation and glycolysis, but to a similar extent in methionine dependent and methionine independent cells. The mitochondrial integrated stress response shared a signature with ferroptosis. Methionine dependent cells displayed a strong ferroptotic signature, which was decreased by half in methionine independent cells. Consistent with ferroptosis, lipid peroxidation was significantly increased in methionine independent cells grown in homocysteine, and viability could be rescued partially but significantly with the inhibitor ferrostatin. Therefore, we propose that methionine stress induces ferroptotic cell death in methionine dependent cancer cells.

cancer biology↗

bioRxiv2020

Inhibition of tryptophan-2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells

Aberrant expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy and immune response in gliomas in part through kynurenine (KYN)-mediated activation of the aryl hydrocarbon receptor (AhR). In the current study, we investigated the hypothesis that TDO activation in gliomas has a broad impact upon genome maintenance - promoting tolerance of replication stress (RS) and repair of DNA damage. We report that inhibition of TDO activity attenuated recovery from hydroxyurea (HU)-induced RS and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU), as fork progress was impeded when TDO-deficient glioma cells were treated with BCNU. Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to treatment with BCNU, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced - indicative of increased fork collapse. Restoration of KYN levels protected against some replication-associated effects of BCNU. Inhibition of TDO activity had a strong anti-proliferative effect on glioma-derived cells - enhancing the cytotoxic effects of BCNU. Analysis of results obtained using quantitative proteomics revealed TDO-dependent changes in several signaling pathways - including down-regulation of DNA repair factors and sirtuin signaling. Consistent with these observations, inhibition of TDO diminished SIRT7 recruitment to chromatin, which increased histone H3K18 acetylation - a key mark involved in 53BP1 recruitment to sites of DNA damage. Cells lacking TDO activity exhibited defective recruitment of 53BP1 to gH2AX foci, which corresponded with delayed repair of BCNU-induced DNA breaks. Addition of exogenous KYN increased the rate of break repair. The discovery that TDO activity modulates sensitivity to DNA damage by fueling SIRT7/53BP1 localization to chromatin and repair of BCNU-induced DNA damage highlights the potential for tumor-specific metabolic changes to influence genome stability and may have implications for glioma biology and treatment strategies.

cancer biology↗

bioRxiv2019

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome using Tandem Mass Tags

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance, but also post-translational modifications (PTMs) that regulate protein activity. We implemented, and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine timepoints. We report that basic pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS3. We quantified 2,275 proteins across 3 biological replicates, and found that differential expression peaked near 90 minutes following heat shock (with 868 differentially expressed proteins at 5% FDR). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

genomics↗

bioRxiv2018

Metaproteomics Reveals Potential Mechanisms by which Dietary Resistant Starch Supplementation Attenuates Chronic Kidney Disease Progression in Rats

BackgroundResistant starch is a prebiotic metabolized by the gut bacteria. It has been shown to attenuate chronic kidney disease (CKD) progression in rats. Previous studies employed taxonomic analysis using 16S rRNA sequencing and untargeted metabolomics profiling. Here we expand these studies by metaproteomics, gaining new insight into the host-microbiome interaction.\n\nMethodsDifferences between cecum contents in CKD rats fed a diet containing resistant starch with those fed a diet containing digestible starch were examined by comparative metaproteomics analysis. Taxonomic information was obtained using unique protein sequences. Our methodology results in quantitative data covering both host and bacterial proteins.\n\nResults5,834 proteins were quantified, with 947 proteins originating from the host organism. Taxonomic information derived from metaproteomics data surpassed previous 16S RNA analysis, and reached species resolutions for moderately abundant taxonomic groups. In particular, the Ruminococcaceae family becomes well resolved - with butyrate producers and amylolytic species such as R. bromii clearly visible and significantly higher while fibrolytic species such as R. flavefaciens are significantly lower with resistant starch feeding. The observed changes in protein patterns are consistent with fiber-associated improvement in CKD phenotype. Several known host CKD-associated proteins and biomarkers of impaired kidney function were significantly reduced with resistant starch supplementation. Data are available via ProteomeXchange with identifier PXD008845.\n\nConclusions- Metaproteomics analysis of cecum contents of CKD rats with and without resistant starch supplementation reveals changes within gut microbiota at unprecedented resolution, providing both functional and taxonomic information. Proteins and organisms differentially abundant with RS supplementation point toward a shift from mucin degraders to butyrate producers.

systems biology↗