Brownfield, Douglas

Abstract CCAAT/enhancer-binding protein alpha (C/EBPα) is a critical lineage-defining transcription factor and tumor suppressor. Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C/EBPα has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C/EBPα. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C/EBPα-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C/EBPα, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C/EBPα, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C/EBPα, offering a novel strategy for mechanistic investigation and future therapeutic targeting.

Background: Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis characterized by chronic inflammation and immune dysregulation. Single-cell RNA sequencing (scRNA-seq) studies of human atherosclerotic plaques have identified distinct transcriptional programs within plaque-associated immune cells. We sought to determine whether transcriptional features described within these plaque-resident cells can be identified in peripheral blood mononuclear cells (PBMCs). Methods: PBMCs from patients with PAD (n = 4) and healthy controls (HC) (n = 5) were profiled using scRNA-seq using the Parse Biosciences platform, yielding approximately 135,000 high-quality cells. Data analysis was conducted in R, using Seurat for quality control, dimensionality reduction and clustering. Cell-type annotation was performed using Azimuth reference-based mapping. Plaque-associated inflammatory and metabolic gene programs were curated from published human plaque single-cell atlases. Results: Gene expression differences between HC and PAD cells were visualized using UMAP plots. CD14+ mononuclear phagocytes (MNPs) exhibited the most prominent and consistent transcriptional differences between HC and PAD samples (Figure 1). Within CD14+ MNPs, genes involved in lipid handling and macrophage metabolic function demonstrated a higher median percentage of expressing cells in PAD compared with HC (Figure 2). Further analysis of AQP9 and CD36, genes implicated in macrophage metabolic activation and lipid uptake in atherosclerotic plaques, with z-score normalization and subject-level averaging revealed modest but consistent increases in relative expression in PAD across subjects. These findings support the relevance of these circulating transcriptional shifts to PAD pathophysiology. Conclusions: Circulating CD14+ MNPs in PAD have detectable transcriptional features consistent with potential engagement of plaque-associated myeloid activation programs. In PAD, increased expression of CD36, which encodes the immune cell scavenger receptor for oxidized LDL, and AQP9, associated with unstable plaques and the endothelial-to-mesenchymal transition, suggest altered immunometabolism states in circulating MNPs. Although effect sizes are modest, they are consistent across subjects and statistically robust. These findings highlight PBMC scRNA-seq as a minimally invasive approach to interrogate systemic immune programs relevant to PAD pathobiology.

Abstract Metabolic vulnerabilities in cancer have been targeted primarily to suppress tumor growth, but less is known about the metabolic requirements for tumor cell invasion. Here we report that lipid catabolism by cytosolic and lysosomal lipases supports pancreatic cancer cell invasion through both overlapping and distinct functional and metabolic mechanisms. Lysosomal acid lipase (LAL)-dependent lipid droplet catabolism promotes invadopodia formation and stabilization, enabling extracellular matrix degradation. In addition to modulating cellular energetics, lipidomics revealed that lipid droplet catabolism regulates cholesterol and membrane phospholipid levels. Using spatially resolved biosensors and cholesterol imaging, we found that lysosomal lipid catabolism occurs at invadopodia and sustains local ATP and membrane cholesterol. These findings identify spatially organized lipid catabolism as a mechanism that couples local energetics and membrane remodeling during the earliest steps of pancreatic cancer cell invasion.

Summary Physical forces shape cell behavior, yet how they integrate with signaling to control fate and disease remains unclear. The alveolar epithelium is patterned by FGF signaling and mechanical stretch, but how these cues specify AT1 and AT2 cells is poorly understood. Here we show that cell membrane tension (CMT) is a conserved regulator of epithelial fate in mouse and human lungs. CMT drops before differentiation and is spatially patterned, defining where bipotent progenitors acquire AT1 or AT2 identity. Lower CMT enhances FGFR2 endocytosis and ERK signaling to drive AT2 differentiation and permits architectural remodeling that enables stretch-mediated YAP/TAZ nuclear entry for AT1 maturation. β-catenin elevates CMT cell-intrinsically independent of its role in canonical WNT transcription, while embedding, osmotic compression, or fibroblast wrapping elevate CMT extrinsically. Combined intrinsic and extrinsic tension traps alveolar epithelial cells in a KRT8⁺ transitional state seen in fibrotic lungs. Membrane tension thus integrates physical and molecular cues linking morphogenesis to fibrosis. Graphical Abstract In Brief We identify a conserved drop in cell membrane tension (CMT) that gates epithelial fate transitions across mouse and human lungs. In bipotent progenitors, reduced CMT promotes Alveolar Type 2 (AT2) fate via FGFR2 endocytosis and ERK signaling, as well as Alveolar Type 1 (AT1) fate by enabling architectural remodeling required for YAP/TAZ nuclear entry. We further show that intrinsic β-catenin and extrinsic confinement cues, including mesenchymal contact, converge to elevate CMT, restricting differentiation and—in adult AT2s—inducing a KRT8⁺ transitional state associated with fibrosis. Highlights Cell membrane tension drops before differentiation and is required for AT1 and AT2 fate acquisition. Reduced tension enhances FGFR2 endocytosis and ERK signaling to drive AT2 specification. Reduced tension permits architectural remodeling and YAP/TAZ nuclear entry required for AT1 maturation. Intrinsic β-catenin and extrinsic confinement elevate CMT to restrict differentiation and together strongly induce a KRT8⁺ transitional state associated with fibrosis.

Introduction: Atherosclerotic arterial disease is the leading cause of death throughout the world. One of the most advanced forms of atherosclerosis, peripheral arterial disease (PAD), affects over 8.5 million people in the United States and over 230 million globally. This disease begins from an inflammatory lesion, known as the fatty streak, formed by mononuclear phagocytes (MNPs), a broad cell type classification that includes monocytes, dendritic cells, and macrophages. Development of more efficacious treatments for PAD requires that we better understand the cellular and molecular processes by which MNPs form these inflammatory lesions. Methods: To compare gene expression of MNPs, we performed scRNAseq on PBMCs from an initial cohort of 4 patients with PAD, mild claudicants to minor tissue loss (Rutherford 1-5), and 5 healthy individuals, defined as those without any chronic diseases and a hsCRP <2.0 mg/L. We conducted single cell RNA library generation using Parse Bio and sequenced to a depth of 70k reads per cell and downstream bioinformatic and differentially gene expression analysis via R and Seurat. Results: Gene expression differences in healthy versus PAD cells were visualized on a UMAP plot. Aligning our cells to the human PBMC Cell Atlas, we found that the cell type with the greatest transcriptional change (healthy vs. PAD) was CD14 + MNPs. Next, we screened for differentially expressed genes within this CD14 + MNP cluster between healthy and PAD subjects and identified 854 upregulated and 730 downregulated genes (log 2 fold change ≥ 0.25 and p < 0.05, Welch’s t-test). Two of the most significantly upregulated genes in PAD CD14 + MNPs are FAM20A and TMTC1. FAM20A encodes a psuedokinase important in calcium homeostasis as well as bone mineralization and that is upregulated in advanced atherosclerotic plaques. TMTC1 encodes for an endoplasmic reticulum protein also involved in calcium homeostasis, vascular homeostasis, and endothelial cadherin egress. Finally, one gene we found downregulated in PAD is CD83, a molecule implicated in resolution of inflammation, that when downregulated promotes an inflammatory state. Conclusion: Based on these findings we hypothesize that in PAD there exists a distinct transcriptional program along the CD14 + MNP cell type lineage that is pro-inflammatory and that may play a role in disease progression and response to intervention.

Abstract RATIONALE: Alveolar Type 2 (AT2) cells play a critical role in maintaining alveolar function and facilitating repair processes in the lung. Despite recent advancement on transcriptomic mapping of cell identities and AT fate selection, the mechanisms governing AT2 fate plasticity remain elusive. Notably, in AT2-derived pulmonary diseases such as COPD and IPF, disruptions in AT2 differentiation are observed, suggesting alterations may exist in the regulation of fate plasticity. Therefore, a refined model describing the regulation of AT2 fate plasticity is needed, along with an assessment of the model's conservation across murine and human AT2s. METHODS: We performed scRNA-sequencing, ATAC-seq, and bioinformatic analysis on samples across murine lung development and validating findings by immunostaining of embryonic lungs. We conducted organotypic cultures as well as genetically engineered mouse model experiments to assess changes in AT2 fate plasticity in. Further, we established a Mayo Clinic patient lung cell biorepository from patient tissue derived AT2s. In parallel, we performed long read scRNA-sequencing for isoform discovery, organotypic culture and immunostaining in human AT2 organoids, to characterize the presence of similar regulatory mechanisms. RESULTS: Throughout the late embryonic and early perinatal period, we found a window of fate plasticity exists in AT2s whereby they retain the capacity to give rise to AT1s, and that its being closed over time by the bZIP transcription factor C/EBPα, as its genetic deletion in AT2s results in regaining fate plasticity. By ATAC-seq and ChIP-seq, we found C/EBPα binds closely to the Dlk1 locus, a Notch signaling regulator implicated in AT2-to-AT1 conversion that is upregulated following Cebpa deletion. Following adult lung injury, we found C/EBPα downregulation is required for re-accessing AT2 fate plasticity and is likely mediated by the dominant negative C/EBP family member CHOP. Finally, we observe Cebpa loss also activates a “defender” AT2 state enriched in interferon stimulated antipathogenic gene expression that is distinct from its reparative state and propose AT2s can toggle between these states following infection to robustly protect and repair alveoli.Further, we validated the presence of C/EBPα and absence of Dlk1 in human AT2s isolated from healthy adult patients, which persists following in vitro expansion. We conclude that AT2 cells window of fate plasticity is dictated by a molecular circuit involving C/EBPα suppressing and Dlk1. Future works aim to remove C/EBPα in human AT2s to reopen their fate plasticity for cell therapy.

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- &gt;PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract RATIONALE: The alveolar epithelium consists of two cell types (AT2 and AT1) derived from a common Distal Progenitor (DP). Understanding how the epithelium of alveoli functions and becomes differentiated is essential for treatment of lung diseases. One possible regulator of cell fate is cell membrane tension (CMT), a biophysical property measured by the energy required to deform the membrane and known to be an important regulator of cell movement and endocytic processes. AT1 fate selection is largely driven and reinforced by YAP (Yes-associated protein), a mechano-sensitive transcription factor gated from entering the nucleus by the size of nuclear pores, which increases upon stretch. Here, we investigated whether and how CMT regulates the transduction of stretch-dependent YAP signaling and the remodeling into AT1 cells. METHODS: DPs were isolated from E15.5 mice and cultured on Matrigel for use in differentiation experiments. Immunostaining for AT1 and AT2 markers, Phalloidin, DAPI and YAP were used to assess YAP localization and architectural changes. We generated transgenic mice containing a pan-epithelial Cre (TgNkx2.1-Cre), a Cre-dependent driver of ltta (TigreAi140), and a TetOff regulated construct expressing YAP1 (Rosa26teto-Yap1), resulting in constitutive Yap1 expression in Cre-expressing DPs. CMT is measured both with optical trap technology and with the use of FlipTR dye, which uses Fluorescence Lifetime Imaging Microscopy (FLIM) to determine CMT. RESULTS: To demonstrate the role of CMT in timing AT1 fate, we found that CMT lowers in DPs concomitantly with differentiation, and that flattening cells (likely AT1s) have even lower CMT than round cells (likely AT2s). Using organotypic culture, we found that raising CMT with MBCD, PEG, or Matrigel embedding largely stops differentiation of DPs. Surprisingly, even when YAP is overdriven in mice in E15.5 distal progenitors, early precocious AT1 differentiation is not observed, suggesting YAP signaling is not alone sufficient to drive AT1 fate selection in DPs with higher CMT. We reproduced this finding when culturing DPs under high CMT conditions-promoting high YAP/TAZ levels via LATS inhibition was also not sufficient to select for AT1 fate. Finally, we found that high CMT disrupts nuclear YAP localization, nuclear-actin attachment, and cytoskeletal remodeling that is typically when DPs remodel towards AT1 fate. We propose that high baseline CMT in DPs prevents cellular remodeling, specifically that it suppresses the formation of actin-nuclear associations that imbue the nucleus with stretch sensitivity, thereby preventing an increased sensitivity to stretch and thus higher YAP signaling.

Abstract Prevalence of chronic lung diseases, such as pulmonary fibrosis and chronic obstructive pulmonary disease, have been found to increase with age. Pulmonary disease can occur from failure to maintain alveolar type (AT) 1 and alveolar type 2 cells located in the epithelium of the lung. It is currently known that AT2 cells are the facultative stem cells of the lung, and can transform into AT1 cells which make up the structure of the alveoli. This process happens throughout embryonic development, but AT2 cells can also replenish AT1 cell populations post lung injury. This research investigates whether this process that normally occurs during development can also occur in mature AT2 cells, and studies this question using genetically engineered mouse models at a postnatal stage. Using a double inhibition mechanism, we attempted to increase cellular proliferation by administering an agonist and conducted cellular lineage tracing with an SpcCreER lox system via tamoxifen-induced GFP expression. Through lineage labeling, we saw AT1 cells arising from AT2 cells that became plastic, mirroring the plasticity observed in development. Further research is needed to determine if this agonist can be used to epigenetically unlock mature AT2 cells. This result would provide a mechanism to temporarily induce plasticity in a patient’s own AT2 cells in an attempt to regenerate their lungs in disease.