ABSTRACT:Covalent cross-linking of fibrin by the plasma transglutaminase coagulation factor XIII (FXIII) is a key determinant of blood clot stability and function. FXIII-catalyzed formation of ε-N-(γ-glutamyl)-lysyl cross-links is restricted to the fibrin γ-chain and α-chain and follows thrombin-driven fibrin polymerization. Fibrinogen is also cross-linked by tissue transglutaminase (TG2) in a reaction favoring intramolecular and intermolecular α-γ cross-linking. Emerging evidence indicates that fibrinogen is a relevant substrate of TG2 in conditions of acute tissue damage. Remarkably, beyond detection of prototypical FXIII-directed cross-links (ie, α-α, γ-γ), we identified entirely novel covalent cross-links involving the fibrinogen β-chain (ie, β-α, via FGB-Q82). Addition of TG2 to in vitro clotting reactions and analysis of fibrin(ogen) in reducing conditions revealed loss of β-chain polypeptide paired with formation of high-molecular weight β-chain species. Mass spectrometry-based cross-linking proteomic analysis of in vitro clots recapitulated the precise TG2-directed β-chain cross-links observed in clots made using plasma from patients following traumatic injury. The results indicate in vitro and ex vivo cross-linking of the fibrin β-chain and highlight a novel example of TG2 emerging as a relevant plasma transglutaminase.

BACKGROUNDPlasma heparan sulfate, a glycosaminoglycan released during endothelial glycocalyx degradation, predicts sepsis mortality. Chondroitin sulfate is a circulating glycosaminoglycan not specific to glycocalyx degradation; its relevance to sepsis is unknown.METHODSWe studied the associations of plasma chondroitin sulfate with (a) mortality in patients with sepsis-associated hypotension and (b) the relative effectiveness of a randomly assigned liberal versus restrictive intravenous fluid resuscitation strategy. We selected 574 patients enrolled in the Crystalloid Liberal or Vasopressors Early Resuscitation in Sepsis trial using an outcome-enriched sampling strategy. We used liquid chromatography-mass spectrometry to quantify plasma chondroitin sulfate. In comparison, we measured hyaluronic acid as a glycocalyx degradation marker and IL-6 as an inflammatory biomarker. We conducted Cox proportional hazards regression analyses to examine associations of baseline biomarker concentrations with mortality and resuscitation strategy effectiveness. We used inverse probability of selection weights and generalized raking to account for the nonrepresentative sampling design.RESULTSPlasma chondroitin sulfate, hyaluronic acid, and IL-6 were associated with mortality within 90 days. As baseline chondroitin sulfate increased, subsequent randomization to a restrictive strategy was increasingly beneficial (P = 0.022): treatment effect hazard ratio (restrictive versus liberal) for mortality was estimated as 1.49 (95% CI, 0.98-2.27), 1.30 (95% CI, 1.00-1.69), 1.09 (95% CI, 0.82-1.44), 0.88 (95% CI, 0.66-1.16), and 0.71 (95% CI, 0.52-0.97) for 10th, 25th, 50th, 75th, and 90th percentiles of baseline chondroitin sulfate.CONCLUSIONPlasma chondroitin sulfate predicts sepsis mortality and may modify the response to a subsequent liberal versus restrictive intravenous fluid resuscitation strategy.TRIAL REGISTRATIONClinicalTrials.gov NCT03434028.FUNDINGNIH grants R01HL149422 and R01HL094786.

Biomaterials-based tissue engineering aims to recapitulate native tissue architecture and function for both clinical repair and advanced in vitro models. While improvements in biomaterials have been made, including granular hydrogels and ECM-derived scaffolds, current biomaterials lack intentional design choices for effective translation, including regulatory considerations, practical extrusion delivery, and biomimetic characteristics. Here, we develop and characterize a library of granular ECM (gECM) biomaterials for five key tissues (cartilage, bone, skin, liver, and kidney), in which ECM particles are densely packed within a hyaluronic acid hydrogel. We optimize tissue processing methods that preserve proteomic content and structure while also aligning with scale-up manufacturing and regulatory guidelines. We show that gECM hydrogels can be molded, extruded, and 3D-printed while retaining their shape, and they stabilize at physiological temperature and pH. Lastly, we demonstrate that bulk gECM mechanics are driven by tissue type, and gECM hydrogels support viability, proliferation, and tissue-specific cellular activity. Together, these findings establish gECM hydrogels as a translational and biomimetic platform for clinical tissue repair and complex in vitro models.

Osteochondral defects remain a major clinical challenge due to the limited regenerative capacity of cartilage and the complexity of the osteochondral interface. Here, we present a human-derived granular extracellular matrix (gECM) hydrogel platform designed for translational osteochondral repair. Using otherwise discarded human donor tissues, we developed cartilage and bone gECM hydrogels under current good manufacturing practice workflows. These materials are shear-thinning, immediately hold their form, and crosslink under physiological conditions to form stable constructs. Proteomic analysis confirmed that cartilage and bone gECM retain distinct tissue-specific biochemical signatures, while mechanical characterization demonstrated tissue-relevant stiffness, with bone gECM hydrogels exhibiting greater stiffness than cartilage gECM hydrogel. Particle packing density primarily governed viscosity, whereas tissue type contributed strongly to bulk stiffness. Together, these findings establish a scalable, human-derived gECM platform that integrates tissue-specific structural and mechanical cues, and advances a clinically translatable strategy for osteochondral repair.

The transcriptional coactivators YAP (yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are key regulators of cell proliferation, apoptosis, and differentiation, thereby maintaining tissue homeostasis and controlling organ size. Although their roles in epithelial cancers and fibrosis are well-established, their involvement in the physiological regulation of the dermal extracellular matrix (ECM) by fibroblasts is less understood. In this study, we investigated the role of Yap/Taz during postnatal development of the dermal ECM. During postnatal growth, mouse skin steadily undergoes significant surface expansion. Postnatal deletion of Yap/Taz in dermal fibroblasts, the primary cells responsible for dermal ECM homeostasis, significantly impairs dermal ECM maturation, as evidenced by marked deficiencies in collagen expression, deposition, and organization. Isolated fibroblasts from Yap/Taz-knockout mice show reduced expression of Yap/Taz target genes (Ccn1, Ccn2, Col1a1), which is rescued by reintroduction of active Yap/Taz. RNA sequencing, spatial transcriptomics, and proteomics of Yap/Taz-knockout skin reveal substantial downregulation of ECM-related genes, including type I (Col1a1, Col1a2) and type III (Col1a3) collagens, which together constitute more than 90% of the skin's collagen content. Mechanistically, deletion of Yap/Taz impairs mouse dermal maturation, at least in part, through the suppression of TGF-β/Smad signaling, the primary pathway governing fibroblast collagen synthesis. These findings demonstrate that YAP/TAZ are essential for postnatal dermal ECM homeostasis.

Protein-l-isoaspartate O-methyltransferase (PIMT), encoded by PCMT1, is a repair enzyme that corrects isoaspartyl lesions, preserving protein structure and function. While indispensable for neuronal integrity, its role in red blood cells (RBCs) and transfusion outcomes is incompletely understood. Here, we show that novel erythroid-specific Pcmt1 knockout mice display profound remodeling of one-carbon metabolism, accumulation of repair intermediates, and destabilization of glycolytic and cytoskeletal proteins, yet maintain lower lipid peroxidation and normal post-transfusion recovery. Analysis of 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study (REDS)-III Red Blood Cell Omics (RBC Omics) study revealed that common PCMT1 variants associate with hemolysis phenotypes and regulate PIMT protein level, as gleaned by protein quantitative trait loci (pQTL) analyses. The nonsynonymous rs4816 (V120I) allele, enriched in donors of Asian or African ancestry, emerged as a beneficial variant: carriers exhibited lower osmotic hemolysis, altered peptide methylation flux, and higher PIMT protein levels. Recombinant expression confirmed that the I120 variant displays preserved global folding, but greater catalytic activity than the canonical V120 enzyme. Transfusion outcome data showed that PCMT1 genotype influences hemoglobin increments and bilirubin responses in recipients. These findings identify PIMT as a novel determinant of RBC storage biology and establish rs4816 as a protective allele, with broader implications for donor diversity, transfusion efficacy, and proteome maintenance in aging.

The Saccharomyces cerevisiae silent information regulator (SIR) complex performs all core heterochromatin functions. It first drives histone deacetylation in an iterative, spreading manner, then stably incorporates with nucleosomes to compact and epigenetically repress the chromatin. How this switch between dynamic spreading and stable compaction occurs has remained poorly understood, partly due to limited structural data on the intact complex. Using crosslinking mass spectrometry, we identified an uncharacterized intersubunit interaction connecting these two states: the Sir2 deacetylase interacts with the scaffolding subunit Sir4 through its coiled-coil domain, which also contacts the Sir3 compaction subunit. This interaction hub contains conserved Sir2 residues that can adopt multiple conformations, including orientation toward the active site, alongside co-evolved residues that enable species-specific Sir4 interactions. Mutations disrupting this hub disrupt heterochromatic repression in vivo and affect the deacetylation activity, directly linking catalysis to compaction. Our findings reveal how a multifunctional complex stages a stepwise transition to achieve epigenetic gene repression.

Abstract Background: Obesity, affecting 42.4% of U.S. adults, correlates with higher tumor grade, increased metastasis, and reduced survival in breast cancer (BC). Our prior work revealed obesity-induced secretion of collagen-crosslinking enzymes, basement membrane proteins, and the matricellular protein galectin-3 (LGALS3) in the breast. These extracellular matrix (ECM) changes were positively associated with the fibroblast population in obese breast tissue. Elevated LGALS3 expression predicts poorer overall survival in estrogen receptor-positive (ER+) BC. We hypothesize that obesity amplifies LGALS3 fibroblast signaling to disrupt ECM composition and organization, supporting BC progression. Methods: Rag1-/- mice were fed a high-fat high-sugar (HFHS) or low-fat low-sugar (LFLS) diet for 16 weeks to establish obese and lean phenotypes. ER+ UCD65 BC cells were implanted bilaterally into the mammary fat pad alone or with CD146neg (HS5) fibroblasts, generating four groups: LFLS, HFHS, LFLS + CD146neg, and HFHS + CD146neg. All mice received 1 mg of estrogen at implantation and remained on their assigned diets for eight weeks. Tumors, plasma, lungs, and mammary fat pads were collected at endpoint for analyses. LGALS3 was knocked down in CD146neg fibroblasts and overexpressed in CD146pos (HS27) fibroblasts for in vitro studies. Conditioned media was generated under starvation conditions and concentrated 20x for western blotting. Results: Obese mice implanted with CD146neg fibroblasts exhibited a 2.7-fold increase in tumor growth and 8.8-fold increase in metastasis compared with lean controls. CD146neg fibroblasts significantly enhanced angiogenesis in both lean and obese settings but did not affect lymphangiogenesis. Obesity significantly disrupted the collagen organization of the mammary fat pads. In obese mice, with CD146neg fibroblasts, increased collagen deposition, macrophage infiltration, and circulating levels of LGALS3. Genetic manipulation of LGALS3 showed that it specifically regulates collagen I secretion without altering intracellular levels. This loss of secretion triggered unfolded protein response activation and reduced TGF-β signaling, further supporting a secretion-focused mechanism. LGALS3 was diet-responsive, with time-restricted feeding and intermittent fasting lowering circulating levels by 2.1 and 2.7-fold, respectively. Conclusions: Obesity synergizes with CD146neg fibroblasts to accelerate BC aggression through LGALS3-mediated ECM remodeling, supported by a novel post-transcriptional control of collagen secretion. LGALS3 can serve as a therapeutic target and is modifiable by dietary strategies to mitigate obesity-associated BC risks, potentially improving outcomes for millions of at-risk patients. Citation Format: Ellen E. Bamberg, Kiran Vinod-Paul, Amy L. Pyo, Kirk C. Hansen, Carol A. Sartorius, Paul S. MacLean, Peter Kabos, Heather Brechbuhl. Fibroblast galectin-3 remodels the obese breast tumor microenvironment to promote cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2097.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymatic disorder, affects over 500 million people worldwide and is often linked to exercise intolerance due to oxidative stress, but its true impact on physical performance remains unclear. This study aimed to evaluate the physiological and metabolic effects of G6PD deficiency on endurance capacity. Using humanized mice carrying the African G6PD variant [V68M; N126D] (hG6PDA-), we show that despite reduced pentose phosphate pathway activity, these mice exhibit a 10.8% increase in treadmill critical speed (CS)-suggesting enhanced endurance capacity. Multi-omics profiling across red blood cells, plasma, skeletal muscle, spleen, kidney, and liver reveals metabolic adaptations, including elevated glycolysis, fatty acid oxidation, and increased mitochondrial activity, alongside heightened oxidative phosphorylation in muscle and accelerated red blood cell turnover in the spleen and liver. These findings indicate that systemic metabolic reprogramming may offset antioxidant deficiencies, potentially conferring a performance advantage. Given that G6PD deficiency affects up to 13% of African Americans and is associated with cardiovascular health disparities, our results challenge conventional exercise restrictions and highlight the need for personalized exercise guidelines for affected individuals.

Tissue-engineered grafts that mimic articular cartilage show promise for treating cartilage injuries. However, engineering cartilage cell-based therapies to match zonal architecture and biochemical composition remains challenging. Decellularized articular cartilage extracellular matrix (dECM) has gained attention for its chondro-inductive properties, yet dECM-based bioinks have limitations in mechanical stability and printability. This study proposes a rapid light-based bioprinting method using a tyrosine-based crosslinking mechanism, which does not require chemical modifications of dECM and thereby preserves its structure and bioactivity. Combining this resin with Filamented Light (FLight) biofabrication enables the creation of cellular, porous, and anisotropic dECM scaffolds composed of aligned microfilaments. Specifically, we focus on the effects of various biopolymer compositions (i.e. hyaluronic acid, collagen I, and dECM) and inner architecture (i.e. bulk light vs FLight) on immune response and cell morphology, and we investigate their influence on nascent ECM production and long-term tissue maturation. Our findings highlight the importance of FLight scaffolds in directing collagen deposition resembling articular cartilage structure and promoting construct maturation, and they emphasize the superiority of biological-rich dECM over single-component materials for engineering articular cartilage, thereby offering new avenues for the development of effective cartilage tissue engineering strategies.

BACKGROUND: Postpartum hemorrhage is difficult to predict, is associated with significant maternal morbidity, and is the leading cause of maternal mortality worldwide. The identification of maternal biomarkers that can predict increased postpartum hemorrhage risk would enhance clinical care and may uncover mechanisms that lead to postpartum hemorrhage. OBJECTIVE: This retrospective case-control study employed agnostic proteomic profiling of maternal plasma samples to identify differentially abundant proteins in controls and postpartum hemorrhage cases. STUDY DESIGN: Maternal plasma samples were procured from a cohort of >60,000 participants in a single institution's perinatal repository. Postpartum hemorrhage was defined as a decrease in hematocrit of >= 10% or receipt of transfusion within 24 hours after delivery. Postpartum hemorrhage cases (n=30) were matched by maternal age and delivery mode (vaginal or cesarean) with controls (n=56). Mass spectrometry was used to identify differentially abundant proteins using integrated peptide peak areas. Statistically significant differences between groups were defined as P<.05 after controlling for multiple comparisons. RESULTS: By study design, cases and controls did not differ in race, ethnicity, gestational age at delivery, blood type, or predelivery platelet count. Cases had slightly but significantly lower predelivery and post- delivery hematocrit and hemoglobin. Mass spectrometry detected 1140 proteins, including 77 proteins for which relative abundance differed significantly between cases and controls (fold change >1.15, P<.05). Of these differentially abundant plasma proteins, most had likely liver or placental origins. Gene ontology term analysis mapped to protein clusters involved in responses to wound healing, stress response, and host immune defense. Significantly differentially abundant proteins with the highest fold change (prostaglandin D2 synthase, periostin, and several serine protease inhibitors) did not correlate with predelivery hematocrit or hemoglobin but identified postpartum hemorrhage cases with logistic regression modeling revealing good-to-excellent area under the operator receiver characteristic curves (0.802-0.874). Incorporating predelivery hemoglobin with these candidate proteins further improved the identification of postpartum hemorrhage cases. CONCLUSION: Agnostic analysis of maternal plasma samples identified differentially abundant proteins in controls and postpartum hemorrhage cases. Several of these proteins are known to participate in biologically plausible pathways for postpartum hemorrhage risk and have potential value for predicting postpartum hemorrhage. These findings identify candidate protein biomarkers for future validation and mechanistic studies.

The dystrophin-glycoprotein complex (DGC) is composed of peripheral and integral membrane proteins at the muscle cell membrane that link the extracellular matrix with the intracellular cytoskeleton. While it is well established that genetic mutations that disrupt the structural integrity of the DGC result in numerous muscular dystrophies, the 3D structure of the complex has remained elusive. Two recent elegant cryoEM structures of the DGC illuminate its molecular architecture and reveal the unique structural placement of sarcospan (SSPN) within the complex. SSPN, a 25 kDa tetraspanin-like protein, anchors β-dystroglycan to the β-, γ- and δ-sarcoglycan trimer, supporting the conclusions of biochemical studies that SSPN is a core element for DGC assembly and stabilization. Here, we advance these studies by revealing that SSPN provides scaffolding in δ-sarcoglycanopathies, enabling substitution of δ-sarcoglycan by its homolog, ζ-sarcoglycan, leading to the structural integrity of the DGC and prevention of limb-girdle muscular dystrophy R5. Three-dimensional modeling reveals that ζ-sarcoglycan preserves protein-protein interactions with the sarcospan, sarcoglycans, dystroglycan, and dystrophin. The structural integrity of the complex maintains myofiber attachment to the extracellular matrix and protects the cell membrane from contraction-induced damage. These findings demonstrate that sarcospan prevents limb-girdle muscular dystrophy R5 by remodeling of the sarcoglycan complex composition.

Caffeine is the most widely consumed psychoactive substance globally, yet its peripheral physiological effects remain incompletely understood. Leveraging comprehensive data from 13,091 blood donors in the REDS RBC-Omics study, we identified caffeine as a significant modulator of red blood cell (RBC) storage quality and transfusion outcomes. Elevated caffeine levels were reproducible across multiple donations from 643 recalled donors, selected based on their extremes in hemolytic propensity. Both in the screening and recalled cohorts, higher caffeine levels were associated with disrupted RBC metabolism, characterized by reduced glycolysis, depletion of adenylate pools or 2,3-bisphosphoglycerate, and increased markers of oxidative stress and osmotic fragility, including kynurenine accumulation. These observations were recapitulated in plasma and RBC of eight volunteers upon consumption of a cup of coffee independently of brewing method (Chemex vs. espresso). Clinically, elevated caffeine levels correlated with increased hemolysis and lower post-transfusion hemoglobin increments, an effect especially pronounced in recipients transfused with RBC from donors carrying common polymorphisms in the ADORA2B gene, a key regulator of RBC metabolism in hypoxia. These human findings were mechanistically validated using a murine model deficient in ADORA2b, which demonstrated impaired glycolytic flux, compromised antioxidant defenses (including caffeine-dependent direct inhibition of recombinantly expressed glucose 6-phosphate dehydrogenase), and decreased transfusion efficacy (lower hemoglobin increments, higher bilirubin after transfusion), effects further exacerbated by caffeine exposure during storage. Our study positions caffeine consumption as a modifiable factor in blood transfusion practice, advocating for precision strategies that integrate genetic and exposome factors, and identifies metabolic interventions to enhance blood quality and clinical outcomes.

Background Many snake venoms have been shown to possess thrombolytic activity. However, it remains unclear if actions on other clot-stabilizing proteins beyond fibrin chains contribute significantly to venom-induced thrombolysis because the clot-wide targets of venom proteases and the mechanisms responsible for thrombolysis are not well understood. Objectives Here, we utilized a high-throughput, time-based thrombolysis assay in combination with untargeted peptidomics to provide comprehensive insight into the effects of venom from 5 snake species on blood clot degradation. Methods We compared thrombolytic profiles across venoms with variable levels of proteases and generated venom-specific fingerprints of cleavage specificity. We also compared the specific effects of venoms that possess a range of thrombolytic activity on fibrin chains and other clot-bound proteins involved in clot structure. Results Protease-rich venom more effectively degraded blood clots. Venoms with higher thrombolytic activity demonstrated an enhanced ability to target multiple sites across fibrin chains critical to clot stability and structure, as well as clot-stabilizing proteins including factor XIII, fibronectin, and vitronectin. Conclusion Collectively, this study significantly expands our understanding of the thrombolytic and fibrinolytic effects of snake venom by determining the full suite of clot-specific venom targets that are involved in clot formation and stability. This has important implications for the treatment of snake envenomation, the bioprospecting of therapeutically useful molecules, and the development of research tools for investigating hematologic disorders.

IntroductionAdvanced thyroid cancer, including papillary (PTC) and anaplastic thyroid cancer (ATC), are the leading causes of endocrine cancer deaths. Thus, there is a critical need to identify novel therapeutic targets to improve standard of care. Focal Adhesion Kinase (FAK) is overexpressed and phosphorylated in thyroid cancer and drives thyroid cancer growth, invasion, and metastasis. FAK is a nonreceptor tyrosine kinase that is autophosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling, resulting in the recruitment of SRC proto-oncogene and downstream signaling pathways. FAK is predominately localized at the plasma membrane but has recently been shown to accumulate in the nucleus as well as the nucleolus to drive tumor growth. The nucleolus is a membraneless subnuclear organelle that is involved in ribosomal biogenesis through the transcription, processing, and assembly of ribosomal RNA (rRNA). The role of FAK in ribosome biogenesis is currently unknown.MethodsNuclear/nucleolar FAK localization and function were studied using genetic and pharmacological approaches. High resolution microscopy was used to study the subcellular localization of FAK. Functional and biochemical assays including transformation and clonogenic assays, polysome profiling, and nascent protein synthesis assays were utilized to assess cell growth and survival. Protein-protein interactions of FAK were determined using a proximity dependent biotinylation (BioID) proteomics approach.ResultsWe have found that pY397 FAK accumulates in the nucleolus of advanced thyroid cancer cells and that autophosphorylation of FAK at pY397 and FAK kinase activity are important for nucleolar accumulation of FAK. Furthermore, knockdown of nucleophosmin 1 (NPM1), an important structural component of the nucleolus, reduced pY397 FAK nucleolar accumulation. Functionally, we showed that nuclear FAK and FAK kinase activity are necessary for anchorage independent growth. We demonstrated that targeted degradation of FAK results in decreased protein synthesis with a specific decrease in the 60S ribosomal subunit. Using a BioID proteomics approach, we showed that autophosphorylated FAK interacts with a network of nucleolar proteins including nucleolar protein 56 (NOP56) which is a core small ribonucleoprotein (snoRNP) important for 60S ribosome biogenesis. Finally, we found that pY397 FAK co-localizes with NOP56 and that knockdown of NOP56 phenocopies FAK depletion.ConclusionsOverall, these findings highlight a novel function for FAK in promoting ribosome biogenesis and suggest that nucleolar FAK represents a promising therapeutic target.

ABSTRACT:The transglutaminase coagulation factor XIII (FXIII) is critical for the stability and function of intravascular fibrin clots. Prorepair extravascular fibrin(ogen) deposits are potentially subject to cross-linking by FXIII and other transglutaminases not typically resident in plasma. However, the impact of these alternative modifiers on fibrin(ogen) structure and function is not known. We tested the hypothesis that tissue transglutaminase (TG2) modifies FXIII-directed fibrin(ogen) cross-linking in vitro and within injured tissue. Global proteomic analysis after experimental acetaminophen (APAP)-induced acute liver injury revealed that intrahepatic fibrin(ogen) deposition was associated with hepatic TG2 levels that exceeded that of FXIII. Mass spectrometry-based cross-link mapping of in vitro fibrin matrices uncovered, to our knowledge, the first evidence of synergistic fibrin(ogen) α-α cross-linking catalyzed by both transglutaminases. Fibrin(ogen) cross-linking was increased in livers from patients with APAP-induced acute liver failure. APAP-challenged TG2-/- mice displayed an altered pattern of FXIII-dependent fibrin(ogen)-γ and fibrin(ogen)-α chain cross-linking aligned with the impact of TG2 on fibrin cross-linking in vitro. This shift in fibrin(ogen) cross-linking exacerbated pathologies including hepatic necrosis and sinusoidal congestion. The results, to our knowledge, are the first to indicate that TG2 impacts FXIII-directed fibrin(ogen) cross-linking, both in vitro and in vivo. The results suggest that TG2 functions to dynamically alter the structure of extravascular fibrin(ogen) to mitigate liver damage, a novel mechanism likely applicable across types of tissue injury.

Background Previously, we demonstrated that bone marrow stromal factors protect FLT3-mutant AML cells from FLT3 inhibitor (FLT3i)-induced death by restoring mTORC1-dependent translation of essential oxidative phosphorylation genes (Park et al., 2022). While FLT3 plus mTORC1 inhibition synergistically eliminates AML cells and prevents relapse in vivo, mTORC1 inhibition alone has modest effects on translation (~50%) and viability (<10%). This suggests that there are compensatory mTOR-independent mechanisms of translation. Our initial findings identified CDK1 as a key mediator of mTOR-independent translation. Importantly, CDK1 inhibition suppressed protein synthesis at drug concentrations that had minimal effects on the cell cycle, revealing a noncanonical role in translation. This study aims to define how AML cells maintain translation despite mTORC1 inhibition and whether this contributes to resistance against FLT3- and mTORC1-targeted therapies. Methods Pulse SILAC was performed on MOLM14 cells (FLT3-ITD AML cell line) to track nascent protein synthesis over a 5-hour window starting 4 hours after treatment, using Everolimus to inhibit mTORC1 and RO-3306 to inhibit CDK1. Pathway enrichment analyses were conducted using MetaboAnalyst and ENRICHR (Reactome, KEGG, WikiPathways). Global translation was quantified using O-propargyl-puromycin (OPP) incorporation, and cell viability/cycle status assessed via propidium iodide (PI) flow cytometry. Results To quantify nascent protein synthesis, or lack thereof, under CDK1 or mTORC1 inhibition, we performed pulse SILAC, a mass spectrometry-based method tracking incorporation of heavy-labeled amino acids into newly synthesized proteins, over a 5-hour window in MOLM14 cells, using RO-3306 and Everolimus to inhibit CDK1 and mTORC1, respectively. Prior OPP-incorporation assays showed that CDK1 inhibition reduces translation by ~40% within 3–6 hours, independent of cell cycle arrest, while by 9 hours, combined CDK1/mTORC1 inhibition nearly abolished translation, with modest effects on the cell cycle. We performed principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) from SILAC results, which revealed clear separation among vehicle, CDK1i, and mTORC1i treatment groups based on global profiles of nascent proteins. These distinct clustering patterns indicate significant shifts in protein expression induced by each treatment condition. Pathway enrichment analyses of differences in nascent proteins revealed that CDK1 inhibition specifically suppressed pathways related to RNA splicing, nuclear export, and transcript maturation, implicating CDK1 as a key regulator of RNA processing and transcript stability. Interestingly, CDK1 inhibition also triggered compensatory upregulation of mTORC1 pathway proteins, suggesting feedback adaptation. In contrast, mTORC1 inhibition specifically promoted DNA repair pathways such as mismatch repair and homologous recombination, while repressing the protein expression of glycolysis, pyruvate metabolism, and apoptotic signaling components. These results suggest a coordinated shift toward a survival state under mTOR suppression. Analysis of alterations in common for CDK1- and mTORC1- inhibited treatment groups revealed downregulation of RNA processing, splicing, and protein turnover, demonstrating non-redundant contributions to these key pathways. Meanwhile, analysis of overlapping but oppositely regulated proteins showed that CDK1 and mTORC1 also exert distinct, and at times antagonistic, control over stress response pathways such as DNA repair, mitochondrial biogenesis, and translation. Thus, their balance may shape how AML cells adapt under therapeutic pressure. Conclusion Our integrated proteomic and pathway analyses reveal that CDK1 and mTORC1 regulate distinct yet intersecting stress adaptation programs in FLT3-ITD AML. Importantly, our analyses reveal novel, noncanonical roles for CDK1 beyond its established function in cell cycle regulation in maintaining the protein expression of mRNA splicing, transcript maturation, and nuclear export components, highlighting a critical role beyond cell cycle control. These findings expose a critical vulnerability in the translational dependence of FLT3-ITD AML cells, highlighting the need to understand how translation is sustained independently of mTOR to anticipate resistance and guide future FLT3i and mTORC1i combination therapies aimed at preventing relapse.

Transforming growth factor β (TGFβ) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGFβ release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGFβ functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGFβ ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGFβ receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGFβ directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGFβ generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.