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Abstract
Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle has been reported to generate potent short-term pacemaker function in various animal models. Here, we investigated the impact of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcomes to those of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activity nor augmented Hcn2-mediated pacing. In conclusion, at functionally non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.
Authors
Jianan Wang, Mathilde R. Rivaud, Mischa Klerk, Arie R. Boender, Ruud N. Visser, Rinske Sparrius, Hee Young Lee, Karel van Duijvenboden, Huiling Zhou, Yuting Yang, Emiel J.M. Kramer, Kyung Ho Park, Larry C. Park, Silke Schrödel, Christian Thirion, Eric Ehrke-Schulz, Anja Ehrhardt, Osne F. Kirzner, Klaus Neef, Hanno L. Tan, Arie O. Verkerk, Vincent M. Christoffels, Gerard J.J. Boink
Abstract
De novo heterozygous variants in CELF2 have recently been associated with a rare neurodevelopmental disorder, yet the mechanisms linking specific variants to distinct clinical phenotypes remain poorly understood. Here, we reported a cohort of 18 individuals and provided evidence that variants causing CELF2 mislocalization, but not protein-null variants, were associated with seizures. Using proband-derived human cortical neurons and transgenic mouse models, we demonstrated that CELF2 underwent activity-dependent nucleocytoplasmic shuttling in excitatory neurons and that its cytoplasmic retention caused neuronal hyperactivity, elevated seizure susceptibility, and learning and memory deficits. We further found that cytoplasmic CELF2 regulated mRNAs critical for synaptic function and neuronal excitability and implicated in epileptic seizures and intellectual disability. Drug screening further identified AKT signaling as a key regulator of CELF2 nucleocytoplasmic shuttling and a candidate target for reversing neuronal hyperactivity. Together, our findings expand the clinical and genetic spectrum of CELF2-related neurodevelopmental disorders and establish a variant-specific mechanism that links CELF2 mislocalization to neuronal hyperactivity, seizures, and cognitive impairment.
Authors
Michelle Hua, Mohamad-Reza Aghanoori, Melissa J. MacPherson, Yi Ren, Shehani V. Siripala, Yifan Yang, Yvonne Yan Yan Or, Malea Nguyen, Robert Duba-Kiss, Daniel Feng, Laura Williams, Christopher J. Gafuik, GengYi Wang, Chloe Quelin, Boris Keren, Sarah Schuhmann, Georgia Vasileiou, Alexia Bourgois, Antonio Vitobello, Christophe Philippe, Zornitza Stark, Richard J. Leventer, George McGillivray, Frederic Tran Mau-Them, Marine Tessarech, Clément Prouteau, Phillis Lakeman, Mahdi M. Motazacker, Donald R. Latner, Raymond C. Caylor, Yvette van Ierland, Eloise Prijoles, Angie Lichty, Evangelos Theodorou, David A. Sweetser, Edward Steel, Jan Cobben, Majed J. Dasouki, Daniel G. Calame, Bertrand Isidor, Benjamin Cogné, Mitchell Kesler, Brooke Rackel, Isabel Clark, Deborah M. Kurrasch, G. Campbell Teskey, James Ellis, Guiqiong He, Scott D. Ryan, Douglas J. Mahoney, A. Micheil Innes, Jonathan R. Epp, Guang Yang
Abstract
Chromatin remodeling is a dynamic epigenetic process that alters chromatin structure to gauge gene accessibility, enabling precise spatiotemporal gene expression, with disruptions often underlying neurodevelopmental disorders (NDDs), although the mechanistic underpinning remains incompletely understood. Despite essential roles in chromatin remodeling processes such as DNA methylation, and histone acetylation and deposition, DMAP1 has not been implicated in human disease. We identified 20 individuals from 16 families with a syndromic NDD carrying homozygous or compound heterozygous variants in DMAP1. Neural-specific knockdown of its Drosophila ortholog, dDMAP1, caused pupal lethality, structural defects in the mushroom body (MB), decreased dendrite length, abnormal social behavior and mechanical-induced seizures. Human reference DMAP1 could largely compensate for the loss of dDMAP1 in knockdown flies, whereas patient variants failed to restore or differentially rescued the phenotypes, confirming their pathogenicity with differing severity. Transcriptome profiling of dDMAP1 knockdown fly brains nominated Cbl and SF1 as downstream targets. Their overexpression rescued the aforementioned lethality and MB defects. Finally, a DNA methylation episignature was identified, leading to the molecular diagnosis of an additional patient. Our findings demonstrate that biallelic inactivating variants in DMAP1 cause a syndromic NDD, expanding the short list of recessive disease-causing genes within the epigenetic machinery.
Authors
Qin Wang, Andrew K. Sobering, Christian Tirrito, Sadegheh Haghshenas, Tina Duelund Hjortshøj, Konrad Platzer, Silke Redler, Michael E. March, Leticia S. Matsuoka, Hang Xi, Josiah Zoodsma, Yuanhua Chen, Mari Mori, Marco L. Leung, Nathalie Couque, Alain Verloes, Antoine Pouzet, Noor A.A. Giesbertz, Marleen E.H. Simon, Ashley K. Yearwood, Dominique L. Assing, Tzung-Chien Hsieh, Jing-Mei Li, Michael A. Levy, Jennifer Kerkhof, Haley McConkey, Jessica Rzasa, Carolyn Lauzon-Young, Raashda A. Sulaiman, Firdous Abdulwahab, Hanan E. Shamseldin, Naif A.M. Almontashiri, Manal Afqi, Vettaikorumakankav Vedanarayanan, Maria J. Guillen Sacoto, Ingrid M. Wentzensen, Nadirah S. Damseh, Rivka Birnbaum, Babeth van Ommeren, Saskia M.J. Hopman, Maha S. Zaki, Gehad Elmakkawy, Erum Afzal, JiHye Kim, Stephanie Efthymiou, Henry Houlden, Ambreen Nusrat, Mathias Toft, Uzma Abdullah, Zafar Iqbal, Shannon Terek, Fowzan S. Alkuraya, Elizabeth J. Bhoj, Reza Maroofian, Bekim Sadikovic, Hakon Hakonarson, Yuanquan Song, Dong Li
Abstract
Obesity is associated with impaired wound healing, but the mechanisms linking excess adiposity to aberrant tissue repair remain unresolved. Heterotopic ossification (HO) is a severe example of pathologic tissue repair in which mesenchymal progenitor cells (MPCs) undergo aberrant osteochondral differentiation within soft tissue, leading to joint contractures and pain. Here, we show that accumulation of dietary omega-6 (ω-6) lipids in the injury site is a key mechanism linking obesity to HO. Specifically, in mice fed a high-fat diet (HFD), injured tissues were enriched in linoleic and arachidonic acids, providing substrate for myeloid cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production. PGE2 then drove a transcriptional program in mesenchymal progenitor cells that promoted osteochondral differentiation. An isocaloric, low linoleic acid HFD reduced HO despite comparable obesity, demonstrating that dietary lipid composition, rather than adiposity alone, drove pathological repair. Clinical data mirrored these findings, showing that obesity conferred increased HO risk, and COX-2 inhibition reduced HO exclusively in obese patients. Together, these findings identify injury site ω-6 lipid enrichment as the key signal linking the diet to MPC reprogramming, pointing to dietary lipid modulation as an actionable strategy to limit HO in obesity.
Authors
Stefanie L. Moye, Monisha Mittal, Tarun Srinivasan, Sneha Korlakunta, Chase A. Pagani, Ayelet Dar, Oromo Geshow, Dylan Feist, Lauren G. Zacharias, Zhao Li, Aaron W. James, Gerta Hoxhaj, Andrew M. Smith, Katherine A. Gallagher, Thomas P. Mathews, Robert J. Tower, Benjamin Levi
Abstract
Enhanced TGFβ signaling caused by mutations in Fibrillin-1 (FBN1) in patients with Marfan syndrome (MFS) leads to myxomatous degeneration of the mitral valve (MDMV). MDMV can result in mitral valve prolapse, severe regurgitation, and sudden cardiac death. However, it remains unknown whether lymphatic vessel (LV) dysfunction contributes to MDMV development in MFS. Here, we show that lymphangiogenesis in murine mitral valves (MVs) begins postnatally. However, this process is inhibited in a mouse MFS model, Fbn1 mutant (Fbn1C1039G/+) mice, accompanied by disrupted lymphatic cell-cell junctions, impaired lymphatic drainage, and an abnormally widespread distribution of MHCII+ infiltrating macrophages. Treatment of Fbn1 mutant mice with VEGF-C156S, a selective VEGFR3 agonist, stimulates the ERK and Akt pathways, increases LV density in MVs, and ameliorates MDMV. Fbn1 mutant MVs display disorganized valvular endothelial cells (VECs) and decreased expression of the anti-inflammatory modulator Zfp36 (zinc finger protein 36) in VECs and immune cells. Treatment with FTY720 (Fingolimod), a ZFP36 activator and S1P antagonist, rescues MDMV phenotypes in Fbn1 mutant mice by reducing immune cell infiltration and restoring lymphatic cell junctions and drainage. These findings suggest that the Fbn1 mutation causes LV hypoplasia and defective lymphatic drainage in MVs, driven in part by pro-inflammatory VECs, leading to MFS-related MDMV.
Authors
Can Tan, Ziyou Ren, Shreya Kurup, Xianpeng Liu, Zhi-Dong Ge, Shodai Suzuki, Pritika Jakka, Cheryl Tang, M. Luisa Iruela-Arispe, Tsutomu Kume
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
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ISSN: 0021-9738 (print), 1558-8238 (online)