Lőrinc Sándor Pongor

Folate is co-administered with fluoropyrimidines in colorectal cancer to potentiate thymidylate synthase inhibition, yet its therapeutic effects are paradoxically inconsistent across disease and treatment settings. Here we show that one-carbon metabolism remodels epithelial identity before treatment and thereby alters fluoropyrimidine response. Folate restoration shifted colorectal cancer cells along a polarized epithelial axis from a stem-like toward a differentiated state, whereas folate deprivation favored the opposite configuration. These pretreatment states differed markedly in 5-fluorouracil sensitivity. Folate restoration increased sensitivity across colorectal cancer cell lines and patient-derived organoids, whereas folate deprivation or methotrexate increased therapy-induced senescence. State-dependent sensitivity was not explained by proliferation alone, as stem-like cells were least responsive under the same conditions in which their S-phase occupancy was highest. Mechanistically, folate restoration increased methylation potential, supported EZH2-dependent H3K27me3 remodeling, reorganized chromatin accessibility and transcription-factor engagement, and reduced canonical WNT activity. In human colorectal tissue, stem-like program activity was higher and differentiation program activity lower in carcinoma than in normal mucosa, with adenomas intermediate, a pattern opposite to folate-induced remodeling. Folate is therefore not merely a cofactor for thymidylate synthase inhibition but an instructive metabolic regulator of the epithelial state that stratifies fluoropyrimidine response.

Polycomb (PcG) bodies are nuclear foci formed by polycomb protein complexes that contain PcG-bound DNA and are implicated in gene regulation during development and differentiation, although their precise molecular function remains unclear. Using tyramide signal amplification sequencing, we provide a comprehensive view of genomic regions associated with PcG bodies, including specific centromeric and telomeric sites. These regions are enriched for the repressive marks H3K27me3 and H3K9me3, depleted of the active mark H3K4me3, and display low chromatin accessibility. We find a high density of replication origins around PcG bodies, consistent with the replication factor origin recognition complex–associated protein (ORCA)/LRWD1 localizing at these sites. ORCA interacts with the polycomb-repressive complex, stabilizes the H3K27 methyltransferase, and facilitates H3K27me3 deposition at specific chromatin sites. Loss of ORCA affects chromatin organization around PcG bodies, leading to decompaction of the repeat regions, and enhanced initiation from replication origins. Our results suggest that ORCA associates with PcG-bound chromatin to maintain a repressive environment that regulates replication origin firing timing.

Small cell lung cancer (SCLC) is a highly aggressive malignancy, accounting for approximately 15% of all lung cancer cases. Characterized by low immunogenicity, SCLC may utilize epigenetic mechanisms to evade immune detection. In this study, we demonstrate that entinostat, a class I histone deacetylase inhibitor, upregulates immune-related genes in human SCLC cells. In vivo, we confirmed that entinostat treatment increased the expression of immune checkpoint ligands and antigen presentation machinery in Myc-driven tumors in a Rb1/Trp53/MycT58A (RPM) SCLC mouse model while shifting tumors from a neuroendocrine (NE)-high phenotype to a NE-low phenotype and was associated with increased T-cell infiltration. Notably, combining entinostat with anti–PD-1 immunotherapy suppresses tumor growth and significantly prolongs survival in RPM allograft models. These findings underscore the potential of entinostat to reprogram the NE status of SCLC, enhance immune checkpoint blockade efficacy, and improve therapeutic outcomes.

Human cortical interneurons differ from their rodent counterparts in intrinsic membrane properties, yet the mechanisms regulating excitability across physiologically relevant membrane potentials remain poorly defined. Here, we investigated inwardly rectifying potassium (Kir) channel control of subthreshold excitability in parvalbumin-expressing (Pvalb) interneurons from human and mouse neocortex. Using whole-cell recordings, dynamic clamp, patch sequencing, immunofluorescence, and computational modeling, we show that membrane hyperpolarization induces a proportional decrease in input resistance mediated by Kir channels in both species, despite higher baseline input resistance in human neurons. Transcriptomic and anatomical analyses revealed somatic membrane expression of four major Kir channel subtypes with moderate interspecies differences. Kir activation suppresses intrinsic excitability through combined voltage-dependent and shunting inhibition, an effect occurring during inhibitory postsynaptic potentials evoked by neurogliaform cells. Together, these findings show that homologous Pvalb neurons in humans have evolved toward a conserved, archetypal excitability phenotype, despite substantial differences in baseline excitability between species.

Experimental validation of copy-number of the MYC locus in NCI-H889, DMS-114, NCI-H446 and RA022 patient derived cell lines and number of ecDNA and HSR positive cells in the RA022 patient derived cell lines

Fusion landscape and ecDNA RLF-MYCL fusions, related to Figure 3.

Heterogeneity in patient derived samples, related to Figure 5

Enhancer-enhancer interaction analysis derived from H3K27ac HiChIP in the NCI-H889 and NCI-H524 cell lines. Interaction strength of proximal enhancers was calculated from the “.hic” files generated by the JuiceBox suite.

EcDNA in SCLC, related to Figure 1

Amplified genes identified from cell-free ChIP-seq of patient plasma samples.