Olamide Animasahun

Rapid repair of genotoxic therapy induced DNA damage mediates treatment resistance in glioblastoma (GBM). The role of non-malignant cell types in the tumor microenvironment in accelerating DNA repair in neoplastic cells is poorly understood. Using spatial transcriptomics, immunofluorescence, metabolomics, patient tissues and preclinical models, we show that tumor associated macrophages (TAMs) in GBM promote DNA repair and treatment resistance in neoplastic cells through the secretion of acetylated amino acids. These acetylated amino acids are consumed by GBM cells, leading to enhanced acetyl Co-A levels, histone acetylation and nucleotide synthesis. Interrupting histone acetylation via inhibition of the acetyltransferase KAT5 breaks these metabolic links and reverses the protective capacity of microenvironment-derived acetylated amino acids and TAMs. Blocking the exchange of acetylated amino acids and their downstream effects is a potential strategy for the treatment of GBM.

Solute Carrier Family 13 Member 5 (SLC13A5) encodes the sodium-dependent citrate cotransporter NaCT, which mediates citrate transport across cell membranes. Pathogenic variants in SLC13A5 cause developmental and epileptic encephalopathy 25 with amelogenesis imperfecta, DEE25; OMIM #615905, a debilitating autosomal recessive disorder. To better define the role of NaCT in ameloblast function and enamel mineralization, we used CRISPR/Cas9 genome editing to generate Slc13a5^R337* knock-in mice that terminate NaCT translation at the Arg337 codon, which is homologous to the human SLC13A5^R333* variant associated with DEE25. We compared enamel phenotypes among wild-type, Slc13a5^+/+; heterozygous, Slc13a5^+/R337*; and homozygous, Slc13a5^R337*/R337* mice using light microscopy, in situ hybridization, immunohistochemistry, backscattered scanning electron microscopy (bSEM); and focused ion beam–scanning electron microscopy (FIB-SEM) with quantitative imaging of organelles and matrix. Citrate bioassays were performed on serum, long bones, such as the femur and tibia, and developing mouse first molars, including enamel organ epithelium, mineralized tooth matrix, and pulp mesenchyme, to assess citrate levels during the presecretory, secretory, and maturation stages of enamel formation. In addition, first molars collected at postnatal days 0, 3, 5, and 12 were analyzed to characterize glycolytic and TCA cycle-related metabolic signatures. Homozygous Slc13a5^R337*/R337* mice exhibited severe defects during the secretory and maturation stages of amelogenesis. Most notably, Slc13a5^R337*/R337* ameloblasts failed to develop a Tomes’ process, detached from the enamel matrix surface, and produced a thin, poorly mineralized crust on the dentin surface rather than organized enamel ribbons. Despite the absence of normal enamel deposition, ameloblasts initially appeared viable and did not become dysplastic until the late secretory stage. Cellular and subcellular analyses revealed increased secondary lysosomes and intracellular accumulation of enamel matrix proteins, consistent with impaired matrix processing or secretion. Citrate concentrations were elevated in serum and long bones at both 7 and 35 weeks of age. Citrate was elevated in secretory-stage Slc13a5^R337*/R337* molars at days 0 and 3, the enamel organ epithelium (including ameloblasts), the pulp mesenchyme (including odontoblasts), and mineralizing dentin and enamel matrices. These levels gradually declined at day 5 and into the enamel maturation stage (day 12). GC-MS-based analysis of central carbon metabolites revealed increased intracellular accumulation of citrate, malate, and pyruvate, suggesting altered energy metabolism and reduced metabolic efficiency in Slc13a5^R337*/R337* mice. Together, these findings indicate that loss of NaCT function in the ameloblasts causes citrate accumulation, which impairs hydroxyapatite formation. Consequently, only a thin, structurally defective mineral crust forms on the dentin surface, while mineral nodules develop ectopically within the maturation-stage enamel organ epithelium. We conclude that regulating citrate concentration is essential for proper appositional growth of enamel.

Supplementary Figure S1 shows the generation of THP1-derived macrophages and preparation of stearic acid–treated conditioned media.

Supplementary Figure S13 shows the percentage distribution of cytokine-producing cells in untreated and stearic acid–stimulated THP1-derived macrophages.

Supplementary Figure S4 shows the correlations between microbial abundance and metabolite presence using Spearman’s rank correlation in affected and unaffected lobes.

Supplementary Figure S3 shows the HAllA analysis illustrating associations between OTU expression and metabolite levels in unaffected lobes.

Supplementary Figure S9 shows association plots between microbial taxa (OTUs) and cytokines in unaffected lobes.

Supplementary Figure S10 shows association plots between cytokines and metabolites in unaffected lobes.

Supplementary Figure S2 shows the agar layer setup used in the anchorage-independent colony formation assay.