Journal: bioRxiv

Article Title: RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

doi: 10.64898/2026.02.04.703672

Figure Lengend Snippet: A: Overview of the DP study design. Peripheral blood samples were collected from patients with VS (n = 13), VLAD (n = 24), and HC (n = 10) at each outpatient visit for RNA-seq, UBA1 mutation analysis, VAF analysis, and plasma storage for ELISA. RNA-seq was performed separately during the discovery and validation phases. Clinical data were prospectively collected. DP, deep phenotype; VS, VEXAS syndrome; VLAD, VEXAS-like autoinflammatory disease; HC, healthy controls; VAF, variant allele frequency. B: Identification of RNASE1 using DP. Genes positively correlated with VEXASCAF, DEGs between VS and VLAD, and VS and HC. Genes common to all comparisons are ranked by their correlation with VEXASCAF. DEGs were defined as FC > 2, FC < 1/2, and FDR < 0.05. VEXASCAF, VEXAS Current Activity Form; DEGs, differentially expressed genes. C: Scatter plots illustrating the correlation between RNASE1 expression and VEXASCAF in both discovery and validation samples. D: Comparison of RNASE1 expression levels among HC (n = 10), VS (n = 79), BD (n=49) and VLAD (n = 36) based on longitudinal RNA-seq analysis. Outliers (Vo) were defined as values meeting the criterion: Vo ≤ Q1 − 3 × IQR or Vo ≥ Q3 + 3 × IQR, where Q1 and Q3 are the first and third quartiles, and IQR is the interquartile range. Outliers were excluded from the analysis. BD, Behçet’s disease. E: Plasma RNASE1 protein levels measured by ELISA in HC (n = 8), VS (n = 28), and VLAD (n = 34) samples. F: Heatmap showing associations between VEXASCAF score, RNASE1 gene, VAF of UBA1 mutation, and various clinical parameters in patients with VS. The clinical symptoms assessed using the VEXASCAF scoring system are marked with asterisks. “Eye” includes ocular inflammation and periorbital edema; “Ear” and “Nose” indicate auricular and nasal chondritis, respectively. Values represent Spearman’s correlation coefficient (r) with P values < 0.05. *P < 0.05 , **P < 0.01 , ***P < 0.001, ns: not significant. Box-and-whisker plots represent the median, IQR, and 1.5× IQR. Statistical comparisons were performed using the two-sided Mann–Whitney U test, and correlations were evaluated using two-sided Spearman’s rank correlation.

Article Snippet: Human serum and plasma ribonuclease 1 (RNASE1) levels were measured using ELISA kits purchased from Sino Biological Inc. (Cat. No. SEK13468; Beijing, China).

Techniques: RNA Sequencing, Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Variant Assay, Activity Assay, Expressing, Comparison, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

doi: 10.64898/2026.02.04.703672

Figure Lengend Snippet: A: Serum RNASE1 levels before and after prednisolone (PSL) dose escalation in the patients with VEXAS syndrome (VS). Changes in PSL dosage, C-reactive protein (CRP) levels, and serum RNASE1 concentrations are shown for four patients with VS who underwent PSL dose increases owing to worsening disease activity. RNASE1 levels significantly decrease following PSL escalation. B: Uniform Manifold Approximation and Projection (UMAP) plot of 253,296 cells in peripheral blood mononuclear cells (PBMCs) from Yokohama City University (YCU) (HC; n = 1, VS; n = 2) and Mizumaki et al (HC; n = 5, VS; n = 9). Cell type annotations for each cluster are indicated. Mo, monocyte; cMo, classical Mo; ncMo, non-classical Mo; DC, dendritic cells; cDC, conventional DC; pDC, plasmacytoid DC; Neu, neutrophil; Baso, basophil; NK, natural killer; n, naïve; Treg, regulatory T; p, proliferating; MAIT, mucosal-associated invariant T; PC, plasma cell; Eryth, erythrocyte; Plt, platelet. C: UMAP plots of PBMCs derived from HC (n = 6) and VS patients (n = 11), colored according to RNASE1 expression levels in each dataset. The total cell numbers were downsampled to 50,000 cells per group. D: Comparison of RNASE1 expression levels in peripheral blood monocytes between HC and VS groups using bulk RNA sequencing. Left: Data from YCU (HC: n = 3, VS: n = 5); Right: Independent dataset from Beck, et al (HC: n = 3, VS: n = 4). *P < 0.05 , **P < 0.01 , ***P < 0.001, ns: not significant. Error bars indicate the standard error of the mean ( D ). Statistical significance was assessed using a two-sided paired t -test ( A ) and a two-sided t-test ( D ).

Article Snippet: Human serum and plasma ribonuclease 1 (RNASE1) levels were measured using ELISA kits purchased from Sino Biological Inc. (Cat. No. SEK13468; Beijing, China).

Techniques: Activity Assay, Clinical Proteomics, Derivative Assay, Expressing, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

doi: 10.64898/2026.02.04.703672

Figure Lengend Snippet: A: Principal component analysis (PCA) of RNA sequencing data from patients with VEXAS syndrome (VS) who participated in deep phenotyping (DP) (n = 79), stratified by UBA1 genotype. B: RNASE1 expression levels in transcripts per million (TPM) in patients with VS who participated in DP stratified by UBA1 genotype. Outliers (Vo) were defined as values meeting the criterion: Vo ≤ Q1 − 3 × IQR or Vo ≥ Q3 + 3 × IQR. A graph that excluded these outliers was generated. C: VEXASCAF score of patients with VS stratified by UBA1 genotype. D: Variant allele frequency (VAF) (%) of patients with VS stratified by UBA1 genotype. E: Schematic overview illustrating genotype-phenotype correlation and disease progression in VS. (Top) Different UBA1 genotype clones (M41V, M41T, M41L, and wild-type [WT]) are associated with varying degrees of inflammation and cell death. (Bottom) Individuals with low UBA1 mutant VAF and the M41L variant tend to exhibit low pathogenicity, whereas the M41V and M41T variants are associated with VS development. In contrast, moderate to high VAF is associated with the high pathogenicity of M41V and M41T, whereas M41L may lead to VS symptoms at moderate VAF. P values were determined using the two-sided Kruskal-Wallis test, followed by Dunn’s post-hoc test for multiple group comparisons ( B - D ). Box-and-whisker plots display the median, interquartile range (IQR), and 1.5× IQR.

Article Snippet: Human serum and plasma ribonuclease 1 (RNASE1) levels were measured using ELISA kits purchased from Sino Biological Inc. (Cat. No. SEK13468; Beijing, China).

Techniques: RNA Sequencing, Expressing, Generated, Variant Assay, Biomarker Discovery, Clone Assay, Mutagenesis, Whisker Assay

Journal: bioRxiv

Article Title: RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

doi: 10.64898/2026.02.04.703672

Figure Lengend Snippet: A: Schematic diagram illustrating the VEXAS syndrome (VS) cell line generation method. In addition to UBA1 mutations associated with VS (M41V, M41T, and M41L), we generated a control cell line with a silent mutation near the UBA1 Met41 position, which is referred to as “Unmutated.” B: Immunoblot analysis of UBA1, UBA6, and polyubiquitin proteins. GAPDH was used as a loading control. Experiments were performed on day 19 after the switch to Tet-On (Dox +, 20 ng/mL) or Tet-Off (Dox −) conditions. Dox, doxycycline. C: Results of XBP1 splicing measurements show an increased percentage of spliced XBP1 with genotype-specific differences. Cell lines were used for experiments on day 18 after switching. D: Annexin V/propidium iodide assays showed an increase in Annexin V-positive dead cells exhibiting membrane damage under Tet-Off conditions, with differences observed among genotypes. Assays were conducted 14 days after switching. E: mRNA expression levels of IL1B under Tet-On/Off conditions, measured on day 20. F: mRNA expression levels of RNASE1 under Tet-On/Off conditions, measured on day 28. G: RNASE1 mRNA expression in M41L VS cells cultured under serum starvation, measured on day 31 post-switching. Cell lines were cultured for six days in media containing varying fetal bovine serum (FBS) concentrations, starting from day 25 post-switching. Unmutated cells are labeled as “M41: M”. Data are representative of more than three independent repeat experiments ( B ). Data were pooled from two to four independent experiments using different clones ( C-G ). *P < 0.05 , **P < 0.01 , ***P < 0.001, ns: not significant. Error bars indicate the standard deviation (SD). P values were determined using a two-sided t -test ( C-G ).

Article Snippet: Human serum and plasma ribonuclease 1 (RNASE1) levels were measured using ELISA kits purchased from Sino Biological Inc. (Cat. No. SEK13468; Beijing, China).

Techniques: Generated, Control, Mutagenesis, Western Blot, Membrane, Expressing, Cell Culture, Labeling, Clone Assay, Standard Deviation

Journal: bioRxiv

Article Title: RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

doi: 10.64898/2026.02.04.703672

Figure Lengend Snippet: A: mRNA expression levels of IFI27, MX1, and CXCL10 were measured after treatment with 30 µM Necrostatin-2 racemate (Nec-1s), 10 µM GSK’872, 10 µM SP600125, 10 µg/mL anifrolumab, or a 10 µg/mL human IgG1 isotype control. Experiments were conducted on M41V cells 18 days after switching. B: mRNA expression levels of IL1B and RNASE1 were measured after treatment with the same inhibitors as in ( A ). Experiments were conducted on M41V cells 22 days after switching. C: The proportion of spliced XBP1 decreased upon treatment with GSK’872. The experiments were conducted on M41V/L/T cells 18 days after switching. D: mRNA expression levels of IFI27, MX1, and CXCL10 decreased after treatment with GSK’872. The experiments were conducted on M41V/L/T cells 18 days after switching. E: mRNA expression levels of IL1B and RNASE1 decreased after treatment with GSK’872. The experiments were conducted on M41V/L/T cells 22 days after switching. *P < 0.05 , **P < 0.01 , ***P < 0.001, ns: not significant. Data are shown as means ± SD. P values were determined using a two-sided Kruskal-Wallis test, followed by Dunn’s post hoc test for multiple group comparisons ( A, B ), and a two-sided t-test ( C-E ). Data were obtained from two independent clones per genotype and pooled from two to four independent repeat experiments ( A-E ).

Article Snippet: Human serum and plasma ribonuclease 1 (RNASE1) levels were measured using ELISA kits purchased from Sino Biological Inc. (Cat. No. SEK13468; Beijing, China).

Techniques: Expressing, Control, Clone Assay