Biology subjects

Thompson, C. B.

Publications and source records attributed to Thompson, C. B..

9 recordsLinked to original sources

bioRxiv2026

Random mtDNA mutagenesis maps genotype-phenotype associations across metabolic contexts and lineages

Mitochondrial DNA (mtDNA) mutations cause inherited disease, accumulate with age, and recur in tumors, yet their effects remain unresolved because existing models interrogate one engineered allele at a time. We developed mtDIVE, a random C[->]T mutagenesis platform for pooled mitochondrial forward genetics in human epithelial cells and zebrafish embryos. mtDIVE generates genotypes ranging from single substitutions to >100 mutations per molecule, impairs respiration, and causes lineage-specific developmental delay. Coupling single-cell transcriptomics with mtDNA genotyping revealed that selection on mutant genomes and their transcriptional consequences depend on metabolic state and developmental lineage. Obligate OXPHOS eliminates highly mutated cells by apoptosis, whereas glycolytic conditions promote their competitive displacement. Individual loci exert modest but non-redundant effects that predict cell state beyond aggregate mutation burden, and cancer-recurrent variants are enriched among mutations linked to reduced mitochondrial transcription. Thus, mtDIVE links mtDNA genotypes to cellular phenotypes, separating the consequences of mutation identity from mutational load.

genomics↗

bioRxiv2026

Respiration-Deficient Cells Require Pyruvate Carboxylase to Suppress Asparagine Auxotrophy

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

cell biology↗

bioRxiv2026

Mitochondrial ATP production promotes T cell differentiation and function by regulating chromatin accessibility

Immune elimination of chronic infection or cancer requires cytotoxic CD8+ T cells that adopt and maintain an effector phenotype. Cytotoxic T cell function is a bioenergetically demanding process and T cells subjected to chronic antigen exposure have compromised effector function despite high rates of glycolysis. Here we report the ability of the short-chain -hydroxy acid, D--hydroxybutyrate, to act as a signaling molecule that increases mitochondrial ATP production and drives the conversion of proliferating T cells into cytotoxic effector cells. DAHB signaling switches ATP production from glycolysis to oxidative phosphorylation supported by fatty acid oxidation, even in glucose-replete media. This conversion suppresses both AMPK phosphorylation and the integrated stress response (ISR) in activated T cells while significantly elevating the level of the phosphagen, phosphocreatine (PCr). Both the PCr bioenergetic reserve and oxidative phosphorylation were required for T cell effector differentiation. DAHB-induction of CD8-effector gene transcription was coupled to bioenergetics by enhanced ATP-dependent remodeling of chromatin accessibility at effector gene loci. DAHB enhanced CD8+ T cell antitumor activity both in vitro and in vivo, and DAHB treatment of transferred T cells led to persistent in vivo antitumor effects. Together, these findings link cellular bioenergetics to the regulation of chromatin accessibility and gene expression required to support effector function.

immunology↗

bioRxiv2023

Lactate activates the mitochondrial electron transport chain independent of its metabolism

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

cell biology↗

bioRxiv2022

A tissue injury repair pathway distinct but parallel to host pathogen defense

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces interferons (IFNs) to activate resistance mechanisms. Applying unbiased phylogenetic analysis, we show that interleukin-24 (IL24) is among the closest evolutionary homologs to the IFN family and shares a common ancestral origin. However, in contrast to IFNs, IL24 induction occurs specifically in barrier epithelial progenitors after injury and is independent of microbiome or adaptive immunity. Surprisingly, Il24 ablation impedes not only epidermal proliferation and re-epithelialization, but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic Il24 induction in homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, sustained Il24 expression depends upon both IL24 receptor/STAT3 signaling and also hypoxia-stabilized HIF1, which converge following injury. Thus, parallel to the IFN-mediated innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL24-mediated tissue repair.

immunology↗

bioRxiv2022

Oncogenic IDH mutations increase heterochromatin-related replication stress without impacting tumor mutation burden

Oncogenic mutations in the metabolic enzyme isocitrate dehydrogenase 1 and 2 (IDH1/2) have been found in a number of liquid and solid tumors. Their pathogenic mechanism of action involves production of 2-hydroxyglutarate (2HG), an oncometabolite that acts in part by inhibiting members of a family of dioxygenases that modulate chromatin dynamics. Recent work has suggested that mutant IDH (mIDH) and 2HG also impact sensitivity to inhibitors of poly-ADP ribose polymerases (PARP) but the molecular basis for this sensitivity is unclear. Unlike PARP inhibitor-sensitive BRCA1/2 tumors which exhibit impaired homologous recombination, IDH-mutant tumors have a silent mutational profile and lack mutational signatures associated with impaired homologous recombination. Instead, 2HG-producing IDH mutations lead to heterochromatin-dependent slowing of DNA replication and increased replication stress, resulting in DNA double strand breaks. This replicative stress manifests as replication fork slowing but the breaks are repaired without a significant increase in the cellular mutation burden. Faithful resolution of replicative stress in IDH-mutant cells is dependent on poly-ADP ribosylation. Treatment with PARP inhibitors restores replication fork speed but results in incomplete repair of DNA breaks. These findings provide evidence of a requirement for PARP in the replication of heterochromatin and further validate PARP as a potential therapeutic target in IDH-mutant tumors.

cancer biology↗

bioRxiv2022

Hypoxia potentiates the inflammatory fibroblast phenotype promoted by pancreatic cancer cell-derived cytokines

Cancer-associated fibroblasts (CAFs) are a major cell type in the stroma of solid tumors and can exert both tumor-promoting and tumor-restraining functions. This functional heterogeneity is correlated with the existence of transcriptionally distinct subpopulations of CAFs. CAF heterogeneity is observed in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by a remarkably dense and hypoxic stroma that features tumor-restraining myofibroblastic CAFs (myCAFs) and tumor-supporting inflammatory CAFs (iCAFs). While CAF heterogeneity can be driven in part by tumor cell-produced cytokines, other determinants shaping CAF identity and function are largely unknown. In vivo, we found that iCAFs display a hypoxic gene expression and biochemical profile and are enriched in hypoxic regions of PDAC tumors. Hypoxia leads fibroblasts to acquire an inflammatory gene expression signature and synergizes with cancer cell-derived cytokines to promote an iCAF phenotype in a HIF-1 dependent fashion. Furthermore, we show that HIF-1 stabilization is sufficient to induce an iCAF phenotype in stromal cells introduced into PDAC organoid co-cultures and to promote PDAC tumor growth. These findings indicate hypoxia-induced HIF-1 as a regulator of CAF heterogeneity and promoter of tumor progression in PDAC.

cell biology↗

bioRxiv2021

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

molecular biology↗

bioRxiv2021

A mitochondrial long-chain fatty acid oxidation defect leads to uncharged tRNA accumulation and activation of the integrated stress response in the mouse heart

The heart relies mainly on mitochondrial fatty acid {beta}-oxidation (FAO) for its high energy requirements. Cardiomyopathy and arrhythmias can be severe complications in patients with inherited defects in mitochondrial long-chain FAO, reinforcing the importance of FAO for cardiac health. However, the pathophysiological mechanisms that underlie the cardiac abnormalities in long-chain FAO disorders remain largely unknown. Here, we investigated the cardiac transcriptional adaptations to the FAO defect in the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found a prominent activation of the integrated stress response (ISR) mediated by the eIF2/ATF4 axis in both fed and fasted states, accompanied by a reduction in cardiac protein synthesis during a short period of food withdrawal. Notably, we found an accumulation of uncharged tRNAs in LCAD KO hearts, consistent with a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the cardiac ISR in hearts of mice with a muscle-specific deletion of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signaling, in particular the ISR, and may play a role in the associated cardiac pathology.

physiology↗