Presence of Activating STAT3 Mutations in the CD8+ T Cells of Rheumatoid Arthritis Patients

Rheumatoid Arthritis (RA) is a systemic autoimmune disorder afflicting around 1% of the global population. It is treated with a variety of immunosuppressants typically progressing from first line therapies such as low dose methotrexate (MTX) or other disease modifying anti-rheumatic drugs (DMARDs) to second line treatments such as tumor necrosis factor (TNF) inhibitors or other biologics designed to target pro-inflammatory cytokines. Several factors are used to anticipate and measure disease severity including Disease Activity Measures (DAM) and positivity of rheumatoid factor (RF), antibodies to cyclic citrullinated protein (CCP), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Presence of anti-CCP antibody is the most specific of these markers of RA, and its presence may be associated with more severe disease.

Large granular lymphocyte (LGL) leukemia is a rare hematologic malignancy characterized by clonal proliferation of natural killer or CD8+ T cells, somatic activating mutations in the signaling transducer and activator of transcription 3 (STAT3) gene, and clinical features including neutropenia and anemia. Interestingly, LGL leukemia is strongly associated with RA, with as high as 36% of patients exhibiting concomitant RA. There is significant overlap between the diseases. Both RA alone and in combination with LGL leukemia exhibit cytotoxic T cell expansions, HLA-DR4 enrichment, female bias, and RA-associated autoantibodies.

The dominant molecular hallmark of LGL leukemia is somatic activating STAT3 mutation. Most patients exhibit increased activation of STAT3 through phosphorylation, and 50% or more patients have a mutation in STAT3, the most common being missense mutations, Y640F and D661Y. Of note, LGL leukemia patients with one or more STAT3 mutations are more likely to suffer from concomitant RA.

Based on these observations, we hypothesized that STAT3 mutations would be enriched in RA patients compared to the general population and that the presence of mutations may correlate with specific clinical characteristics of RA. In this study, blood samples and demographic data were simultaneously collected from 96 RA patients at the UVA Rheumatology Clinic. CD8+ cells were isolated from blood samples and DNA was extracted and analyzed via droplet digital PCR to detect Y640F, D661Y, and S614R_G618R STAT3 mutations. Figure A shows that roughly 3% (3/96) of the RA samples had a STAT3 mutation in greater than 5% of the DNA analyzed. Additionally, an even larger proportion of RA patients had either Y640F or D661Y mutations at a rate of between 1:100 and 1:1000 cells (10/96 Y640F, 14/96 D661Y). These samples were classified as mutated for the purposes of statistical analysis. Of the 9 healthy control samples analyzed, only one exhibited a mutation of this size (1/9 Y640F, 0/9 D661Y). Correlation with clinical parameters showed similar disease manifestations between WT and STAT3 mutant samples. However, CCP differed between mutational types, with the Y640F group exhibiting higher positivity than the D661Y group (Figure B). Taken together, the increased presence of STAT3 activating mutations in CD8+ cells isolated from RA blood samples as well as the difference in CCP positivity between D661Y and Y640F mutational groups merits further investigation of these pathways in RA.

Feith:Kymera Therapeutics: Honoraria; AstraZeneca: Research Funding; Recludix Pharma: Research Funding. Kimpel:Aurinia Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Landos Biopharma: Membership on an entity's Board of Directors or advisory committees. Loughran:Kymera Therapeutics: Honoraria; Dren Bio, Inc: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Recludix Pharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Prime Genomics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.