Biology subjects

Schwartz, M. A.

Publications and source records attributed to Schwartz, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Discovery of Tcf7 regulators with clonally-resolved CRISPR screens identifies Trim28 as a mediator of CD8 T cell differentiation in tumors

Stem-like TCF7+ CD8 T cells sustain anti-tumor responses and support immune checkpoint blockade. We systematically identified regulators of this cell state using genome-wide CRISPR screens in primary T cells in vitro. Using random barcodes to link clonal relationships with guide identity and transcriptional states in single cells, we inferred differentiation trajectories and differentiation rates of CD8 T cells in tumors, while mitigating confounding clonal bias. We found that Trim28-deficient T cells in tumors were enriched in the TCF7+ cell state, depleted in cycling and terminal effector states, and uniquely generated a tissue-resident memory (TRM)-like state with increased chromatin accessibility at known TRM loci as well as repeat elements. Despite the increase in TCF7+ CD8 T cells, loss of Trim28 did not improve tumor control, likely because of reduced effector differentiation, highlighting the need for tuning the balance and dynamics of stem-like versus effector states for effective tumor clearance.

immunology↗

bioRxiv2026

A community machine learning challenge to predict the effects of gene perturbations on T cell differentiation for cancer immunotherapy

Perturbations of genes with functional importance in T cells could be used to change the distribution of CD8 T cell states to enhance anti-tumor functions for cancer immunotherapies. We launched a world-wide computational challenge to predict the effects of gene perturbations and to devise objective functions for prioritizing gene perturbations that lead to desired T-cell state distributions. We supported the challenge by generating a single-cell Perturb-seq dataset profiling the effect of knocking out 73 individual expert-defined genes in T cells transferred into a mouse melanoma model. We compared the top algorithms developed by participants, and found that performance was primarily determined by the prior data used for gene feature representation, with perturbational data derived features, proving most effective. Experimental validation of the top 61 genes nominated by the algorithms revealed that perturbation of Ndufv2 and Dimt1 reached the defined objective and biased T cell differentiation toward desired states.

bioinformatics↗

bioRxiv2026

Metabolic control of smooth muscle cell phenotype switching in atherosclerosis

The loss of smooth muscle cell (SMC) contractile phenotype contributes to various diseases including atherosclerosis. However, its metabolic basis is not entirely elucidated. Since the transforming growth factor beta (TGF{beta}) signaling is among principal regulators of SMC contractility, we studied metabolic regulation of TGF{beta} signaling in SMCs in vitro and atherosclerotic mouse models and human lesions. We found that TGF{beta} induced Ac-CoA synthetase 2 (ACSS2)-dependent Ac-CoA production, by suppressing pyruvate dehydrogenase kinase 4 (PDK4). This stabilized R-SMADs and TGF{beta} receptor 1, preserving SMC contractile phenotype. SMC-specific PDK4 knockout mimicked the effect of TGF{beta} signaling both metabolically and phenotypically, increasing glucose-derived synthesis of Ac-CoA and SMC contractile phenotype. SMC-specific Pdk4 knockout in ApoE knockout mice reduced atherosclerosis. Furthermore, human specimens demonstrated a strong correlation between PDK4 level and atherosclerosis severity. These findings indicate that continuous TGF{beta} signaling, critical to the maintenance of the normal SMC contractile state and is regulated by PDK4 and carbohydrate metabolism. TeaserReducing PDK4 metabolically restricts aortic plaque growth via TGF{beta}-dependent SMC contractility.

cell biology↗

bioRxiv2026

Deletion of endothelial KLF4 as a model for preeclampsia

Preeclampsia (PE), or gestational hypertension, affects around 5% of pregnancies and leads to approximately 70,000 maternal and 500,000 fetal deaths per year worldwide, with increased cardiovascular and metabolic disease in survivors. PE is associated with elevated circulating levels of the alternative splice isoform of VEGF receptor 1 (sFlt1), defects in placental vasculature, kidney damage and, in severe disease, fetal growth restriction. Current mouse models induce PE via direct expression of sFlt1 or elevation of blood pressure, which bypass the natural risk factors for human disease, such as age, obesity, hypertension and diabetes. These risk factors have in common reduced expression of Kruppel-like factors 2 and 4 (KLF2/4), the endothelial transcription factors that protect against cardiovascular disease. We now report that inducible deletion of KLF4 in maternal endothelium (KLF4iECKO) results in gestational hypertension, elevated sFlt1, defective placental vasculature, kidney damage and fetal growth restriction. KLF4iECKO may thus serve as a mouse PE model suitable for mechanistic analysis and screening of treatments that address upstream risk factors.

cell biology↗

bioRxiv2026

A transcriptional code controlling fluid shear stress-induced gene expression

The formation and health of the vascular system is dependent on fluid shear stress (FSS), a hemodynamic force exerted onto endothelium by flowing blood. FSS strongly induces the endothelial expression of Kruppel-like factor 2 (KLF2), an atheroprotective TF essential for vascular development and homeostasis. Despite its early and crucial role in the cascade of cardiovascular events triggered by FSS, the transcriptional mechanisms by which FSS regulates KLF2 expression remain unclear, although they are known to involve the widely expressed MEF2 proteins. Here, we identified and characterized two FSS-dependent enhancers for KLF2 which collectively recapitulate endogenous endothelial KLF2 expression, and determined the TFs contributing to their regulation. This analysis identified an essential and precisely spaced MEF2-TBP double motif also shared by the FSS-sensitive KLF2 promoter. MEF-TBP double motifs are extremely rare across the genome but were also found within regulatory elements of three other FSS-induced KLF genes, including KLF4. Although normally part of the basal transcriptional machinery, TBP specifically bound all KLF elements at the MEF-TBP double motifs in a FSS-dependent manner. Collectively, this work demonstrates a specific and targetable requirement for combined MEF2-TBP binding during FSS-induced gene activation. Significance statementBlood flow induces a force known as fluid shear stress (FSS) which is required for vascular development and for the health of the mature arterial system. One of the first endothelial responses to FSS is the induction of Kruppel-like transcription factors (KLFs). However, the mechanisms by which FSS activates KLF gene expression are incompletely understood. In this paper, we characterized all regulatory elements involved in driving FSS-induced expression of KLF2. This identified an essential MEF2-TBP double motif that was extremely rare across the genome, yet found within regulatory elements for multiple FSS-responsive KLF genes including KLF2 and KLF4. This MEF2- and TBP-bound motif therefore enables blood flow to specifically activate the cascade of cardiovascular responses necessary for atheroprotective gene expression.

developmental biology↗

bioRxiv2026

CRISPR screens identify targets to rescue age-related T cell dysfunction in cancer

Immune aging is being increasingly recognized as a critical barrier to effective cancer immunotherapy, as the aged tumor microenvironment (TME) drives T cell dysfunction and impairs immune control of cancer1-4. However, the key molecular drivers of this process as well as potential targets to rescue T cell dysfunction in aged tumors remain incompletely understood. Therefore, we performed in vivo single-cell CRISPR screens in CD8 T cells within aged tumors and tumor-draining lymph nodes (tdLNs). We identified Dusp5 and Zfp219 as key regulators of T cell persistence and effector differentiation in aged hosts. Loss of Dusp5, a negative regulator of ERK signaling, increased ERK1/2 phosphorylation and enhanced T cell proliferation in both young and aged tumors. In contrast, loss of Zfp219, a transcriptional repressor, induced epigenetic reprogramming of cytotoxic gene programs, thereby increasing granzyme secretion and enhancing antitumor immunity. Moreover, expression of the human ortholog gene ZNF219 is increased within intratumoral CD8 T cells in older cancer patients. High ZNF219 expression correlates with poorer survival following immune checkpoint blockade (ICB) and reduces persistence of human intratumoral T cells. Notably, Zfp219 ablation synergized with anti-PD-1 blockade in mice to expand effector-like CD8 T cells, leading to significantly enhanced anti-tumor immunity and tumor clearance in aged hosts. Together, these findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction and as potential therapeutic targets to rejuvenate T cell antitumor immunity in older cancer patients.

immunology↗

bioRxiv2025

RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

Immune checkpoint blockade has transformed cancer therapy, yet many patients fail to respond, and few new targets have emerged beyond PD-1 and CTLA-4. Alternative splicing dramatically diversifies the T cell proteome, but the functional roles of most isoforms remain unknown. Here we constructed the first single-cell splicing atlas of human CD8 T cells, capturing dynamic isoform programs across activation and subset differentiation. This revealed distinct splicing footprints that refine conventional transcriptomic states and highlight receptor families with isoform-level regulation. To functionally interrogate these candidates, we developed SpliceSeek, a CRISPR-based pooled screening platform that perturbs splice sites to redirect isoform usage. Using SpliceSeek, we uncovered isoform-specific immune checkpoints whose perturbation enhanced effector function and tumor control, including the LRRN3-203 isoform, which augmented cytokine secretion and antitumor immunity in mice models. Together, our results establish alternative splicing as a targetable layer of immune regulation and demonstrate the potential of isoform-focused screening to expand the landscape of cancer immunotherapy.

immunology↗

bioRxiv2025

Cannabinoid Receptor 2 Activating Antibodies: A Promising Therapeutic Strategy for Macrophage-Driven Fibro-Inflammatory Diseases

Fibrotic diseases, often fueled by chronic inflammation, represent a major unmet medical need. A critical driver of this process is the activation of macrophages, which secrete pro-inflammatory cytokines and chemokines, leading to immune cell recruitment and collagen deposition by myofibroblasts. Agonizing the cannabinoid receptor 2 (CB2), expressed on macrophages, offers a potential therapeutic avenue to suppress this activation. Despite widespread interest in CB2 modulation, challenges remain in the development of small molecule agonists, including insufficient specificity and poor drug-like properties. Antibodies are highly specific with favorable pharmacokinetics and bioavailability, but GPCR agonist antibodies have been difficult to discover. Here we present first-in-class CB2 agonist antibodies, AB120 and AB150. These antibodies are CB2 specific, G-alpha biased, and effectively decrease the expression of macrophage activation markers and key pro-inflammatory cytokines, including IL-6, IL-1{beta} and TNF-. Furthermore, using an ex vivo human precision-cut liver slice (hPCLS) model of liver fibrosis, we observe that treatment with these CB2 agonist antibodies not only reduces inflammatory markers but also decreases collagen expression, indicating a potential to halt or reverse fibrosis. These novel CB2-specific agonist antibodies hold promise as therapeutics for a range of fibrotic and inflammatory conditions driven by chronic macrophage activation and demonstrate the potential of GPCR antibody agonists to drug this challenging class of targets. Significance StatementFibrotic diseases driven by chronic inflammation lack effective treatments. CB2 agonism modulates macrophage activation to reduce pro-inflammatory cytokines suggesting a novel avenue of treatment, but small molecule CB2 agonists have faced substantial limitations due to off-target effects. We introduce two novel CB2-specific agonist antibodies with potent anti-inflammatory and anti-fibrotic effects in vitro and in a human ex vivo liver fibrosis model. These antibodies provide a promising new therapeutic strategy and highlight the advantages of antibodies as GPCR agonist drugs.

pharmacology and toxicology↗

bioRxiv2025

Latrophilin-2 Orchestrates Endothelial Flow-Mediated Smad1/5 Activation via Endoglin and Shank3

Fluid shear stress (FSS) from blood flow critically determines vascular morphology and function. FSS activates the Smad1/5 pathway maximally at physiological FSS through the BMP9/10 receptors Alk1 and Endoglin to promote vascular stability. We now report that the adhesion G protein-coupled receptor Latrophilin-2 (Lphn2), which mediates flow activation of the canonical junctional complex, is required for flow-mediated Smad1/5 activation in endothelial cells. Lphn2 physically associates with Endoglin and is required for Smad1/5 activation by FSS but not soluble BMP9. This regulation is independent of G-proteins but requires Lphn2s C-terminal PDZ binding motif. Endothelial-specific Lphn2 knockout reduces Smad1/5 activation in mice. In zebrafish embryos, loss of flow or Lphn2 knockout similarly reduced endothelial Smad1/5 activation, with no additive effect, indicating that Lphn2 mediates flow-induced activation in vivo. APEX2 proximity labeling revealed that the PDZ domain-containing scaffold protein Shank3 associated with Lphn2 under flow. Shank3 knockdown blocked flow-induced Smad1/5 activation in vitro and Shank3 global knockout mice exhibited reduced endothelial nuclear Smad1/5 and increased susceptibility to arteriovenous malformations. Lphn2 thus mediates flow-induced Smad1/5 activation and vessel stabilization through the scaffold protein Shank3, independently of G proteins. Together with the accompanying paper on Notch1, we conclude that Lphn2 confers FSS sensitivity to three of the major FSS pathways via distinct mechanisms.

cell biology↗

bioRxiv2025

Latrophilin mediates flow activation of Notch to control vascular endothelial phenotype and atherosclerosis

Fluid shear stress (FSS) is a major determinant of endothelial cell (EC) phenotype, with physio-logical laminar FSS promoting arterial identity and stability, and disturbed FSS promoting atherosclerosis. We previously identified the adhesion G protein-coupled receptor (GPCR) Latrophilin-2 (LPHN2) as a junctional protein required for FSS activation of the PECAM1/VE-cadherin/VEGFR2/PlexinD1 junctional mechanosensory pathway, which regulates EC alignment to laminar blood flow and promotes EC inflammatory activation and atherogenesis under disturbed flow. Here, we demonstrate that Lphn2 endothelial cell-specific knockout hyperlipidemic mice develop larger atherosclerotic plaques than controls--opposite from the previously reported Pecam1 knockout model. Transcriptomic analysis revealed that LPHN2 contributes more than half of flow-responsive gene expression and suppresses pro-inflammatory pathways. Critically, LPHN2 is essential for FSS-induced Notch1 activation to suppress inflammation, via a GPCR-independent mechanism. Active Notch correlates with physical association of LPHN2 with Notch1 activator, {gamma}-secretase, and Notch1 in response to flow. Both the Notch1 intracellular domain (NICD) and transmembrane domain (NTMD) contribute to the anti-inflammatory gene program, with the non-canonical NTMD signaling specifically suppressing the pro-inflammatory YAP pathway. Together with the accompanying paper showing that LPHN2 also mediates flow activation of the Smad1/5 pathway, these data identify LPHN2 as a central mediator of EC flow responses through multiple independent mechanisms in vascular biology and disease.

molecular biology↗

bioRxiv2024

Polycomb Repressive Complex 2 promotes atherosclerotic plaque vulnerability

Key findings1. PRC2 regulates EC shear stress responses. 2. PRC2 governs Klf2/4 suppression downstream of Pcdhg. 3. High PRC2 in ASCVD-prone arterial regions suppresses Klf2/4 to promote ASCVD. 4. Athero-protective Klf2/4 induction upon PRC2 inhibition requires Notch signaling. 5. Tazemetostat, an FDA approved PRC2 inhibitor, slows ASCVD progression and improves markers of plaque stability. Atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality worldwide, is driven by endothelial cell inflammatory activation and counter-balanced by anti-inflammatory transcription factors Klf2 and Klf4 (Klf2/4). Understanding vascular endothelial inflammation to develop effective treatments is thus essential. Here, we identify, Polycomb Repressive Complex (PRC) 2, which blocks gene transcription by trimethylating histone3 Lysine27 in gene promoter/enhancers, as a potent, therapeutically targetable determinant of vascular inflammation and ASCVD progression. Bioinformatics identified PRC2 as a direct suppressor of Klf2/4 transcription. Klf2/4 transcription requires Notch signaling, which reverses PRC2 modification of Klf2/4 promoter/enhancers. PRC2 activity is elevated in human ASCVD endothelium. Treating mice with established ASCVD with tazemetostat, an FDA approved pharmacological inhibitor of PRC2, slowed plaque progression by 50% and drastically improved markers of plaque stability. This study elucidates a fundamental mechanism of vascular inflammation, thus identifying a potential method for treating ASCVD and possibly other vascular inflammatory diseases.

cell biology↗

bioRxiv2024

DNA nanodevice for analysis of force-activated protein extension and interactions

AbstractForce-induced changes in protein structure and function mediate cellular responses to mechanical stresses. Existing methods to study protein conformation under mechanical force are incompatible with biochemical and structural analysis. Taking advantage of DNA nanotechnology, including the well-defined geometry of DNA origami and programmable mechanics of DNA hairpins, we built a nanodevice to apply controlled forces to proteins. This device was used to study the R1-R2 segment of the talin1 rod domain as a model protein, which comprises two alpha-helical bundles that reversibly unfold under tension to expose binding sites for the cytoskeletal protein vinculin. Electron microscopy confirmed tension-dependent protein extension while biochemical analysis demonstrated enhanced vinculin binding under tension. The device could also be used in pull down assays with cell lysates, which identified filamins as novel tension-dependent talin binders. The DNA nanodevice is thus a valuable addition to the molecular toolbox for studying mechanosensitive proteins.

biophysics↗

bioRxiv2024

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and G proteins may serve this role, we performed siRNA screening of G subunits and found that Gi2 and Gq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the G residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.

molecular biology↗

bioRxiv2024

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

bioengineering↗

bioRxiv2024

Short-Term Disruption of TGFβ Signaling in Adult Mice Renders the Aorta Vulnerable to Hypertension-Induced Dissection

Hypertension and transient increases in blood pressure from extreme exertion are risk factors for aortic dissection in patients with age-related vascular degeneration or inherited connective tissue disorders. Yet, the common experimental model of angiotensin II-induced aortopathy in mice appears independent of high blood pressure as lesions do not occur in response to an alternative vasoconstrictor, norepinephrine, and are not prevented by co-treatment with a vasodilator, hydralazine. We investigated vasoconstrictor administration to adult mice 1 week after disruption of TGF{beta} signaling in smooth muscle cells. Norepinephrine increased blood pressure and induced aortic dissection by 7 days and even within 30 minutes that was rescued by hydralazine; results were similar with angiotensin II. Changes in regulatory contractile molecule expression were not of pathological significance. Rather, reduced synthesis of extracellular matrix yielded a vulnerable aortic phenotype by decreasing medial collagen, most dynamically type XVIII, and impairing cell-matrix adhesion. We conclude that transient and sustained increases in blood pressure cause dissection in aortas rendered vulnerable by inhibition of TGF{beta}-driven extracellular matrix production by smooth muscle cells. A corollary is that medial fibrosis, a frequent feature of medial degeneration, may afford some protection against aortic dissection.

pathology↗

bioRxiv2024

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

Postnatal vascular morphogenesis is a dynamic process that initially involves angiogenesis to form the capillary bed, followed by its partial remodeling into arteries. Vascular endothelial growth factor A (VEGF hereafter), the primary inducer of angiogenesis, is also implicated in arterial specification in developmental contexts. However, it is unclear why arterial patterning is spatiotemporally segregated from angiogenesis, while postnatal arterial specification in animal models with blocked VEGF signaling remains unstudied. Here, we report that VEGF does not induce arterial fate in the capillaries, instead serving as a physiological brake to attenuate fluid shear stress (FSS)-driven capillary-to-arterial cell fate transition. Mouse models with disrupted VEGF signaling reveal impaired angiogenesis but intact and ectopic arterialization. Mechanistically, mechanosensitive transcription factor Sox17 determines the FSS-arterial program, while VEGF suppresses Sox17 transcription activity at arterial promoters and enhancers sites. Angiogenic signaling, while essential for the initial capillary morphogenesis, inhibits arterial specification, thereby protecting the capillary bed from premature arterialization driven by flow. These findings establish a new paradigm in which precise spatiotemporal coordination of environmental stimuli orchestrates angiogenesis, arterial patterning and capillary maintenance during vascular morphogenesis.

developmental biology↗

bioRxiv2024

Gamma protocadherins in vascular endothelial cells inhibit Klf2/4 to promote atherosclerosis

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide1. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2/4 anti-inflammatory program in endothelial cells (ECs)2-8. Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation9,10. We previously reported a whole genome CRISPR knockout screen11 that identified novel inducers of Klf2/4. Here we report suppressors of Klf2/4 and characterize one candidate, protocadherin gamma A9 (Pcdhga9), a member of the clustered protocadherin gene family12. Pcdhg deletion increases Klf2/4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2/4 by inhibiting the Notch pathway via physical interaction of cleaved Notch1 intracellular domain (NICD Val1744) with nuclear Pcdhg C-terminal constant domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis. This study identifies a novel fundamental mechanism of EC resilience and therapeutic target for treating inflammatory vascular disease.

cell biology↗

bioRxiv2023

Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation

Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

cell biology↗