Biology subjects

Fillmore, T. L.

Publications and source records attributed to Fillmore, T. L..

6 recordsLinked to original sources

bioRxiv2026

PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

Acute myeloid leukemia (AML) exhibits substantial transcriptional heterogeneity across differentiation states that influences therapeutic response to BCL2 inhibition with venetoclax. While hematopoietic stem cell (HSC)-like AMLs show high sensitivity to venetoclax and monocytic-like AMLs demonstrate resistance, the therapeutic behavior of leukemias harboring both transcriptional programs remains poorly defined. Analysis of a large AML cohort reveals a distinct patient population exhibiting concurrent HSC- and monocyte-like transcriptional signatures, which we term stem-monocytic AML. Ex vivo drug sensitivity profiling demonstrates that stem-monocytic AMLs exhibit venetoclax resistance comparable to pure monocytic disease, despite expressing HSC-like transcriptional features. Using a leukemia cell line model that recapitulates stem-monocytic AML characteristics, we show through immunophenotyping and single-cell lineage tracing that venetoclax preferentially depletes immature blasts while sparing differentiated monocytic populations. Single-cell transcriptomic and chromatin accessibility analyses identify enrichment of myeloid differentiation transcription factors, particularly PU.1, in resistant populations. A targeted CRISPR knockout screen confirms that PU.1 disruption induces differentiation arrest and enhances venetoclax sensitivity primarily in the immature immunophenotypic compartments. Pharmacologic PU.1 inhibition with the small molecule DB2313 synergizes with venetoclax in both cell line models and primary patient samples. These findings establish stem-monocytic AML as a transcriptionally and functionally distinct subtype and nominate combined PU.1 and BCL2 inhibition as a rational therapeutic strategy for improving venetoclax response in this patient population.

cancer biology↗

bioRxiv2025

Phosphoproteomics unveils the signaling dynamics in neuronal cells stimulated with insulin and insulin-like growth factors

BackgroundGiven the role of metabolism in brain health and disease, investigating the role of insulin (INS) and insulin-like growth factors (IGFs) as potential therapeutic strategies for neurodegenerative diseases is currently underway. Yet, the signaling pathways associated with INS and IGFs in the brain remain elusive, particularly for the human brain. Unraveling these pathways is critical for harnessing their therapeutic potential in metabolism-associated brain disorders. MethodsThis study employed phosphoproteomics using human neuroblastoma cell line, SH-SY5Y, to unravel the signaling network of INS, IGF-1, and IGF-2. Briefly, cells were stimulated at 10 and 60-minutes with the ligands, followed by protein extraction, trypsin digestion, tandem mass tag (TMT)-labelling and phosphopeptides enrichment using an immobilized metal affinity chromatography (IMAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data was processed using R statistical software. Protein annotations were obtained from the UniprotKB database, and pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). ResultsPhosphoproteomics performed at 10 and 60 minutes identified 34358 phosphosites of which 3284 were significant at 10 min and 2374 at 60 min (p.adj <0.05) across all three ligands. Ligand stimulation induced modulation in phosphorylation at both the receptor level and downstream signaling targets at serine (S), threonine (T) and tyrosine (Y) residues. Phosphorylation of LIMA1-Y229, a regulator of actin-cytoskeletal function, was the most prominent Y phosphosite across all ligands. IPA identified Rho GTPase, the molecular switches that regulate actin cytoskeletal dynamics, as the most significantly enriched pathway, with IGF-1 predominantly driving phosphorylation of Rho GTPase effectors such as Rho Guanine nucleotide exchange factors (ARHGEFs), Rho GTPase activating proteins (ARHGAPs) and CDC42. Myocardin related transcription factor A (MRTFA), a transcriptional target of Rho GTPase, was increased in ligand-stimulated cells at 10 min, and inhibition of Rho/SRF pathway by CCG1423 prevents nuclear localization of IGF-1-induced MRTFA. ConclusionsThis study demonstrates that INS, IGF-1 and IGF-2 regulate Rho GTPase and MRTFA activation, thereby contributing to the control of actin cytoskeletal dynamics in neuronal cells. Given the role of INS and IGFs in neuronal survival and neurodegenerative conditions, elucidating the mechanisms is of critical importance, as it offers insights into disease pathogenesis and potential therapeutic targets.

biochemistry↗

bioRxiv2025

Sorted-Cell Proteomics Reveals an AT1-Associated Epithelial Cornification Phenotype and Suggests Endothelial Redox Imbalance in Human Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease characterized by inflammation and scarring leading to long-term tissue damage. Previous whole tissue proteomics identified BPD-specific proteome changes and cell type shifts. Little is known about the proteome-level changes within specific cell populations in disease. Here, we sorted epithelial (EPI) and endothelial (ENDO) cell populations based on their differential surface markers from normal and BPD human lungs. Using a low-input compatible sample preparation method (MicroPOT), proteins were extracted and digested into peptides and subjected to Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) proteome analysis. Of the 4,970 proteins detected, 293 were modulated in abundance or detection in the EPI population and 422 were modulated in ENDO cells. Modulation of proteins associated with actin-cytoskeletal function such as SCEL, LMO7, and TBA1B were observed in the BPD EPIs. Using confocal imaging and analysis, we validated the presence of aberrant multilayer-like structures comprising SCEL and LMO7, known to be associated with epidermal cornification, in the human BPD lung. This is the first report of accumulation of cornification-associated proteins in BPD. Their localization in the alveolar parenchyma, primarily associated with alveolar type 1 (AT1) cells, suggests a role in the BPD post-injury response. In the ENDOs, redox balance and mitochondrial function pathways were modulated. Alternative mRNA splicing and cell proliferative functions were elevated in both populations suggesting potential dysregulation of cell progenitor fate. This study characterized the proteome of epithelial and endothelial cells from the BPD lung for the first time, identifying population-specific changes in BPD pathogenesis. New & NoteworthyThe study is the first to perform proteomics on sorted pulmonary epithelial and endothelial populations from BPD and age-matched control human donors. We identified an increase in cornification-associated proteins in BPD (e.g., SCEL and LMO7), and evidenced the presence of multilayered structures unique to BPD alveolar regions, associated with alveolar type 1 (AT1) cells. By changing the nature and/or biomechanical properties of the epithelium, these structures may alter the behavior of other alveolar cell types potentially contributing to the arrested alveolarization observed in BPD. Lastly, our data suggest the modulation of cell proliferation and redox homeostasis in BPD providing potential mechanisms for the reduced vascular growth associated with BPD.

biochemistry↗

bioRxiv2023

Coupling microdroplet-based sample preparation, multiplexed isobaric labeling, and nanoflow peptide fractionation for deep proteome profiling of tissue microenvironment

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity at a cell-type-specific level to better understand and predict the function of complex biological systems, such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverages due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (Microdroplet Processing in One pot for Trace Samples), the multiplexed isobaric labelling, and a nanoflow peptide fractionation approach. The integrated workflow allowed to maximize proteome coverage of laser-isolated tissue samples containing nanogram proteins. We demonstrated the deep spatial proteomics can quantify more than 5,000 unique proteins from a small-sized human pancreatic tissue pixel ([~]60,000 {micro}m2) and reveal unique islet microenvironments.

biochemistry↗

bioRxiv2023

Pumping Iron: A Multi-omics Analysis of Two Extremophilic Algae Reveals Mechanisms of Iron Economy

Marine algae are responsible for half of the worlds primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hyper-saline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multi-omics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a novel family of siderophore-iron uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, an LHCA-related protein found also in other chlorophytes. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms. Significance StatementDespite their small size, microalgae play a huge role in CO2 uptake via photosynthesis, and represent an important target for climate crisis mitigation efforts. Most photosynthesis proteins require iron as a co-factor so that insufficient iron often limits algal CO2 sequestration. With this in mind, we examined a genus of microalgae called Dunaliella that is exceptionally well-adapted to low iron environments. We produced complete genomes, transcriptomes, and proteomes for two species of Dunaliella that hail from radically different environments: one from coastal ocean waters and the other from salt flats. We identified dozens of genes and multiple, complementary strategies that both species utilize for iron-uptake and management that explain Dunaliellas remarkable ability to thrive on low iron.

systems biology↗

bioRxiv2018

Rapidly Assessing the Quality of Targeted Proteomics Experiments Through Monitoring Stable-isotope Labeled Standards

Targeted proteomics experiments based on selected reaction monitoring (SRM) have gained wide adoption in clinical biomarker, cellular modeling and numerous other biological experiments due to their highly accurate and reproducible quantification. The quantitative accuracy in targeted proteomics experiments is reliant on the stable-isotope, heavy-labeled peptide standards which are spiked into a sample and used as a reference when calculating the abundance of endogenous peptides. Therefore, the quality of measurement for these standards is a critical factor in determining whether data acquisition was successful. With improved MS instrumentation that enables the monitoring of hundreds of peptides in hundreds to thousands of samples, quality assessment is increasingly important and cannot be performed manually. We present Q4SRM, a software tool that rapidly checks the signal from all heavy labeled peptides and flags those that fail quality control metrics. Using four metrics, the tool detects problems both with individual SRM transitions and the collective group of transitions that monitor a single peptide. The programs speed enables its use at the point of data acquisition and can be ideally run immediately upon the completion of an LC-SRM-MS analysis.

bioinformatics↗