Meridew, Jeffrey A.

Abstract Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression ( CTSK ) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and remains difficult to treat due to the absence of targeted therapies. While recent advances in immunotherapy, particularly with immune checkpoint inhibitors (ICIs), have transformed the treatment landscape for many cancers, response rates to ICIs remain low for TNBC, largely due to tumor-intrinsic immune evasion mechanisms. Objective: We identify and characterize a novel immune evasion pathway in TNBC driven by deregulation of cancer cell-intrinsic protease-activated receptor 1 (PAR1), a thrombin receptor frequently upregulated in aggressive tumors. Methods and Results: Through integrative bioinformatic analyses, genetic perturbations, xenograft models (immunodeficient and immunocompetent), flow cytometry-based tumor immune profiling, and RNA-seq, we demonstrate that PAR1 activation enhances immune evasion in TNBC. Mechanistically, we show that the paracaspase MALT1 acts as a critical downstream effector of PAR1 signaling and promotes immune escape by driving PD-L1 expression in TNBC cells. Functional assays reveal that genetic depletion of MALT1 or PD-L1 increases TNBC cell susceptibility to T cell-mediated cytotoxicity in vitro and significantly suppresses tumor growth in vivo. Notably, through knock-in and rescue experiments using a catalytically inactive MALT1 mutant, we reveal that MALT1’s scaffolding - rather than protease - function is essential for PD-L1 regulation. Immune cell depletion assays further identify CD4+ T cells, CD8+ T cells, and natural killer (NK) cells as key mediators of the anti-tumor immune response suppressed by the PAR1-MALT1 axis. These findings are supported by human TNBC specimen analyses, where MALT1 expression inversely correlates with T cell activation and positively associates with PD-L1 in PAR1-high, but not PAR1-low, tumors. Conclusion: Our study reveals immune evasion as a novel mechanism of PAR1-driven breast cancer pathogenesis, mediated by a PAR1/MALT1/PD-L1 signaling cascade. These findings bridge a critical gap by positioning MALT1 at the interface between tumor-intrinsic signaling and the immune microenvironment - two areas of CARMA-BCL10-MALT1 (CBM) complex research that have largely evolved in parallel over the years. Importantly, targeting MALT1 may enhance immunotherapy efficacy in TNBC. Given the availability of MALT1 inhibitors in clinical development, our work supports combining MALT1 inhibition with ICIs or other immune-oncology agents as a promising strategy to overcome immune resistance in TNBC. Citation Format: Dong Hu, Prasanna Ekambaram, Zheqi Li, Linda Klei, Maria L. Beecher, Yi Liu, Zongyou Cai, John Little, Jeffrey A. Meridew, Jia-Ying Lee, E. Aubrey Thompson, Tullia C. Bruno, Lidija Covic, Seung-Oe Lim, Anushka Dongre, Heide L. Ford, Mien-Chie Hung, Adrian V. Lee, Steffi Oesterreich, Linda McAllister-Lucas, Peter C. Lucas. A cancer cell-intrinsic PAR1/MALT1/PD-L1 signaling pathway drives immune evasion in triple-negative breast cancer [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 7392.

Abstract Background: Treatment for patients with triple-negative breast cancer (TNBC) relies on non-specific chemotherapy, underscoring the need for precision-based approaches. Our lab discovered that MALT1, the effector protein of the CARMA3-BCL10-MALT1 (CBM) signaling complex, is activated downstream of several oncogenic G protein-coupled receptors (GPCRs) implicated in breast cancer, including the thrombin (PAR1), angiotensin II (AGTR1), and lysophosphatidic acid (LPAR) receptors. When activated, MALT1 functions as a protease to cleave select protein substrates. Based on our finding that CBM activity in TNBC cells promotes tumor growth in immunocompetent but not in immunodeficient mouse models, we hypothesized that MALT1 protease activity induces a pro-tumor secretome that creates an immune-suppressed tumor immune microenvironment (TIME). Methods/Results: We confirmed by western that MALT1 protease is active in multiple human and mouse TNBC cell lines. Next, to identify MALT1-dependent secreted factors, we performed transcriptomic and proteomic analyses comparing TNBC cells treated with or without MALT1 protease inhibitors. We found that several secreted factors, including GM-CSF, CXCL1, and Pentraxin 3, were reduced by MALT1 protease inhibition. To assess the functional impact of the MALT1 protease-dependent secretome, we performed media transfer experiments comparing the effects of conditioned media (CM) from TNBC cells treated with or without MALT1 protease inhibitors. To account for species differences, experiments were performed evaluating human TNBC cell CM added to human PBMCs and mouse TNBC cell CM added to murine bone marrow cells. We found that the MALT1 protease-dependent secretome reduces the expression of iNOS in both human and murine macrophages. For use in validating our findings in human TNBC clinical specimens, we developed a “MALT1 protease active gene signature” using RNA-seq analysis. Conclusions/Future Directions: Our studies indicate that MALT1 protease activity in TNBC cells promotes expression of numerous immune-suppressive factors. In both human and mouse systems, blockade of MALT1 protease alters the tumor cell secretome and promotes an increase in expression of the anti-tumor M1-like marker iNOS in macrophages. Future studies will evaluate how this change in iNOS expression impacts functional properties of TNBC-associated macrophages. We will also assess the influence of the TNBC MALT1-dependent secretome on the TIME in vivo by comparing the effects of intratumoral injection of CM from MALT1 wild-type or MALT1 protease-deficient TNBC cells. Finally, we will utilize our MALT1 activity gene signature to evaluate TNBC clinical specimens for an association between MALT1 activity and tumor immune contexture, with an emphasis on tumor-associated macrophages. Our studies could nominate MALT1 protease as a potential therapeutic target for improving anti-tumor immunity in TNBC. Since multiple MALT1 protease inhibitors are in clinical trial, our findings could be rapidly translated to improve outcomes for patients with TNBC. Citation Format: John W. Little IV, Jeffrey A. Meridew, Dong Hu, Juliana Hofstätter Azambuja, Linda M. McAllister-Lucas, Peter C. Lucas. The GPCR-MALT1 protease signaling axis drives an immunosuppressive secretome in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B046.

Abstract Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers—short, synthetic, single-stranded DNA or RNA molecules—can be ∼6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast (∼100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo , cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

Abstract Repetitive injury is hypothesized to lead to progressive tissue fibrosis and end-stage organ failure. Whether tissue-resident mesenchymal cell populations retain epigenetic memory of prior injuries that contribute to this pathological process is unknown. Here we used a genetic lineage labeling approach to mark the lung mesenchyme prior to injury, then performed multimodal analyses on isolated lung mesenchyme during the initiation, progression, and resolution of the fibrotic response. Our results demonstrate the remarkable epigenetic and transcriptional plasticity of the lung mesenchyme during fibrotic activation and de-activation. Despite this plasticity, we also find that the lung mesenchyme exhibits an enhanced fibrotic program upon reinjury. We identify RUNX1 as a critical driver of both fibrotic activation and fibrotic memory. Comparison of fresh isolated and cultured lung mesenchyme demonstrates that RUNX1 is spontaneously activated in standard culture conditions, previously masking these roles of RUNX1. Targeted knockdown of RUNX1 dampens fibrotic mesenchymal cell activation immediately after cell isolation, but with reduced efficacy after only days of culture, confirming its functional importance to both early activation and long-term memory. Collectively, our findings implicate RUNX1 in the initiation and memory of fibrotic mesenchymal cell activation that together prime enhanced mesenchymal cell responses upon repeated injury.

Abstract Pulmonary fibrosis is increasingly understood to involve dysfunction within and across multiple cellular compartments, with recent attention highlighting the involvement of pulmonary vascular dysfunction in failed repair and progression of fibrosis. Formulation and delivery of lung-targeting lipid nanoparticles (LNPs) may provide a means to selectively target the lung but not systemic vasculature. However, the feasibility and efficacy of such approaches in the fibrotic lung are unknown. We sought to test whether intravenously administered lung-targeting LNPs can safely deliver mRNA to the healthy and fibrotic lung vasculature in young and aged mice and whether delivery of mRNA encoding a matricellular protein could promote fibrosis resolution. We used a selective organ targeting LNP formulation and characterized cell-specificity of delivery after bleomycin-induced lung fibrosis. We then delivered Ccn3 mRNA (encoding cellular communication network factor 3) to aged mice in the setting of established lung fibrosis and evaluated fibrotic regression and vascular repair. The matricellular protein encoded by Ccn3 was previously identified by our group as an important regulator of lung endothelial function. We found that LNP delivery was lung specific and predominantly endothelial targeting in the setting of lung fibrosis. Delivery of Ccn3 mRNA to aged mice via LNPs modestly reduced fibrosis and improved microvascular density in the lungs. Our results support the concept that cell-specific and repair-promoting cargos delivered via lung-targeting LNPs may have utility for treatment of established fibrosis.

Abstract Rationale Extracellular matrix (ECM) accumulation results from an imbalance between ECM deposition and degradation, due to persistently increased ECM synthesis or reduced resorption. Fibroblasts, as key regulators of ECM homeostasis in both health and disease, are essential targets to rebalance ECM turnover. Recent findings indicate that fibroblasts are regulated by immune cells, which can influence ECM deposition. Specifically, myeloperoxidase (MPO), an enzyme produced by neutrophils, has been shown to enhance fibroblast-driven ECM accumulation in various diseases. In patients with radiographic Interstitial Lung Disease (ILD), elevated MPO levels have been correlated with reduced lung capacity (Cantin et al., 1987), while recent studies report increased MPO levels in the bronchoalveolar lavage fluid of ILD patients (Ngo et al., EJROR 2024). Additionally, MPO and related immune dysregulation proteins are found in fibrotic alveoli in Idiopathic Pulmonary Fibrosis (IPF) (Herrera et al., JCI Insight 2022). These findings suggest an association between MPO and lung fibrosis, yet it remains uncertain whether MPO directly influences ECM accumulation in fibrotic progression. Methods Patient tissue and serum samples were collected with consent, and MPO levels were quantified using ELISA and immunofluorescence techniques. Biochemical assays were performed using MPO isolated from human leukocytes, recombinant cathepsin K, and hydrogen peroxide to investigate whether MPO influences cathepsin K activity. Precision cut lung slices (PCLS) were prepared from untreated C57BL/6 mice and treated with transforming growth factor-beta (TGFβ) and/or MPO for four days, with cathepsin K activity assessed using fluorescence assays. Additionally, MPO-knockout and wild-type mice received intratracheal bleomycin, and after 14 days, cathepsin K activity was quantified via in vivo imaging. Collagen content, as an indicator of fibrosis, was measured by quantifying hydroxyproline in harvested lung tissue. Results/Discussion/Conclusion Increased MPO levels were observed in immunofluorescent-stained samples (57%; 4/7) and serum samples (30%; 3/10) from IPF patients. Biochemical assays demonstrated that physiological levels of MPO (0.2 µg/ml) combined with H2O2 reduced recombinant cathepsin K activity by 85%. In PCLS treated with MPO, cathepsin K activity decreased by 50% with or without TGFβ. After bleomycin treatment, wild-type mice showed reduced cathepsin K activity, while MPO-knockout mice maintained activity levels. Both survival and weight loss were similar between wild-type and MPO-knockout mice; however, collagen content was significantly higher in wild-type mice, whereas MPO-knockout mice were protected from bleomycin-induced collagen accumulation. These findings suggest that MPO contributes to ECM accumulation in lung fibrosis, potentially through inhibition of collagenolytic enzymes such as cathepsin K.

Abstract Idiopathic pulmonary fibrosis (IPF) is characterized by the sustained activation of interstitial fibroblasts leading to excessive collagen deposition and progressive organ failure. Epigenetic and metabolic abnormalities have been shown to contribute to the persistent activated state of scar-forming fibroblasts. However, how epigenetic changes regulate fibroblast metabolic responses to promote fibroblast activation and progressive fibrosis remains largely unknown. Here, we show that the epigenetic regulator CBX5 (chromobox protein homolog 5) is critical to the transition of quiescent fibroblasts to activated collagen-producing fibroblasts in response to bleomycin-induced lung injury. Loss of mesenchymal CBX5 attenuated fibrosis development, and this effect was accompanied by the downregulation of pathogenic fibroblast genes, including Cthrc1, Col1a1, and Spp1, and by the upregulation of metabolic genes with antifibrotic activity such as Ppara and Pparg. Single-cell RNA sequencing and immunohistochemistry analyses revealed that CBX5 expression was enriched in pathogenic fibroblasts and fibroblastic foci of IPF lungs. Bulk RNA-sequencing analysis combined with metabolic assessments demonstrated that CBX5 silencing in IPF fibroblasts potently inhibited transforming growth factor–stimulated glycolysis while enhancing AMPK signaling and mitochondrial metabolism. Finally, interruption of the CBX5 pathway in IPF fibroblasts in vitro and in IPF lung explants ex vivo synergistically potentiated the activation of metformin-induced AMP-activated protein kinase activation and inhibited collagen secretion. Collectively, our findings identify CBX5 as an epigenetic regulator linking metabolic maladaptation to the persistent activated state of lung fibroblasts during IPF progression.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency–mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.