An integrated single-cell transcriptomic pipeline identifies ZNF740/BRD3 and Cathepsin S as novel therapeutic targets in chronic active rim smoldering multiple sclerosis

This study identifies ZNF740, Cathepsin S, and DNMT1 as potential new targets in smoldering MS, but further research is needed to establish their clinical relevance.

Summary and findings

This study aimed to identify molecular targets in chronic active rim smoldering multiple sclerosis (MS) using a computational pipeline. The analysis involved 6 MS patients and 4 healthy controls, revealing 394 upregulated candidates and identifying ZNF740, Cathepsin S, and DNMT1 as potential therapeutic targets. No therapeutic claims are made.

Abstract

Abstract Background. Smoldering multiple sclerosis (MS), characterised by slowly expanding chronic active rim (CA-RIM) lesions, is responsible for a substantial proportion of irreversible disability yet has no approved disease-modifying therapy. The molecular drivers of the CA-RIM lesion microenvironment are poorly characterised. Methods. We developed a four-phase computational pipeline integrating bulk RNA-seq differential expression (Phase 1), single-cell variational autoencoder (scVI) atlas construction and cross-modal integration (Phase 2), multi-database target validation (Phase 3), and STRING-DB network medicine proximity scoring (Phase 4). Bulk DEGs from CA-RIM vs. control white matter (GSE108000) were mapped onto a 32,239-cell scVI atlas combining CELLxGENE Census MS data with single-cell CD8⁺ T-cell profiles from the 6 MS patients and 4 healthy controls in GSE193770 (an earlier version of this atlas pooled in 25 unrelated COVID-19 patients from the same GEO submission's merged file; see §2.1 and Data/Code Availability). Results. Phase 1 identified 394 upregulated candidates (log₂FC ≥ 0.5, FDR < 0.1) from 1,065 total CA-RIM DEGs. Phase 2 constructed a 32,239-cell, 11,811-gene atlas with 19 Leiden clusters; ZNF740 was enriched in the CD8⁺ T-cell cluster, while Cathepsin S (CTSS) was most highly expressed in VIP GABAergic cortical interneurons (see Discussion §4.2 for why the CTSS cell-type attribution changed from an earlier analysis). Phase 2 priority scoring ranked DNMT1 first overall (score 0.715, log₂FC = +1.59), ZNF740 second (0.548, STRING partner BRD3 pairwise score 0.960), and CTSS 15th (0.379, ChEMBL pChEMBL 10.0); 20/25 candidates were blood-accessible by GTEx criteria. Phase 4 network proximity scoring identified CTSS as the strongest MS network medicine candidate among the three focal targets (connects to CSF1R and PTPRC seed genes; combined repurposing score 0.040, second overall behind LINGO1), ZNF740 as directly connected to IFNG (STRING score 0.447) and the BRD2/BRD3/BRD4 bromodomain complex, and DNMT1 as connected to STAT3. Conclusions. This pipeline identifies three previously unreported therapeutic axes in smoldering MS: BET bromodomain inhibition via the ZNF740/BRD3 transcriptional complex; CTSS inhibition combined with a blood-accessible CTSS liquid biopsy (cell-type locus of action not yet resolved — see Discussion); and DNMT1-targeted epigenetic reprogramming of CA-RIM lesion CD8⁺ T cells. All three targets are novel, blood-accessible, and supported by orthogonal computational evidence.

Elsewhere in the VIP (Vasoactive Intestinal Polypeptide) corpus