TRANSCRIPTOMIC ANALYSIS REVEALS A SYNERGY OF HYDROXYCHLOROQUINE AND GLUCOCORTICOIDS IN MODULATING B CELL-RELATED IMMUNE PROCESSES

PV113 / #260 Poster Topic: AS12 - Genetics, Epigenetics, Transcriptomics Background/Purpose Hydroxychloroquine (HCQ), glucocorticoids (GC), and their combination are common treatments in autoimmune rheumatic diseases. Their effects at the molecular level and potential synergistic effects remain unclear, which formed the scope of this work, including investigation of specific gene sets that are involved in this potential interaction. Methods We analyzed bulk RNA sequencing data from 591 samples from the PRECISESADS project.[1] The patients were diagnosed with systemic lupus erythematosus (SLE), Sjögren’s disease (SjD), undifferentiated connective tissue disease (UCTD), and mixed connective tissue disease (MCTD), and were grouped into 4 categories based on treatment status: no current exposure to HCQ or GC (off-treatment; n = 244), on HCQ (n = 198), on GC (n = 47), or on HCQ and GC combined (n = 102) (Table 1). Patients were not on any immunosuppressive treatment at the time of sampling. We performed differential gene expression (DGE) analysis across treatment groups (HCQ, GC, and HCQ+GC) with the off-treatment group of patients serving as the comparator. Overrepresentation analysis (ORA) was conducted on genes with amplified effects (absolute log 2 fold change (FC) < 0.5 for the individual treatments; absolute log 2 FC > 0.5 for the combination treatment), using Chaussabel’s gene set modules to identify enriched pathways.[2] The activity of the modules was estimated using gene set variation analysis (GSVA). Statistical comparisons of gene activities within modules between treatment groups (GC vs. HCQ+GC) for steroid dosages (low, medium, high) were conducted using the Mann-Whitney U test. Table 1. Number of samples per patient diagnosis and treatment category. Results Combination of HCQ and GC generated a synergistic molecular response, with a higher number of differentially expressed genes and greater effect size compared to the individual treatments, regarding both differentially overexpressed and downregulated genes. The ORA of genes with amplified effect size pointed to numerous immune-related pathways, consistent with the GSEA analysis results. Notably, B cell-related gene proliferation and activity modules were significantly suppressed (ORA adj. p-value < 0.05, GSEA adj. p-value < 0.05) in the group of patients on combination treatment. The B cell proliferation module was significantly lowered by the addition of HCQ to GC at a daily average dose of 4-6 mg of prednisone equivalents compared to GC alone at the same doses ( p = 0.014 ). Pathways related to DNA damage and DNA replication were also reduced. Conclusions The combination of HCQ and GC results in a synergistic molecular response. ORA and GSEA revealed significant involvement of immune-related pathways, with a notable suppression of B cell-related gene modules. While the suppression of the B cell proliferation module was significantly amplified by the addition of HCQ to GC treatment, the influence of GC dosage requires further investigation. Overall, these findings suggest a synergy at the molecular level when HCQ and GC are administered concurrently in combined regimens, enhancing the modulation of key immune processes. References: [1.] Barturen G. Arthritis Rheumatol 2021;73:1073-85. [2.] Rinchai D. Bioinformatics 2021;37:2382-9.

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