<i> <i>DNMT3A</i> </i> Mutant Clonal Hematopoiesis in Vexas Syndrome Epigenetically Potentiates IL-13 and IL-15 Inflammatory Pathways and Is Associated with Increased Mortality

Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic cells. VEXAS is associated with a high frequency of clonal hematopoiesis (CH- 60%); with the clonal landscape being dominated by DNMT3A mutations. In our prior study (Gutierrez-Rodrigues Blood 2023), we showed that DNMT3A mutations in VEXAS associated with increased mortality, but without a higher risk of MDS/AML. As DNMT3A is a critical epigenetic regulator, we conducted this study to define the epigenetic deregulation and impact on mortality of DNMT3Amt (mutant) CH in VEXAS. Methods: After IRB approval, 88 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 81 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 16,600 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. Genotyping of targeted loci with chromatin accessibility (GoTChA) was conducted on two PB DNMT3Amt samples from CCUS individuals (Izzo et al Nature 2024). Results: Patients' median age was 70 years (range: 56 - 89); 95% were white. Twelve out of 81 patients screened by ECS were found with a DNMT3Amt (15%), with a median variant allele fraction (VAF) of 42% (range: 6-46%). In this cohort, while the presence of any CH mutation did not impact survival, DNMT3Amt CH had an age-independent negative impact on mortality (5.5 years vs 8.7 years, p =0.005), without an increase in risk for MDS/AML. Changes in DNA methylation were profiled in 19 and 9 VEXAS patients without CH (Median Age: 68) or with DNMT3Amt CH (Median Age: 74), respectively. VEXAS samples with DNMT3Amt CH had &amp;gt;4,000 differentially methylated CpGs with 95% of the CpGs being hypomethylated compared to those without CH. Using the Roadmap Epigenomics project, we found that this hypomethylation was enriched at enhancer (Enh) sites and actively transcribed states (TxWk). Pathway analysis revealed that the hypomethylated sites were in or near genes involved in the development and differentiation of leukocytes, with IL-13 and IL-15 being identified as upstream regulators of the methylation changes. Bulk RNA-seq performed on PB of 2 and 3 VEXAS patients with and without DNMT3Amt, respectively, identified &amp;gt;1,500 differentially expressed genes in DNMT3Amt samples, 78% of these being overexpressed. Pathway analysis was enriched in gene pathways involved in the development of leukocytes, with IL-13 and IL-15 once again being identified as upstream regulators. Integration of methylation and transcriptomic data identified upregulation of several genes within the IL-13 and IL-15 pathways, with these regions correlating with a decrease in methylation. To assess if the epigenetic dysregulation is linked to DNMT3A mutations, we performed GoTChA-seq on two DNMT3Amt clonal cytopenia samples (CCUS). The comparison of the number of cutsites in DNMT3A wild type vs mt cells from the same individual demonstrated increased open chromatin within the IL-13 and IL-15 pathways in mt cells, both globally and in a genomic locus specific manner. CITE-seq performed on 3 VEXAS samples (2 DNMT3Amt CH vs 1 no CH) further showed that expression of genes involved in IL-13 and IL-15 pathway is significantly increased in CD4+ and CD8+ T cells from DNMT3Amt samples, suggesting a paracrine regulatory effect of T lymphocytes (mostly UBA1 and DNMT3A wild-type) in VEXAS. Pathway analysis of DEGs in these cells also revealed an enrichment of genes involved in inflammation and lymphocyte proliferation. Conclusions: Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3Amt mediates epigenetic and transcriptional priming associated with upregulation of IL-13/15 pathway in CD4/CD8 T cells, which are mostly DNMT3A and UBA1 wild-type, suggesting that the contribution of T lymphocytes to inflammation may be secondary to CH in the myeloid compartment.