Association of altered folylpolyglutamate synthetase pre-mRNA splicing with methotrexate unresponsiveness in early rheumatoid arthritis

Abstract

Objectives An efficient pharmacological response to MTX treatment in RA patients relies on the retention and accumulation of intracellular MTX-polyglutamates catalysed by the enzyme folylpolyglutamate synthetase (FPGS). We recently identified a partial retention of FPGS intron 8 (8PR) as a prominent splice variant conferring FPGS dysfunction and decreased MTX polyglutamylation in acute lymphoblastic leukaemia. Here, we explored the association between FPGS 8PR levels and lack of MTX responsiveness in RA patients. Methods Thirty-six patients undergoing MTX treatment were enrolled from the Combinatie behandeling Reumatoide Artritis (COBRA)-light trial. RNA was isolated from blood samples at baseline, 13 weeks and 26 weeks of therapy, from patients in either COBRA-light (n = 21) or COBRA (n = 15) treatment arms. RT-qPCR analysis was used to assess RNA levels of FPGS 8PR over wild-type FPGS (8WT). Results In the COBRA-light treatment arm, higher baseline ratios of 8PR/8WT were significantly associated with higher 44-joint disease activity score (DAS44) at 13 and 26 weeks. Higher baseline ratios of 8PR/8WT also trended towards not obtaining low disease activity (DAS <1.6) and becoming a EULAR non-responder at 13 and 26 weeks. In the COBRA-treatment arm, a significant association was observed between high baseline 8PR/8WT ratios and higher DAS44 score at 26 weeks. Higher 8PR/8WT ratios were associated with non-response at week 26 based on both low disease activity and EULAR criteria. Conclusion This study is the first to associate alterations in FPGS pre-mRNA splicing levels with reduced responsiveness to MTX treatment in RA patients. Trial registration ISRCTN55552928.

ATR and CDK4/6 inhibition target the growth of methotrexate-resistant choriocarcinoma

“…1D-E ). Indeed, changes to the mRNA splicing for folylpolyglutamate synthetase (FPGS), the enzyme that polyglutamates MTX to achieve the drug’s intracellular retention, was recently associated with reduced responsiveness to MTX treatment [ 74 ]. More generally, mRNA splicing has been found to impact response to therapy in cancer [ 75 ], making this proposed avenue of research topical.…”

Heterogeneity of RNA editing in mesothelioma and how RNA editing enzyme ADAR2 affects mesothelioma cell growth, response to chemotherapy and tumor microenvironment

“…A recent study has described that altered folylpolyglutamate synthetase ( FPGS ) pre-mRNA splicing is associated with unresponsiveness to antifolate agent methotrexate in rheumatoid arthritis (30). FPGS activity adds polyglutamate to antifolate drugs and increases their retention in cells.…”

“…TMA construction was achieved as previously described (28). Immunohistochemistry was performed as previously described (29) Quantification of intron 8 retaining (inactive) and normally spliced (active) transcripts of folylpolyglutamate synthetase (FPGS) was done using qPCR as previously described (30). The ratio was determined by 2^(-ΔCt) where ΔCt is the difference between the Ct values of inactive and active FPGS.…”

“…FPGS activity adds polyglutamate to antifolate drugs and increases their retention in cells. High levels of intronretaining FPGS transcript leads to reduced activity, hence resistance (30). Because of the dynamic between RNA editing and alternative splicing (37), we analyzed the ratio of the intron 8 retaining (inactive) transcript vs. normally spliced (active) FPGS and observed (Fig.…”

Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

“…With respect to the latter, AS has been proposed as a source of potential biomarkers or as a target for drug development [ 10 – 12 ]. For example, in childhood acute lymphoblastic leukemia (ALL), an A5SS selection in exon 8 of the folate metabolizing enzyme folylpolyglutamate synthetase (FPGS) was shown to be associated with the clinical response to methotrexate (MTX), an anchor drug in the treatment of ALL [ 13 ] and rheumatoid arthritis [ 14 ].…”