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Abstract
Imaging-based single-cell spatial transcriptomics (iSCST) on formalin-fixed, paraffin-embedded (FFPE) tissue enables comprehensive analysis of archived specimens while preserving spatial context, critical to an understanding of ulcerative colitis (UC) pathology. Here, we deployed a robust framework for applying iSCST to clinical FFPE mucosal biopsies from patients with UC, immune checkpoint inhibitor-induced (ICI) colitis and healthy controls. iSCST using custom Xenium gene panels enabled precise detection of diverse cell subsets and disease-specific genes. We mapped transcriptionally distinct fibroblast subsets within mucosal niches, including inflammation-associated fibroblasts (IAFs), and identified colitis-specific neighborhoods formed by IAFs, monocytes, and neutrophils. Transcriptional signatures and spatial neighborhoods uncovered through iSCST were associated with vedolizumab (VDZ) response, with non-responders exhibiting either an innate IAF-monocyte-neutrophil signature or adaptive gut-associated lymphoid tissue (GALT) signature, while responders showed enrichment of an epithelial cellular neighborhood. These signatures were validated in an internal and an external dataset, supporting the existence of two distinct archetypes of treatment resistance to VDZ in UC. This iSCST framework provides a powerful approach for analyzing FFPE tissues, offering insights into colitis-associated cellular networks and identifying biomarkers to enhance patient risk stratification in routine clinical workflows.
Authors
Elvira Mennillo, Madison L. Lotstein, Gyehyun Lee, Julian H. Hou, Vrinda Johri, Donna E. Leet, Christina A. Ekstrand, Jessica Tsui, Jun Yan He, Uma Mahadevan, Walter L. Eckalbar, Ryan M. Gill, Christopher J. Bowman, David Y. Oh, Gabriela K. Fragiadakis, Michael G. Kattah, Alexis J. Combes
Abstract
BACKGROUND. Sepsis is a leading cause of morbidity and mortality in critically ill children, yet heterogeneous immune responses complicate the development of targeted therapies and the host immune factors driving sepsis pathobiology remain unclear. METHODS. We integrated deep immune phenotyping, plasma proteomics, single-cell transcriptomics, and phosphoflow cytometry in a prospective cohort of 88 critically ill children to elucidate the mechanisms underlying immune heterogeneity. RESULTS. Unsupervised clustering of plasma cytokines identified three immunologic subgroups, including a high-severity group (“Group C”) characterized by hypercytokinemia driven by IL-6 and IFN-γ. Group C exhibited distinct alterations in immune cell frequency and activation, with a strong association between hyperinflammatory cytokine signaling and lymphocyte dysfunction. Single-cell RNA sequencing revealed transcriptional signatures of T cell activation and metabolic stress, with suppression of a lymphoid protective gene program across CD8⁺ T cell subsets. Despite increased expression of activation markers, T cell receptor repertoire analysis revealed no dominant clonotypes, consistent with bystander activation. Phosphoflow cytometry demonstrated baseline STAT1/STAT3 hyperactivation in Group C CD8⁺ T cells, which failed to respond to αCD3/αCD28/αCD49d stimulation. CONCLUSIONS. These findings define an IL‑6/IFN‑γ–driven endotype of T cell dysfunction in pediatric sepsis and highlight the JAK/STAT axis as a rational target for immunomodulatory therapy. FUNDING. K12HD047349, K23GM159013, K08AI135091, R01HD095976, Thrasher Research Foundation, Burroughs Wellcome Fund CAMS, Immune Deficiency Foundation, Primary Immune Deficiency Treatment Consortium, Barbara Brodsky Foundation, CHOP Research Institute
Authors
Robert B. Lindell, Samir U. Sayed, Jose S. Campos Duran, Sydney A. Sheetz, Apoorva Babu, Montana S. Knight, Andrea A. Mauracher, Ceire A. Hay, Peyton E. Conrey, Julie C. Fitzgerald, Nadir Yehya, Stephen T. Famularo III, Teresa Arroyo, Richard Tustin III, Hossein Fazelinia, Edward M. Behrens, David T. Teachey, Lisa R. Forbes Satter, Alexandra F. Freeman, Jenna R.E. Bergerson, Steven M. Holland, Jennifer W. Leiding, Scott L. Weiss, Mark W. Hall, Deanne M. Taylor, Rui Feng, E. John Wherry, Nuala J. Meyer, Sarah E. Henrickson
Abstract
Cardiac macrophages are broadly studied as two subtypes, tissue resident C-X3-C motif chemokine receptor 1 positive (CX3CR1+) that are also C-C motif chemokine receptor 2 negative (CCR2–), and monocyte derived CCR2+. Previous systemic loss of function approaches suggested unique roles for each subtype in the heart with CCR2+ being inflammatory and CX3CR1+ being pro-healing. Here we employed a cardiac-specific gain of function approach to selectively enhance either macrophage subtype. A robust increase in basal CCR2+ macrophages in the heart by targeted C-C motif chemokine ligand 2 (Ccl2) expression did not induce inflammation, cause fibroblast activation, or impair cardiac function. However, increased CCR2+ macrophages reciprocally diminished self-renewing tissue resident macrophages and worsened cardiac fibrosis due to pressure overload stimulation. Conversely, augmented expression of colony-stimulating factor-1 (Csf1) in the heart promoted selective expansion of resident CX3CR1+ macrophages, which exerted no pathophysiological consequences at steady-state. However, pressure overload in these mice with expanded CX3CR1+ macrophages showed a CCR2+ macrophage-dependent inflammation leading to exacerbated cardiac dysfunction, simultaneously still protecting from adverse remodeling and cardiac fibrosis. In conclusion, cardiac-specific selective enrichment of macrophage subtypes shows their intricate interplay and unique functional roles in regulating myocardial inflammation and fibrosis during hypertrophy and at homeostasis.
Authors
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
Abstract
Men with advanced prostate cancer are typically treated with androgen deprivation therapy, but most ultimately develop resistance and incurable disease (e.g. castration-resistant prostate cancer (CRPC)). The majority of CRPCs overexpress the epigenetic enzyme EZH2 and harbor alterations in the PI3K pathway, providing two targetable pathways outside of AR. Here we show that EZH2 inhibitors synergize with PI3K, AKT, or mTORC1 inhibitors to kill CRPC in vitro and promote tumor regression in vivo. Strikingly, these agents trigger a catastrophic energy crisis by cooperatively suppressing glycolysis, the TCA cycle, and oxidative phosphorylation prior to cell death. EZH2 and PI3K pathway inhibitors achieve this by respectively inhibiting two key regulators of metabolism, MYC and HIF-1A, while concomitantly derepressing a pro-apoptotic stress sensor. Together, these studies reveal a promising therapeutic strategy for CRPC and demonstrate how metabolic plasticity can be fatally impaired by co-targeting upstream oncogenic nodes that converge on this important process.
Authors
Rhea Sahu, Miriam Enos, Swastika Sharma, Amy E. Schade, Alycia Gardner, Akiko Yoshinaga, Alexandra Indeglia, Eleanor Minogue, Songhua Hu, Kiran Kurmi, Shakchhi Joshi, Daniel R. Schmidt, Samkyu Yaffe, Van T.M. Nguyen, Fang Xie, Steven P. Balk, Matthew G. Vander Heiden, Kristian Helin, Marcia C. Haigis, Karen Cichowski
Abstract
Clonal hematopoiesis (CH) is the age-related expansion of mutated hematopoietic stem cells without hematologic abnormalities. In patients with solid tumors, CH is associated with higher mortality and may evolve to therapy-related myeloid neoplasms; however, the mechanisms by which cancer treatments promote CH dynamics remain largely unknown. Here, we analyzed 392 serial samples from a prospective cohort of breast cancer patients and showed that cytotoxic treatments led to strong therapeutic bottlenecks, resulting in significant reductions in hematopoietic allelic populations and differential clonal selection. Positively selected CH that expanded through dose-dependent therapeutic bottlenecks harbored mutations in TP53, PPM1D, SRCAP, DNMT3A, and YLPM1. Patients with positively selected CH during treatment had the shortest progression-free and overall survival compared to patients with unchanging or negatively selected CH across all therapies. These findings, validated in independent breast cancer and pan-cancer cohorts, provide strong evidence for clinical relevance of monitoring CH during cancer treatment.
Authors
Mona Arabzadeh, Yi-Han Tang, Christelle Colin-Leitzinger, Sadegh Marzban, Daniel Walgenbach, Stefania Morganti, Vaidhyanathan Mahaganapathy, Erika Harper, Mingxiang Teng, Jacob K. Kresovich, Iman Washington, Heather A. Parsons, Judy E. Garber, Jeffrey West, Shridar Ganesan, Hossein Khiabanian, Nancy Gillis
Abstract
Exonic variants in Apolipoprotein-L1 (G1 and G2) are linked to increased risk of kidney disease as well as kidney transplant rejection. Outside of the association of these prevalent variants with African ancestry, underpinning causal mechanisms for rejection are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-APOL1 (G1), or G2-APOL1 (G2). Mice with either variant showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca2+-signaling. G1-CD8+T-cells demonstrated baseline ER-Ca2+ depletion followed by sustained increases in cytosolic-Ca2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca2+ chelation, or store-operated Ca2+ entry inhibition, and were relatively resistant to calcineurin antagonism compared to G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients that had elevated peripheral TCMs before transplantation, developed rejection despite significantly higher tacrolimus levels vs G0/G0 recipients. In summary, we unravel an excitatory mechanism for APOL1 variants in T-cells that causally links them to kidney rejection.
Authors
John Pell, EM Tanvir, Zeguo Sun, Irene Chernova, Anand Reghuvaran, Soichiro Nagata, Mateus T. Guerra, John Choi, Soltan Al Chaar, Hiroki Mizuno, Ke Dong, Xin Tian, Reika Ishibe, Barbara Franchin, Paolo Cravedi, Ashwani Kumar, Gabriel Barsotti, Hongmei Shi, Bony De Kumar, Shinobu Smithson, Wenzhi Song, John Cijiang He, Anita S. Chong, Jordan S. Pober, Stefan Somlo, Ian W. Gibson, Waldemar Popik, Zhongyang Zhang, Joseph Craft, Jamil Azzi, Naoka Murakami, Shuta Ishibe, Peter S. Heeger, Madhav C Menon
Abstract
Klinefelter syndrome (KS), the most common sex chromosome aneuploidy (affecting approximately 1 in 650 live male births), causes severe infertility. The extra X chromosome can impair the development of fetal germ cells, but its effects on somatic cells, especially the Leydig cells, are still not well known. We performed single-cell transcriptome analysis of fetal KS and control testicular cells, and found that two clusters of KS Sertoli cells with the XIST-negative cluster showing distinct gene expression pattern and abnormally increased G2/M ratio. Fetal KS Leydig cells showed increased proliferation and immature differentiation with high level of MAPK signaling pathway and X-linked EIF1AX. Inhibition of MAPK signaling partially rescued overproliferation and defective differentiation and androgen secretion in KS Leydig cells, while overexpression of EIF1AX recapitulated the phenotype of increased proliferation and decline in testosterone synthesis capacity in the Leydig cell line. These findings revealed the early pathological mechanisms of KS somatic cells, and lay the groundwork for developing novel early intervention strategies.
Authors
Tong Yan, Guancheng Chen, Jie Zhang, Wenjing Jia, Nan Lu, Shuping Jin, Haotian Zhang, Yichen Zhao, Lu Jiang, Jing Wu, Qing Liu, Chenghao Situ, Hui Zhu, Yan Li, Quan Wang, Xiaoyu Yang, Chao Qin, Xiaofeng Song, Qing Cheng, Xuejiang Guo
Abstract
Studies with a candidate vaccine deleted in glycoprotein D (ΔgD-2) for herpes simplex virus (HSV) prevention uncovered a role for herpes virus entry mediator (HVEM) in mediating antibody-dependent cell-mediated killing (ADCK) of virally-infected cells. Antibodies elicited by ΔgD-2 passively protect wild-type but not Fc gamma receptor (FcγR) or HVEM knockout (KO) mice. The goals of this study were to identify which cells mediate ADCK and the role of HVEM signaling. Using HVEM ligand and conditional cell-type specific HVEM KO mice combined with in vitro mouse and human cytolytic assays, we demonstrate that ADCK of HSV-infected cells is mediated primarily by neutrophils and requires their expression of HVEM and its ligand, LIGHT. Cytolysis is not associated with granzyme and perforin production but occurs by a trogocytosis-like pathway. Pharmacological inhibition of myosin light-chain kinase (MLCK), which mediates trogocytosis, inhibits cytolysis. Similar results were obtained when human neutrophils were cocultured with HSV-infected cells opsonized with ADCK-containing human immune serum or with breast cancer cells treated with an anti-HER2 trogocytosis mediating antibody. Killing was significantly reduced when an MLCK inhibitor or blocking antibodies to CD16a, HVEM, or LIGHT were added. Together these results define a mechanism of HVEM-enhanced FcγR-mediated neutrophil-dependent ADCK of targets cells.
Authors
Matthew S. Gromisch, Masayuki Kuraoka, Carl F. Ware, Steven C. Almo, Betsy C. Herold
Abstract
Immune checkpoint blockade (ICB), including PD-1/PD-L1 inhibitors, has transformed cancer therapy but benefits only a subset of patients. Understanding how PD-L1 is regulated and identifying strategies to overcome resistance remain critical. Here, we identify SIRT2 as a key positive regulator of PD-L1 across multiple human cancers. Unexpectedly, SIRT2 does not act at the transcriptional level but stabilizes PD-L1 protein by preventing ubiquitin-mediated degradation. Mechanistically, SIRT2 maintains the protein stability of USP22, a PD-L1 deubiquitinase. Loss of SIRT2 reduces USP22 levels, whereas ectopic USP22 fully rescues PD-L1 expression and reverses the enhanced antitumor immunity induced by SIRT2 inhibition. We further show that SIRT2 directly deacetylates USP22 at lysines 382 and 505 within its catalytic domain, promoting USP22 deubiquitinase activity and protecting both itself and its substrates from degradation. Our findings reveal a molecular mechanism by which an acetylation–deacetylation switch dynamically regulates deubiquitinase catalytic activity. Therapeutically, SIRT2 inhibition synergizes with PD-1/PD-L1 blockade and USP22 inhibition to enhance antitumor immunity. Consistently, protein but not mRNA levels of SIRT2, USP22, and PD- L1 positively correlate in human bladder cancer and melanoma. Together, these findings define a SIRT2–USP22–PD-L1 axis driving tumor immune evasion and highlight SIRT2 as a promising target to improve ICB efficacy.
Authors
Na Li, Qiong Gao, Huijun Jia, Guoqing Xue, Yuanzhang Zhou, Shengnan Wang, Suxian Ma, Bingjin Hu, Zhuoyue Zhao, Chen Su, Yinghong Liu, Wenxuan Xi, Zhonghao Li, Donna D. Zhang, Peng Chu, Zhaolin Sun, Deyu Fang
Abstract
Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma in adults and is characterized by a hypoxic and immunosuppressive tumor microenvironment (TME). Bone marrow-derived tumor-associated macrophages (TAMs) dominate the immune landscape in GBM and are recruited to the peri-necrotic niche following the onset of necrosis. CLEC5A has the strongest association with poor clinical outcome among immune-related genes in GBM, and is preferentially expressed in hypoxic, peri-necrotic TAMs. CLEC5A overexpression promotes TAM polarization toward an immunosuppressive phenotype, and secretion of immunoregulatory cytokines. Using an RCAS/tv-a GBM model with bone marrow transplantation from Clec5a-/- donor mice, we demonstrated that CLEC5A loss prolongs survival, delays tumor progression, and attenuates TME immunosuppression. Mechanistically, podoplanin (PDPN) expressed on glioma cells directly engages CLEC5A and triggers downstream Syk-JAK-STAT3 signaling in TAMs. Pharmacologic Syk inhibition suppresses glioma growth, diminishes TAM infiltration and polarization, reverses the immunosuppressive TME, and prolongs survival in vivo. Collectively, our findings indicate that the PDPN-CLEC5A-Syk-STAT3 axis orchestrates TAM polarization and TME immunosuppression in the peri-necrotic niche of GBM, highlighting CLEC5A/Syk as a promising therapeutic target for reversing the immunosuppressive TME and improving outcomes.
Authors
Jiabo Li, Xuya Wang, Luqing Tong, Bo Feng, Ling-kai Shih, Steven M. Markwell, Hannah Nuszen, Tomasz Gruchala, Nicholas G. Lam, Petros Basakis, Erika Ruiz-Yamamoto, Deyu Fang, Roger Stupp, Xuejun Yang, Daniel J. Brat
Abstract
BACKGROUND. Right ventricular failure (RVF) is a major determinant of mortality in pulmonary arterial hypertension (PAH), and hepatic dysfunction predicts adverse outcomes. However, the cell-specific effects of PAH/RVF on the human liver remain poorly defined. METHODS. We performed single-nucleus RNA sequencing of autopsy-derived liver tissue from 5 PAH patients and 4 non-PAH controls and compared these findings with publicly available single-nucleus RNA sequencing datasets from non-alcoholic steatohepatitis (NASH) and Fontan-associated liver disease (FALD). Transcriptomic analyses were integrated with histologic assessment, mitochondrial-enriched proteomics, and correlated with clinical markers of PAH/RVF severity. RESULTS. PAH livers showed cell-specific metabolic, inflammatory, and fibrotic remodeling distinct from NASH and FALD. PAH hepatocytes exhibited a hypoxia-adapted, Warburg-like metabolic phenotype with reduced fatty acid metabolism, gluconeogenesis, cytochrome P450 activity, and ketone metabolism. PAH endothelial cells demonstrated increased glycolytic pathway activity and disrupted adhesion/barrier signaling. PAH hepatic stellate cells displayed HIF-1 and PI3K-Akt pathway activation, and increased IL6 expression, which resulted in central vein fibrotic remodeling. PAH macrophages showed complement activation with reduced JAK-STAT signaling. Finally, HSC HIF-1 activity correlated with clinical markers of PAH/RVF severity. CONCLUSION. PAH induces a distinct metabolic and inflammatory hepatopathy characterized by hepatocyte metabolic reprogramming, HSC activation, and macrophage complement signaling. These findings support PAH-associated hepatopathy as a disease-specific end-organ phenotype linked to RVF severity.
Authors
Madelyn J. Blake, Sally E. Prins, Jeffrey C. Blake, Lynn M. Hartweck, Jenna B. Mendelson, Steeve Provencher, Sandra Breuils-Bonnet, Sebastien Bonnet, Kurt W. Prins
Abstract
Cancers reflect aberrant growth and differentiation of normal cell populations. Biological understanding of small intestine neuroendocrine tumors (SI-NETs) is hampered because their closest normal counterparts, enteroendocrine cells (EECs), constitute tiny fractions of intestinal epithelium. Recent characterization of adult human EEC ontogeny from intestinal stem cells can help overcome that limitation. Transient expression of transcription factor gene ASCL1 normally ensures proper timing and fidelity of well-differentiated EECs, which express NEUROD1. Here we report that SI-NETs resembled mature enterochromaffin cells; however, individual tumor cells co-expressed stem/progenitor genes, harboring each differentiation state along the EEC trajectory except ASCL1+ precursors. We found that enhancers normally active, and others inactive, during EEC differentiation underlie aberrant SI-NET gene activity. SI-NETs uniformly expressed NEUROD1 but lacked ASCL1, owing to inaccessible chromatin and repressive H3K27me3 marking at the ASCL1 locus. Multiple cyclin-dependent kinase inhibitor (CDKi) genes were similarly silenced, other than CDKN1B, the only gene recurrently mutated in SI-NETs. Deletion of CDKN1B altered cell cycle kinetics during human EEC differentiation, and deletions of ASCL1 or CDKN1B activated certain genes that are expressed in SI-NETs but not in the normal EEC trajectory. We propose that a limited CDKi repertoire and absence of ASCL1-dependent constraints on EEC maturation together explain unique SI-NET characteristics.
Authors
Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer Chan, Bradley E. Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani
Abstract
We previously identified a muscular dystrophy caused by biallelic variants in JAG2, whose protein product Jagged2 JAGGED2 (JAG2) is a canonical Notch NOTCH ligand. However, the disease mechanism remains unclear, particularly with respect to muscle stem cell (MuSC) function and muscle regeneration. We examined the consequences of JAG2 deficiency and modeled pathogenic JAG2 variants in vitro and in vivo, the latter in mouse and fly models and with particular attention to the MuSC-muscle endothelial cell (MuEC) niche. We found that both Jag2 deficiency and overexpression of pathogenic JAG2 variants impaired NOTCHNotch signaling and myogenic self-renewal and differentiation. Hypomorphic Jag2 mutant (Jag2sm) mice display depleted MuSCs, corresponding with impaired muscle regeneration in those mice. Co-culture experiments and the examination of cell-type-specific Jag2 conditional knockout mice demonstrated that MuEC-specific Jag2 knockout resulted in reduced MuSC self-renewal, while MuSC-specific Jag2 knockout resulted in reduced myogenic differentiation. Human reference JAG2, but not human pathogenic variants of JAG2, rescued the deficiency of Serrate (Ser), the Drosophila ortholog of JAG2. Therefore, pathogenic variants in JAG2 impair muscle development and regeneration through disrupted cell-autonomous cis-inhibition and non-autonomous trans-activation involving NOTCHNotch signaling dysfunction. Our findings indicate that optimizing JAG2-mediated NOTCHNotch signaling is a potential therapeutic approach for JAG2-related muscular dystrophy.
Authors
Minoru Tanaka, Nam Chul Kim, Isabelle Draper, Hannah R. Littel, Mekala Gunasekaran, Johnnie Turner, Natalya M. Wells, Qasim Mujteba, Yoko Asakura, Peter B. Kang, Atsushi Asakura
Abstract
Dysfunctional intestinal fibrosis is an irreversible complication of Crohn’s disease (CD), The complex heterogeneity of intestinal mesenchymal cells makes it difficult to understand the pathogenesis of intestinal fibrosis. Previously, we identified Meflin as a marker of fibroblast subsets. This study aimed to explore the role of Meflin-positive fibroblasts in intestinal fibrogenesis and investigate the potential of pharmacological control of Meflin expression as a treatment for patients with CD. Our results indicated that Meflin expression was upregulated in fibroblasts at the early stage of fibrosis but was downregulated in established fibrosis in both patients with CD and two different mouse models, which are the chronic dextran sodium sulfate (DSS) model and an interleukin-10-deficient model that spontaneously develops intestinal inflammation. Meflin-deficient mice exacerbated intestinal fibrosis with dysregulated expression of non-canonical Wnt ligand WNT5A and its receptor ROR2. Pharmacologically induced Meflin expression through the administration of a synthetic retinoid reversed intestinal fibrosis in the DSS model and suppressed pro-fibrotic protein secretion in fibroblasts isolated from patients with CD. Our findings indicate that Meflin-positive fibroblasts represent a functional subpopulation that suppresses intestinal fibrosis. Augmentation of Meflin expression shows antifibrotic effects and holds promise as a therapeutic approach for intestinal fibrosis in patients with CD.
Authors
Jingxi Mu, Keiko Maeda, Tadashi Iida, Shinji Mii, Nobutoshi Esaki, Yukihiro Shiraki, Yasuyuki Mizutani, Masanao Nakamura, Takeshi Yamamura, Tsunaki Sawada, Eri Ishikawa, Kentaro Murate, Takashi Hirose, Kazuhiro Furukawa, Akina Oishi, Haruhiko Suzuki, Takayoshi Kishida, Goro Nakayama, Mitsuhiro Fujishiro, Hiroki Kawashima, Atsushi Enomoto
Abstract
BACKGROUND. Minimally invasive biomarkers predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. METHODS. Using patients from a prospective, multi-institutional phase II trial, we performed whole-genome sequencing of 185 longitudinal plasma cell-free DNA (cfDNA) samples from 68 patients with locally advanced, surgically resectable HNSCC who received neoadjuvant and adjuvant pembrolizumab. We developed the regional motif diversity score (rMDS), a fragmentomic metric that quantifies the entropy of cfDNA 5′-end motifs across genomic regions. RESULTS. Unsupervised analysis showed rMDS robustly distinguished responders from non-responders, outperforming established fragmentomic metrics and copy number alterations while remaining independent of technical confounders. Longitudinal rMDS changes localized to regions enriched for immune-, lectin-, and keratinization-related genes — hallmarks of squamous cell carcinoma — reflecting tumor–peripheral immunity interplay during treatment. The most dynamic regions clustered at telomere-proximal loci, suggesting a link between telomere biology and cfDNA fragmentation. An rMDS-based machine learning classifier achieved AUC 0.89–0.99 across validation settings, with the highest accuracy post-treatment, outperforming PD-L1 expression and tumor fraction in matched samples. Predicted responders showed improved disease-free survival (log-rank P = 0.035; HR 2.67, 95% CI 1.03–6.92). CONCLUSION. rMDS represents a biologically meaningful, clinically actionable biomarker for immunotherapy response in HNSCC, supporting integration into future risk assessment frameworks. TRIAL REGISTRATION. ClinicalTrials.gov NCT02641093. FUNDING. NHGRI R56HG012360 and startup funds from Cincinnati Children’s Hospital Medical Center, Northwestern University, and Robert H. Lurie Comprehensive Cancer Center (Y.L.); Science Olympiad Alumni Research Grant, Science Olympiad USA Foundation (R.B.); Merck Sharp & Dohme Corp. (T.W.D.).
Authors
Ravi Bandaru, Hailu Fu, Haizi Zheng, Jocelyn Liang, Li Wang, Shuchi Gulati, Benjamin H. Hinrichs, Mingxiang Teng, Bin Zhang, Masha Kocherginsky, De-Chen Lin, David A. Hildeman, Francis P. Worden, Matthew O. Old, Neal E. Dunlap, John M. Kaczmar, Maura L. Gillison, Dalia El-Gamal, Trisha Wise Draper, Yaping Liu
Abstract
The global prevalence of metabolic dysfunction-associated steatohepatitis (MASH) is rising, driven by a complex interplay of metabolic disturbances, inflammation, and fibrosis, yet effective treatment options remain limited. This study examined the relationships among intestinal microbial dysbiosis, ammonia production, and hepatic CD8+ T cell activity in MASH, and assessed the therapeutic potential of DT-109, a glycine-based tripeptide. We investigated the gut-liver axis across human cohorts and both non-human primate and mouse MASH models. Multi-omics approaches were used to characterize ileal microbiota, ammonia levels, and hepatic immune and metabolic pathways. Causality was verified through microbiota transplantation, C. perfringens NirA-knockout mutants, and functional validation in vitro and in vivo. The efficacy of DT-109 was evaluated in non-human primates and mice. Our results revealed a significant increase in the ammonia-producing gut bacterium C. perfringens, which led to elevated intestinal ammonia and disruption of the intestinal barrier in MASH. Elevated ammonia levels triggered FosB-mediated upregulation of chemokine C-C motif ligand 5 (CCL5) in CD8+ T cells, which in turn drove T cell cytotoxicity in the liver. Notably, DT-109 effectively lowered C. perfringens abundance, reduced intestinal ammonia, restored intestinal barrier integrity, and alleviated CD8+ T cell dysregulation in MASH. These results identify a distinct mechanism in which gut-derived ammonia drives CD8+ T cell-mediated MASH and demonstrate that DT-109 effectively targets this axis by inhibiting C. perfringens and reducing ammonia, ultimately ameliorating MASH.
Authors
Pengxiang Qu, Shusi Ding, Yanru Zhang, Yang Zhao, Erfei Song, Liangshuo Hu, Ruike Ding, Wenbin Cao, Yiting Hou, Jia Qi, Juan Zhao, Chenjing Duan, Shuangqing Liu, Chong Shen, Ying Zhao, Yanhong Guo, Zuowen Zheng, Shiwei Luo, Huizhong Hu, Liang Bai, Sihai Zhao, Bo Wang, Shuixiang He, Yi Wu, Xuelian Xiong, Qiutong Wu, Weiwang Gu, Oren Rom, Aimin Xu, Lemin Zheng, Jifeng Zhang, Enqi Liu, Y. Eugene Chen
Abstract
Cellular plasticity is a hallmark of cancer, enabling tumor cells to alter identity and evade therapeutic pressure. In invasive mucinous adenocarcinoma of the lung (IMA), NK2 homeobox 1 (NKX2-1) loss triggers a pulmonary to gastric switch marked by aberrant activation of hepatocyte nuclear factor 4 alpha (HNF4α), a master regulator of gastrointestinal/hepatic differentiation. We show that HNF4α promotes IMA growth and activates a gastric pit cell-like program. Loss of HNF4α enables forkhead box A1/A2 (FoxA1/2) transcription factors to bind de novo sites and activate alternative, non-gastric identities in IMA. HNF4α also establishes a mucinous program associated with tolerance to KRAS blockade, and loss of HNF4α enhances response to KRASG12D inhibition. Mechanistically, HNF4α blocks cell cycle exit in drug-tolerant persister cells and promotes activity of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). NRF2 activation partially rescues effects of Hnf4a deletion on KRASG12D inhibition, whereas NRF2 inhibition enhances sensitivity to KRASG12D blockade. Thus, HNF4α is a key regulator of growth, identity, and primary response to KRASG12D inhibition in IMA.
Authors
Headtlove Essel Dadzie, Yangsook Song Green, Soledad Camolotto, Henry U. Arnold, Matthew Gumbleton, Minzhe Guo, Mari Mino-Kenudson, Yutaka Maeda, Benjamin T. Spike, Eric L. Snyder
Abstract
Immune checkpoint inhibitor-induced inflammatory arthritis (ICI-IA) significantly impairs cancer therapy and patient quality of life, yet its pathogenic mechanisms remain unclear. Through integrated single-cell multi-omics analysis of paired peripheral blood, synovial fluid, and tumor samples from longitudinal ICI-IA cohorts and matched controls, we identified a unique regulatory T-cell (Treg) population co-expressing CD137 and IL-6R (AtpTreg). These cells exhibited reduced immunosuppressive capacity while aberrantly producing high level of IL-17 and promoting proinflammatory responses of synoviocytes. AtpTreg exhibits shared clonotypes and phenotypes across tissue compartments. Notably, AtpTreg frequency correlates with increased arthritis severity yet paradoxically associates with improved overall survival. Anti-IL6R therapy reduced AtpTreg levels, corresponding with improved arthritis outcomes and quality of life, without compromising anti-tumor immunity. Our findings define a pathogenic Treg subset in ICI-IA and validate IL-6R blockade as a mechanism-based therapeutic strategy, bridging mechanistic discovery to clinical translation. This study is registered at ClinicalTrials.gov (NCT07357636).
Authors
Yifei Ma, Nianqi Liu, Yan Li, Denghan Zhang, Shaohui He, Jun Lv, Yongluo Jiang, Guangmin Jian, Jingyao Zhang, Pengfei Zhu, Yue Ma, Jiacai Lin, Jin Li, Tong Wu, Yiwei Xu, Xiajie Lyu, Youlong Wang, Yiming Li, Yu Si Niu, Zhenyun Guo, Churong Lin, Ningnan Fang, Wei Jiang, Lihong Wang, Mengqin Yuan, Shenyue Wang, Shulin Huang, Qi Huang, Jinjian Li, Jun Lu, Bocen Chen, Guanqing Zhong, Haizhou Liu, Fadian Ding, Shangeng Weng, Rui Li, Ao Zhang
Abstract
Based on the observation that loss-of-function mutations of KMT2C and KMT2D (KMT2C/D) are enriched and co-occur in gastric adenocarcinoma, we developed genetically engineered mouse model (GEMM) to conditionally knock out Kmt2c and Kmt2d in gastric epithelial cells. We observed that Kmt2c/d loss led to nuclear dysplasia, cellular crowding, and expansion of cells with mixed gastric lineage markers. When combined with Pten deletion, Kmt2c/d loss drove rapid development of muscle-invasive gastric adenocarcinoma as early as 3 weeks post Cre-mediated gene deletion. The adenocarcinoma exhibited decreased expression of gastric lineage markers and increased expression of intestinal differentiation markers, phenocopying human intestinal type gastric adenocarcinoma. Bioinformatic integration of single cell RNA-seq of our GEMMs and human gastric cancer datasets shows co-clustering of normal and of cancerous gastric epithelial cells. Kmt2c/d knockout in gastric epithelium reduced protein synthesis but upregulated transcription of ribosomal proteins, rendering the cells to be hypersensitive to mTORC1 inhibitors. Additionally, Kmt2c/d knockout increased MHC-I molecule expression and enhanced antigen presentation. Combination of mTORC1 inhibition and anti-PD1 immunotherapy markedly suppressed tumor growth in immune-competent mice. Together, these findings reveal the role of Kmt2c/d loss in gastric cancer initiation and suggest the potential therapeutic strategies for KMT2C/D-deficient gastric cancer.
Authors
Naitao Wang, Dan Li, Tao Zhang, Mohini R. Pachai, Dana M. Schoeps, Yudi Bao, Woo Hyun Cho, Makhzuna N. Khudoynazarova, Kae Kristoff, Marion Liu, Laura Tang, Yelena Y. Janjigian, Ping Chi, Yu Chen
Abstract
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease marked by progressive motor deficits and Purkinje cell (PC) degeneration, driven by polyglutamine expansion in ataxin-1. While oligodendroglial dysfunction precedes PC loss, its direct contribution toward SCA1 pathogenesis remains unclear. Here, using an oligodendroglia-specific SCA1 conditional knock-in mouse model, we demonstrate that mutant ataxin-1 in oligodendrocytes is sufficient to drive aspects of SCA1-related pathology, including dysregulated myelination, PC axonal shrinkage, and torpedo formation, ultimately impairing motor coordination. Transcriptomic analysis uncovers cerebellar oligodendrocyte subtypes with distinct gene expression signatures and aberrant abundance that contribute to demyelination. This, compounded by a progressive decline in the neuroprotective functions of a cerebellar-specific oligodendrocyte subtype, establishes a critical link between demyelination, axo-myelinic dysfunction, and axonal pathology in SCA1. Upstream transcriptional regulator analysis in oligodendroglia identifies TCF7L2 and HTT as key mediators of oligodendroglial dysfunction in SCA1, suggesting shared pathogenic mechanisms with other polyglutamine diseases. Collectively, these findings establish oligodendroglia as key mediators of SCA1 pathogenesis and underscore their critical role in preserving PC axonal integrity.
Authors
Changwoo Lee, Rosalie M. Grijalva, Leon Tejwani, Eunwoo Bae, Alison Chase, Hannah Ro, Hannah Kim, Victor Olmos, James P. Orengo, Janghoo Lim
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)