Cell Death & Differentiation

Colorectal cancer (CRC) exhibits substantial intratumoral heterogeneity, with differences in oncogenic driver mutations and apoptotic competence contributing to therapeutic resistance. Here, we investigated whether combined celecoxib and NDGA treatment (C+N) induces redox-mediated cell death via the p62/KEAP1 axis, offering a therapeutic approach that can overcome genetic diversity in CRC. We used two genetically distinct CRC cell lines, HCT116 and HT29, harbouring KRAS and BRAF mutations respectively, with differing p53 status, as models of tumor heterogeneity. C+N exhibited profound cytotoxicity in both cell lines, independent of COX-2/5-LOX expression and apoptotic competence. Additionally, C+N induced marked oxidative stress with mitochondrial ROS, lipid peroxidation, and labile ferrous iron accumulation, visualized as distinct JC-1 green mitochondrial puncta and FerroOrange red iron puncta in both cell lines. The antioxidant NAC reversed C+N-induced death, but inhibitors of apoptosis, autophagy, necroptosis, or ferroptosis did not, indicating a mechanism distinct from these pathways. Mechanistically, the combination induced p62 Ser349 phosphorylation, promoting KEAP1 degradation and NRF2 stabilization independently of ULK1-mediated autophagy; however, NRF2 stabilization was uncoupled from HO-1 induction. Our work reveals that C+N induces a ULK1/autophagy-independent, redox-mediated cell death mechanism that circumvents apoptotic competence, highlighting redox disruption as a genotype-independent therapeutic strategy to overcome CRC heterogeneity.

The modulation of iron metabolism is increasingly explored as a therapeutic strategy in cancer, particularly through the induction of ferroptosis. Given the central role of ferritin as a major intracellular iron buffer, targeting ferritin represents an attractive strategy to disrupt iron homeostasis; however, the lack of pharmacological approaches capable of selectively targeting ferritin currently limits the therapeutic exploitation of this vulnerability. MMRi62, initially developed to target the MDM2-MDM4 axis, has been proposed to induce ferroptosis via ferritin degradation. Here, we revisited this hypothesis in the context of medulloblastoma (MB). Contrary to this proposed mechanism, we found that MMRi62 does not trigger ferroptosis but instead induces robust apoptotic cell death. This effect is observed in both p53-mutant DAOY and p53 wild-type HD-MB03 cells, as well as in c-Myc/OTX2-driven medulloblastoma-like tumours genetically induced in brain organoids. Although ferritin degradation occurs upon treatment, genetic dissection demonstrates that neither ferritin itself nor ferritinophagy are required for MMRi62-induced cytotoxicity. Together, these findings rule out ferritin-dependent mechanisms as primary drivers of cytotoxicity. Instead, we uncovered that MMi62 induces a profound rewiring of iron metabolism consistent with a canonical iron starvation response, accompanied by a marked reduction in intracellular iron levels. Consistently, the UV-visible spectroscopic data were in agreement with the proposed iron-chelating properties of MMRi62. Taken together, our findings identify iron depletion-driven apoptosis, rather than ferroptosis, independently of ferritin degradation, as the primary mechanism of MMRi62 cytotoxicity in MB, refining its mode of action and highlighting iron homeostasis as a therapeutic vulnerability.

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.

PhenoAge, a blood-based biological aging clock, is associated with cancer outcomes, a finding taken as evidence that biological aging shapes prognosis. In 3,067 patients undergoing primary tumor resection, PhenoAge acceleration rose with tumor stage. One or two of its eight blood components, differing by cancer type, carried the prognostic signal, and replacing its general-population weights with equal weights did not weaken it. In cancer, PhenoAge may reflect disease burden rather than biological aging.

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

Background: The organization of pancreatic cancer care may be associated with survival. Using health administrative claims data, we reconstructed the pathway from diagnosis to first-line therapy and examined associations of multidisciplinary team review and treatment at specialized centers with overall survival. Methods: Longitudinal historical cohort study using anonymized health administrative claims data from six Czech health insurance funds, covering approximately 17% of patients diagnosed with pancreatic cancer nationally between 2018 and 2020. Adults with an incident ICD-10 C25 record confirmed by imaging and histopathology were followed from the index date (the date of the first qualifying imaging or histopathological examination) until death, censoring, or 31 December 2021. Overall survival was analyzed using Kaplan-Meier methods and Cox regression adjusted for age and first-line therapy type. Results: Of 1,255 patients, 920 (73.3%) received first-line therapy and 266 (21.2%) died untreated. Among treated patients, 436 (47.4%) underwent multidisciplinary team review, 335 (36.4%) underwent surgery, and 667 (72.5%) were treated at a complex oncological center. Surgery was associated with longer overall survival than chemotherapy (age-adjusted hazard ratio [HR] 0.32, P < 0.001). Treatment at a complex oncological center was associated with longer overall survival (adjusted HR 0.77, 95% confidence interval [CI] 0.64-0.92, P = 0.004; crude median 532 versus 394 days). Multidisciplinary team review was not significantly associated with overall survival (adjusted HR 0.86, 95% CI 0.73-1.01, P = 0.073). Conclusions: Treatment at a complex oncological center was associated with longer survival, while fewer than half of patients had a recorded multidisciplinary team review. How care is organized, not only which therapy is delivered, is associated with outcome. The observational design and absence of key clinical covariates limit causal interpretation.

Neutrophils infiltrate the liver as metabolic dysfunction-associated steatotic liver disease (MASLD) progresses from simple steatosis to steatohepatitis, but whether neutrophil effectors merely mark this transition or cause it is unknown. Here we show that human neutrophil peptides (HNPs), or alpha-defensins, are drivers rather than bystanders of disease. In patients surgically treated for hepatic hemangioma who displayed overt MASLD, the abundance of HNPs in their liver biopsy samples localized to diseased tissue correlates with the histological severity of steatosis. In vitro, HNPs enter hepatocytes to induce multimerization of voltage-dependent anion channel 1 (VDAC1) in the mitochondrial outer membrane, causing mitochondrial dysfunction and lipid accumulation. Under lipid overload, but not on a standard diet, HNP1-transgenic mice develop increased adiposity and steatohepatitis, which is reversed by an orally available prodrug that inhibits HNP-induced VDAC1 multimerization. These findings link neutrophil innate immunity to hepatocyte mitochondrial dysfunction, recast host-protective HNPs as pathogenic effectors when hepatocyte mitochondria are lipid-burdened, and establish the HNP-VDAC1 axis as a therapeutic target in MASLD.

Communication between peroxisomes and mitochondria is essential for cellular metabolic homeostasis, yet how peroxisomal import stress impacts mitochondria function during aging and cellular senescence remains poorly defined. Using a genome-wide CRISPR screening in HEK293 cells under peroxisome import stress, we identified SCAF1 (SR-related CTD-associated factor 1) a known canonical nuclear pre-mRNA splicing factor, as an essential regulator of mitochondrial homeostasis. Under peroxisome stress SCAF1 undergoes proteolytic processing and translocates to the mitochondria, where its N-terminal region acts as an autonomous repressor module that blocks mitoribosomal subunit joining. Consequently, SCAF1 depletion accelerates subunit joining and elevates oxidative phosphorylation protein levels, whereas its overexpression in IMR90 fibroblast cells triggers robust cellular senescence characterized by increased senescence associated {beta} gal staining. Together, our findings uncover a stress-responsive peroxisome-to-mitochondria signaling axis mediated by SCAF1 translocation. This pathway directly modulates mitoribosome assembly to maintain translational homeostasis, providing a precise molecular mechanism for how upstream peroxisomal decline drives downstream mitochondrial dysfunction and cellular senescence.

Innate immune responses must be tightly controlled to maintain immuno-homeostasis. However, the regulatory mechanisms underlying the activation and early stages of the innate immune response remain poorly defined. Here, we found the soluble C-type lectin Clec18a is up-regulated in multiple inflammatory diseases, including sepsis and influenza, and secreted within minutes upon activation in multiple immune cells, including macrophages, dendritic cells (DCs), and T cells. Clec18a-myeloid-knockout mice exhibited reduced survival and exacerbated inflammation than the wild-type. However, exogenous recombinant Clec18a administration alleviated inflammation both in vitro and in vivo. Mechanistically, we identified macrophage-inducible C-type lectin (Mincle) as the receptor of Clec18a. Clec18a-Mincle binding triggers Trpm7-dependent calcium influx, which activates the ubiquitin E3 ligase Cbl and leads to Mincle-Cbl-MyD88-Trpm7 complex formation and degradation. Cbl deficiency, calcium inhibition and Trpm7 knockdown abrogated Clec18a-mediated immune suppression. Thus, our study revealed Clec18a as a critical checkpoint on the initiation of innate immune response through an intrinsic crosstalk between CLR and TLR signaling.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic {beta}-cells. While anti-CD3 therapy can delay disease progression and preserve residual {beta}-cell function, disease reversal will likely require both immune modulation and {beta}-cell regeneration. We found that the combination of harmine and exendin-4 (H+E) reduced inflammation-induced human {beta}-cell apoptosis, suppressed cytokine signaling and immunogenicity pathways, and improved {beta}-cell function. Although H+E alone did not reverse diabetes in NOD mice, low-dose anti-CD3 followed by H+E normalized blood glucose, increased insulin levels, improved glucose tolerance, expanded {beta}-cell mass, and enhanced diabetes remission. These effects were associated with reduced pro-inflammatory T-cell responses, increased regulatory T cells, and greater expression of exhaustion-related T-cell markers, without broad lymphocyte depletion. Similar immunomodulatory effects were observed in activated human PBMCs. Transcriptomic analyses identified the lncRNA SNHG6 as a key mediator of H+E action; SNHG6 protected {beta}-cells from cytokine-induced stress, apoptosis, and immunogenicity. Together, these findings demonstrate that H+E promotes {beta}-cell recovery and resilience while reducing {beta}-cell immunogenicity, enabling remission of recent-onset T1D when combined with anti-CD3 therapy. SNHG6 emerges as a novel regulator of {beta}-cell protection during inflammation.

The endoplasmic reticulum (ER) chaperone BiP/GRP78 sits at the center of the proteostasis network, coordinating unfolded protein response (UPR) signaling and supporting oncogenic programs of plasticity, metastasis, immune evasion, and chemoresistance. While BiP undergoes liquid-liquid phase separation in vitro, whether mammalian cells dynamically tune this physicochemical behavior to prevent functional exhaustion or pathological condensation under sustained ER stress has remained unclear. Here, we identify the E3 ubiquitin ligase TRIM21 as a phase-state regulator that suppresses BiP's liquid-to-solid phase separation, clearing, and preventing insoluble aggregates-thereby controlling BiP's localization and availability to sustain the adaptive UPR under ER stress. We found that during ER stress, BiP is prone to liquid-to-solid phase separation, a transition that renders the chaperone dysfunctional. TRIM21 is recruited to BiP, where it directly polyubiquitinates BiP at specific K547/553/554/556 sites with K6-, K27-, K33-, and K63-linked chains; VCP recognizes these marks and extracts BiP from the ER lumen via ER-associated degradation (ERAD), coupling ubiquitin tagging to retrotranslocation. This preserves chaperone availability and sustains adaptive UPR-a homeostatic mechanism broadly relevant to UPR-driven diseases. In vivo, genetic ablation of Trim21 in KrasG12D/+; Pdx1Cre/+ mice triggers aberrant BiP solidification while curtailing acinar-to-ductal transdifferentiation and tumorigenesis; notably, TRIM21 co-localizes with BiP condensates in pre-malignant human pancreatic lesions, underscoring the clinical relevance of this regulatory axis. Our findings define the TRIM21-BiP axis as a phase-control system at the interface of proteostasis and stress adaptation, offering a new framework with translational potential.

Biliary Atresia (BA) is the leading cause of pediatric liver transplantation. Given the heterogeneity of the disease, outcomes after diagnosis remain unpredictable. A multiscale atlas of BA hepatic tissue established early lymphatic dysplasia in infants that later proceed toward liver failure and transplantation (severe BA). An architecturally distorted, immature and CCL21-high lymphatic vasculature marked severe BA, along with an expanding cholangiocyte compartment pivoting toward immune-reactive MMP7/SOX9-high states. Hepatocyte-to-biliary transdifferentiation emerged broadly across BA- and non-BA-cholestatic injuries, while dedifferentiation and acute-phase-reactions were specific to severe BA. Functionally, lymphatic endothelial tissue, isolated from severe BA livers, induced an analogous LGR5- and SOX9-positive epithelial progenitor response when assembled with cholangiocytes as organoids. Further experiments revealed that lymphatic endothelium signals ectopically on liver epithelium via CCL21-CCR7-axis after BA-specific inflammatory priming. CCL21-depletion strategies mitigated the BA-specific MMP7-program. In sum, a maladaptive lymphatic endothelial dysplasia perturbs intrahepatic epithelium in severe BA and offers CCL21 as a possible target for epithelial stability.

Caspase-11 (Casp11) is a murine zymogen that becomes active upon sensing cytosolic lipopolysaccharide (LPS), enabling it to form the non-canonical inflammasome and execute an inflammatory cell death known as pyroptosis. The lipid A acylation state of LPS is thought to be the primary regulator of Casp11 activation. However, our data demonstrate that the polysaccharide moiety of LPS, and particularly the polysaccharide phosphates, are critical for eliciting maximal Casp11 activation. Accordingly, the polysaccharide was required for Rhodobacter sphaeroides LPS to act as an inflammasome antagonist. Furthermore, isolated polysaccharide was sufficient to antagonize Casp11 activation. Mechanistically, our findings support a two-step model of non-canonical inflammasome activation where the polysaccharide recruits Casp11 to LPS and the lipid A acylation state governs oligomerization efficiency. These findings provide new insight into the molecular basis of LPS-driven Casp11 activation, with implications for the design of future therapies that target the non-canonical inflammasome.

During apoptosis, the permeabilization of the mitochondrial inner membrane (MIM) through still-unclear mechanisms releases mtDNA into the cytosol, triggering the inflammatory cGAS/STING pathway under low caspase activity. Here, we report that, in apoptosis, the active form of the pore-forming protein Gasdermin E (GSDME-N) damages mitochondria before plasma membrane disruption. We visualize GSDME-N pore-like nano-assemblies in the MIM of apoptotic cells and of isolated mitochondria, which we bridge with the cryo-EM structure of the GSDME-N pore in mitochondria-like membranes. Deep membrane insertion of the anchor domain, which acts as a determinant of GSDME-N cardiolipin binding preferences, supports a role in pore formation. Notably, GSDME depletion results in a reduction in cristae swelling, MIM extrusion and mtDNA release during apoptosis. Subsequently, this decreases STING activation and inflammatory responses under caspase inhibition. Our findings reveal that GSDME mediates MIM permeabilization and mtDNA release during apoptosis and define the mechanism of GSDME-mediated membrane damage.

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease, and liver fibrosis is the principal determinant of disease progression and clinical outcome. We recently showed that non-apoptotic caspase-8 in hepatocytes is upregulated in MASH and leads to the induction and secretion of meteorin, which promotes hepatic stellate cell (HSC) activation and liver fibrosis. We address here the major mechanistic question arising from this study, namely how non-apoptotic caspase-8 induces pro-fibrotic meteorin in hepatocytes in MASH. Methods: Mechanistic studies were performed using AML12 hepatocytes; primary mouse and human hepatocytes; and diet-induced mouse models of MASH with hepatocyte-specific caspase-8 deletion. YY1 post-translational modifications were identified by LC-MS/MS and investigated using phosphorylation-defective and phosphomimetic YY1 mutants. Genetic and biochemical approaches were used to interrogate the caspase-8 DED-SRC-GSK3b; pathway, and its relevance was assessed in human MASH liver specimens. Results: In vitro mechanistic studies showed that the amino-terminal death effector domain (DED) of caspase-8 activated a SRC-GSK3b signaling cascade that promoted phosphorylation of YY1 at Ser247, resulting in YY1 nuclear localization and Metrn induction. Disruption of the conserved DED RXDLL motif impaired SRC activation, YY1 phosphorylation, and Metrn expression. Hepatocyte YY1 overexpression increased Metrn expression and fibrosis in wild-type MASH mice but not in hepatocyte-caspase-8-deficient mice. In contrast, phosphomimetic YY1-S247D restored these endpoints even when hepatocyte caspase-8 was lacking. Studies in primary mouse and human hepatocytes and human MASH liver specimens supported conservation of this signaling pathway. Conclusions: These findings identify YY1 Ser247 phosphorylation as a critical molecular event linking non-apoptotic hepatocyte caspase-8 signaling to METRN induction and MASH fibrosis and define a previously unrecognized DED-SRC-GSK3b-YY1 signaling axis with potential therapeutic relevance.

Stem cell-derived islets (SC-islets) are a promising source for replacing the loss of insulin-secreting {beta} cells in insulin-dependent diabetes patients, but compared to their primary human counter parts, SC-islets are considered functionally immature and do not immediately regulate blood glucose after transplantation. To better understand the molecular underpinnings of {beta} cell maturation, we genetically engineered human pluripotent stem cells (hPSCs) to control the induction of the {beta} cell-maturing transcription factor MAFA or a MAFA S64F mutant variant associated with premature {beta} cell aging and senescence in SC-islets. We employ a combination of in vitro assays, multi-omics analyses, and murine in vivo transplantation studies to demonstrate that MAFA remodeling of the SC-islet transcriptional landscape promotes SC-islet biogenesis and metabolic maturation giving rise to greater SC-islet resilience and insulin secretory capacity. Parallel MAFA S64F studies highlight the importance for compensatory mechanisms for sustaining accelerated oxidative metabolism and enhanced insulin secretion without inducing intracellular stress responses and islet dysfunction. Overall, these results reveal MAFA-governed transcriptional changes that drive SC-islet functional maturation, which provides the foundation for maturing SC-islets in vitro, preserving {beta} cell integrity, and improving SC-islet cell replacement therapies.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

Severe COVID-19 infection in patients with cancer is characterized by a unique pattern of immunologic dysfunction including muted adaptive immune responses. The molecular drivers of immune dysfunction in cancer patients with severe COVID-19 remain unclear, and therapeutic strategies to overcome immune dysfunction in this setting have not been identified. We performed integrated proteomic and metabolomic profiling of matched cohorts of cancer and non-cancer patients with or without COVID-19 and identified dysfunctional cysteine metabolism as uniquely associated with severe COVID-19 in cancer patients. Treatment of patients with cancer and steroid-refractory COVID-19 with N-acetylcysteine in a prospective clinical trial (NCT04374461) improved clinical outcomes compared with disease severity-matched hospitalized patients during the period immediately preceding clinical trial initiation. N-AC treatment reduced circulating markers of innate inflammation, decreased severe disease-associated MHC-II low monocytes, and increased circulating CD8+ T cell abundance, activation, and effector differentiation. Mechanistically, N-AC reduced prostaglandin E2-driven interactions between suppressive monocytes and T cells, which we confirmed was sufficient to limit T cell expansion and effector differentiation in a dose- and avidity-dependent fashion. Moreover, N-AC reduced the activity of inhibitory, redox sensitive transcription factors such as KLF6, enabling clonal expansion and effector T cell differentiation. We confirmed these observations in two murine models of severe respiratory viral infection, in which N-AC treatment significantly enhanced lung-infiltrating CD8+ T cell abundance. These findings establish cysteine supplementation as a viable therapeutic strategy to reverse redox-driven immune dysregulation in severe respiratory viral infection, particularly in the high-risk cancer population.

Sporadic disease accounts for about 90% of amyotrophic lateral sclerosis (ALS) cases, and its biological heterogeneity is a major contributor to the failure of trials that enroll patients under a single diagnosis. Because sporadic ALS lacks a shared genetic anchor, identifying biological signals is critical for effective patient stratification. We analyzed induced pluripotent stem cell derived motor neurons from 619 AnswerALS participants using transcriptomics, proteomics, and whole-genome sequencing and identified CCT2 as the first single-gene marker of a molecular state within sporadic ALS, providing a measurable basis for trial enrollment according to biology. We first anchored the analysis in known ALS genotypes. C9orf72 and SOD1 motor neurons did not converge on a common signature but retained genotype-specific differences that bulk postmortem tissue compresses. Scoring 391 sporadic patients along three transcriptomic axes (bioenergetic stress, cell cycle, and inflammation) resolved three states. One state was marked by CCT2, a TRiC chaperonin subunit, which distinguished it from the quiescent state with an area under the curve of 0.892. CCT2 was also elevated at the protein level and associated with the rate of ALSFRS-R decline, although this association weakened after adjustment for culture composition. Upstream regulator analysis placed this state downstream of MYC and mTORC1 activation. No germline test across 360 whole genomes explained these states. Validation using Target ALS data showed that the relationship between CCT2 and the cell cycle program was also present in postmortem cortex enriched for layer 5b projection neurons.