Cell biology
Abstract
Background: The molecular landscape of lung adenocarcinoma (LUAD) is often illustrated as a driver-oncogene “pie chart,” but identical mutations exhibit heterogeneous signaling shaped by co-mutations, transcriptional programs, and lineage context. We propose a lineage-integrated signaling framework using an EGFR mutation signature (mSig). Methods: We defined EGFR mSig using differentially expressed genes in EGFR-mutant (mt) LUADs. Semi-supervised clustering and machine learning models were used to test reproducibility in different combinations of datasets. We analyzed molecular subtypes, lineage markers, co-occurring mutations and EGFR copy number alterations in EGFR mSig-defined subtypes of LUAD. Results: EGFR mSig showed robust classification performance (AUROC = 0.83-0.95; mean NPV = 96.3%). Validated gene expression subtypes and lung lineage markers were closely aligned with EGFR mSig status. Most EGFR mSig(+) tumors, including many without EGFR mutations belonged to Bronchioid subtype. A subset of canonical RAS mutations were mSig(+) and mirrored the EGFR mutation pattern. EGFR wild-type (WT)/mSig(-) tumors were enriched for non-Bronchioid subtypes and had co-mutations in TP53 or RAS/RAF/RTKs. We highlighted a parsimonious collection of coordinated mutations identified including RAS, KEAP1, STK11, TP53, and CDKN2A, supportive of prior reports. Conclusions: A novel EGFR mSig that captures the transcriptional footprint of EGFR activation revealed a subset of EGFR WT LUADs with “mt-like” features. mSig refines LUAD taxonomy beyond mutation-only pie-chart models by incorporating lineage and co-mutation context. Lineage-directed stratification with co-alteration identifies clinically relevant groups across EGFR and RAS states and highlights new treatment opportunities for patients currently considered “oncogene-negative.” Funding: NCI U01CA272541, R01CA262296, U24CA264021, UG1CA233333, R01CA211939.
Authors
Minjeong Kim, Wisut Lamlertthon, Heejoon Jo, Yan Cui, Miyeon Yeon, Hyo Young Choi, Katherine A. Hoadley, Matthew P. Smeltzer, Michele C. Hayward, Matthew D. Wilkerson, Liza Makowski, D. Neil Hayes
Abstract
Moderate hyperoxia (30–60% O₂) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via airway smooth muscle (ASM), a key regulator of bronchoconstriction, bronchodilation, and remodeling. Understanding how O2 exposure drives long-term bronchial changes in prematurity is critical for developing therapies for airway disease across the lifespan. Premature lungs have immature antioxidant defenses, potentially due to disrupted mitochondrial dynamics, increasing susceptibility to O2-induced oxidative stress. Thus, mitochondrial homeostasis is highly relevant to ASM dysfunction and airway disease. We propose that hyperoxia in prematurity promotes mitochondrial dysfunction, and that the gasotransmitter hydrogen sulfide (H₂S) mitigates O2-induced mitochondrial damage in developing ASM. Human fetal ASM (fASM) were exposed to moderate hyperoxia to investigate the effects of exogenous H₂S donors (GYY4137, AP39) and stabilization of cystathionine β-synthase (CBS), an H₂S biosynthetic enzyme, on mitochondrial structure and function. Hyperoxia impaired fASM mitochondrial integrity, while H₂S donors in particular, or CBS stabilization attenuated adverse O2 effects on mitochondrial morphology, reactive oxygen species, respiration, calcium regulation, and contractility. These findings highlight the therapeutic potential of H₂S in the premature lung exposed to moderate hyperoxia.
Authors
Colleen M. Bartman, Michael Thompson, Samantha K. Hamrick, Niyati A. Borkar, Daniel Pfeffer-Kleemann, Preetham Ravi, Marta Schiliro, Yak Nak, Christian Vivar Ramon, Li Drake, Y. S. Prakash, Christina Pabelick
Abstract
Authors
Caitlin M. Pavelec, Leigh A. Bradley, Priyanka Rawat, Luke S. Dunaway, Maya Bolger-Chen, Bethany A. Gholson, Jonathan R. Lindner, Brant E. Isakson, Norbert Leitinger, Matthew J. Wolf
Abstract
While the pathogenic role of autoantibodies targeting the podocyte protein THSD7A in membranous nephropathy (MN) is well described, the consequences of autoantibody binding for podocyte homeostasis and the function of THSD7A remain unclear. Here, we induced an MN model in control and podocyte-specific Thsd7a knockout (Thsd7a–/–) mice using rabbit anti-THSD7A antibodies, followed by transcriptome and proteome analyses. Anti-THSD7A antibodies in WT mice caused significant loss of key slit diaphragm (SD) proteins such as nephrin and NEPH1, without transcriptional downregulation. Glomeruli showed substantial transcriptomic and proteomic reconfiguration indicative of extensive podocyte injury, including disruptions in podocyte adhesion, cytoskeletal dynamics, and marked upregulation of ubiquitin-proteasome system components, cathepsins and ADAM proteases. Notably, experiments in C3-deficient mice revealed that proteolytic activation and SD protein loss are driven by complement-independent pathways. While Thsd7a–/– mice only displayed a mild phenotype under basal conditions, they were completely protected from MN development upon anti-THSD7A antibody transfer. Finally, interactomic analysis identified a protein complex including THSD7A and integrin α3, linking THSD7A complexes to pathogenic regulation of cytoskeleton, adhesion, and membrane signaling in MN. Thus, anti-THSD7A antibodies induce profound molecular reconfiguration, including dysregulated proteolytic systems via a complement-independent pathway, revealing potential therapeutic targets in MN.
Authors
Ming Huang, Moritz Lassé, Silke Dehde, Felicitas E. Hengel, Fatih Demir, Anja M. Billing, Ning Song, Larissa Seifert, Oliver Kretz, Florian Grahammer, Ulf Panzer, Sebastian Brähler, Tobias B. Huber, Gunther Zahner, Markus M. Rinschen, Nicola M. Tomas
Abstract
Fanconi anemia (FA) is the most common bone marrow failure (BMF) syndrome. Beyond a role in DNA repair, FA genes have a role in suppressing DNA-RNA hybrids, termed R-loops, which can be generated via RNA polymerase (RNAP)-mediated transcription. However, how these processes, including a role in fate determination of hematopoietic stem cells (HSCs), are related to BMF is largely unknown. Additionally, single FA gene knockouts in mice do not recapitulate most phenotypes observed in FA patients. Thus, we generated a mouse model for FA by introducing heterozygous Setd2, which restricts RNAP-dependent transcription. Here, we show that FA patient-derived cells and Setd2+/– Fanca–/– HSCs share increased R-loop as well as dsRNA levels, and a ribosomal biogenesis defect. Further, Setd2+/– Fanca–/– HSCs display cell cycle arrest, mitotic errors and BMF phenotypes. Importantly, utilizing our Setd2+/– Fanca–/– mice, we discovered that Juglone, a pan RNAP inhibitor, reduces R-loop and dsRNA and reverses ribosomal biogenesis defects and mitotic errors, thereby rescuing BMF. In conclusion, this study establishes a novel mouse model that underscores a key role for R-loop formation, ribosomal biogenesis defects and mitotic errors in HSCs in driving BMF in Fanconi anemia. We also introduce a potential therapeutic avenue based upon pan-inhibition of RNA polymerases utilizing Juglone.
Authors
Michihiro Hashimoto, Xiaomin Feng, Jie Bai, Huimin Zeng, Tian Li, Jue Li, Terumasa Umemoto, Paul R. Andreassen, Gang Huang
Abstract
Mitochondrial dysfunction devastates the heart in major cardiovascular diseases, yet the mechanisms governing mitochondrial quality control remain elusive. We discovered that TIGAR (TP53-induced glycolysis and apoptosis regulator) deficiency established profound cardiac protection through developmental epigenetic programming of Parkin expression. Using whole-body and cardiomyocyte-specific TIGAR knockout mice, we demonstrated remarkable cardioprotection following myocardial infarction with maintained ejection fraction, and complete resistance to diet-induced cardiac hypertrophy despite comparable weight gain. TIGAR deficiency triggered dramatic increases in Parkin expression across all somatic tissues except testes, where Parkin levels remained extraordinarily high (100-fold greater than cardiac levels) regardless of TIGAR status, revealing tissue-specific regulatory mechanisms. This protection was entirely Parkin-dependent, as double knockout mice lost all cardioprotective benefits. Crucially, adult TIGAR manipulation failed to alter Parkin levels, demonstrating that this pathway operated exclusively during critical developmental windows to program lifelong cardiac resilience. Whole-genome bisulfite sequencing identified reduced DNA methylation in Prkn intron 10 as the key regulatory mechanism, with CRISPR deletion dramatically increased Parkin expression in multiple cell lines. Our findings reveiled how early cardiac metabolism programmed lifelong cardiac function through epigenetic mechanisms, and identifyied developmental metabolic programming as a potential therapeutic target for preventing both ischemic heart disease and metabolic cardiomyopathy.
Authors
Yan Tang, Stanislovas S. Jankauskas, Li Liu, Xujun Wang, Alus M. Xiaoli, Fajun Yang, Gaetano Santulli, Daorong Feng, Jeffrey E. Pessin
Abstract
Heart failure (HF) persists as the primary cause of death among patients recovering from acute myocardial infarction (AMI). Protein ubiquitination has been implicated as a key modulator of HF pathogenesis, yet the role of ubiquitination in the Aldh2 rs671 mutant—the most common single-nucleotide variant in human populations—remains poorly understood. We discovered TRIM21 as a previously unrecognized E3 ubiquitin ligase for the ALDH2 rs671 mutant and elucidated its mechanistic involvement in HF progression. Using Aldh2 bone marrow chimeric mice to model AMI, we observed that wild-type mice transplanted with Aldh2 rs671 donor bone marrow developed severe myocardial fibrosis and markedly reduced cardiac systolic function two weeks post-infarction compared to controls. This phenotype arose from defective macrophage efferocytosis caused by myeloid-specific Aldh2 rs671 mutation. Through high-resolution mass spectrometry proteomics, we identified TRIM21 as the E3 ligase targeting ALDH2. TRIM21 catalyzed K48-linked ubiquitination at ALDH2 lysine 73. Macrophage-specific Trim21 knockdown via AAV-shTrim21 reversed both the exacerbated cardiac fibrosis and systolic dysfunction by restoring macrophage efferocytosis. These findings delineate the upstream E3 ubiquitin ligase and the ubiquitination site of ALDH2, revealing a potential therapeutic target for HF.
Authors
Tianrui Han, Xin Wen, Yunyun Guo, Xiangkai Zhao, Jian Zhang, Yuguo Chen, Feng Xu
Abstract
Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the gene encoding huntingtin. Since accumulation of mutant huntingtin (mHtt) leads to dysfunction of numerous cellular pathways and toxicity, reducing levels of the mutant protein represents a key therapeutic objective in HD. We found that ubiquitination of mHtt by E3 ubiquitin ligase Nedd4 promotes clearance of the mutant protein. Knockdown of Nedd4 increased toxicity of mHtt in mouse primary neurons and in a fly model of HD, suggesting the protective role of Nedd4. Importantly, levels of Nedd4 were decreased in mHtt-expressing neurons through impaired mTORC1 activity, suggesting a feedback loop of mHtt accumulation and Nedd4 reduction that leads to accumulation and, ultimately, toxicity of mHtt. These findings suggest that restoring Nedd4 activity may offer a novel therapeutic opportunity for HD.
Authors
Hyunkyung Jeong, Yiyang Qin, Fangke Xu, Katarina Trajkovic, Myung Jong Kim, Nicolas Marotta, Kana Hamada, Ravi Allada, Su Yang, Dimitri Krainc
Abstract
Angiopoietin-2 (ANGPT2) is known to destabilize vascular barriers in most peripheral organs; however, its role in the brain vasculature remains poorly understood. To investigate its physiological function within the brain vasculature, we analyzed constitutive Angpt2 knockout (KO) mice in adulthood. We showed that loss of ANGPT2 leads to region-specific vascular malformations and blood-brain barrier (BBB) dysfunction, resulting in differential permeability to 1 kDa and 70 kDa fluorescent tracers. Notably, overt vascular malformations appeared only in select brain regions that allowed leakage of both tracers. These malformations were characterized by dilated, intertwined, and sprouting endothelial cells, surrounded by reactive perivascular cells, along with high levels of astrocyte- and neuron-derived vascular endothelial growth factor A (VEGFA) and elevated expression of the vascular receptors VEGF receptor 2 (KDR) and neuropilin 1 (NRP1). Other cortical areas without obvious malformations exhibited significant leakage of the 1 kDa tracer. We also demonstrated that different cell types took up the tracers after passing the BBB. Our findings identified ANGPT2 as an important factor involved in the regulation of cerebrovascular architecture, barrier integrity, and endothelial–parenchymal interactions, and uncovered surprising differences in the leakage patterns and cellular uptake of two widely used BBB tracers.
Authors
Weihan Li, Elisa Vázquez-Liébanas, Chanaëlle Fébrissy, Florent Sauvé, Jianhao Wang, Doğan Emre Sayıner, Pia Buslaps, Amanda Norrén, Michael Vanlandewijck, Liqun He, Marie Jeansson, Lars Muhl, Maarja Andaloussi Mäe
Abstract
Pancreatic cancer is a highly innervated gastrointestinal disease in which sympathetic nerves play a critical role in modulating tumor growth and the tumor microenvironment (TME). While recent studies suggest that sympathetic nerves influence various TME components, including lymphoid and myeloid immune cells, their interactions with cancer-associated fibroblasts (CAFs) remain poorly understood. CAFs are a hallmark of pancreatic tumors and are known to upregulate axon guidance and neuroactive cues, suggesting a potential feedback loop with tumor-innervating nerves. Here, we investigated the bidirectional crosstalk between sympathetic nerves and CAFs in human and mouse pancreatic tumors. Using a chemo-genetic ablation model, we selectively eliminated pancreatic sympathetic nerves and found that denervation significantly reduced tumor size in female mice. To further dissect this interaction, we established co-culture systems with immortalized pancreatic fibroblasts and primary sympathetic neuron explants, identifying key transcriptional changes driven by CAF-sympathetic nerve signaling. Our findings demonstrated that sympathetic signaling enhanced CAF activation and extracellular matrix remodeling, while activated CAFs, in turn, induced transcriptional programs in sympathetic neurons associated with nerve injury response. These results establish CAFs as central mediators of the tumor-supportive role of sympathetic nerves, offering further insights into the neural regulation of pancreatic cancer progression.
Authors
Ariana L. Sattler, Parham Diba, Kevin Hawthorne, Carl Pelz, Joe Grieco, Tetiana Korzun, Bryan Chong, M.J. Kuykendall, Rosalie C. Sears, Daniel L. Marks, Mara H. Sherman, Teresa A Zimmers, S. Ece Eksi
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