Background: Low-dose methotrexate (MTX) serves as the first-line treatment for rheumatoid arthritis (RA). Conversely, for hematological malignancies, high-dose MTX treatment is indicated in combination with folinic acid/leucovorin to prevent untoward toxicity. For both disease modalities, efficient pharmacological response critically depends on the retention and accumulation of intracellular MTX. The latter process is mediated by the enzyme folylpolyglutamate synthetase (FPGS) through MTX conversion into MTX-polyglutamates (MTX-PG). As such, decreased FPGS activity has been recognized as a mode of MTX resistance, but the underlying molecular mechanism has not yet been pinpointed [1,2]. Recently, aberrant pre-mRNA splicing of FPGS in acute lymphoblastic leukemia cells was associated with reduced FPGS activity, decreased MTX-PG accumulation and lower overall survival. Specifically, a partial retention of FPGS intron 8 (8PR) was identified as a prominent splice variant causing FPGS dysfunction . Objective(s): To investigate whether the expression of the FPGS premRNA variant partial retention of intron 8 is associated with MTX unresponsiveness in RA patients over 3-6 months of MTX therapy. Method(s): Patients were enrolled from the COBRA-light trial comprising MTX treatment . After informed consent, blood was obtained from 38 patients in either the COBRA (n=15) or the COBRA-light (n=21) treatment arm  and collected in Paxgene tubes for RNA isolation. Two patients were excluded due to insufficient sample quality. Quantitative PCR techniques were used to assess the ratios of FPGS 8PR over wild type FPGS (8WT) expression in whole blood RNA. Remission was defined as a DAS44 < 1.6. Result(s): Baseline ratios of 8PR/8WT were associated with a decline in DAS44 at T3 (p=0.001) and T6 (p=0.13) in the COBRA-light patients. T3 ratios were also associated with DAS44 at T6 (p=0.001) and DELTADAS44 at T6 (p=0.26). Logistic regression analysis showed that in COBRA-light patients baseline ratios of 8PR/8WT were associated (p=0.05) with the probability of T3 remission but not T6 remission. T3 ratios of 8PR/8WT in COBRA-light patients were similarly associated with T6 remission but not with T3 remission. COBRA patients also showed a significant association between baseline 8PR/8WT ratios and DAS44 at T6 (p=0.04) while T3 ratios of 8WT/8PR were significantly associated with DAS44 at T3 (p=0.03) and T6 (p=0.05). However, analysis of COBRA patients showed no significant associations between baseline 8PR/8WT ratios and T3/T6 remission, nor associations between T3 ratios and T3/T6 remission. Conclusion(s): These results demonstrate that a higher expression of an intron 8 retention of FPGS mRNA is associated with a higher DAS44 at T3 and T6 in COBRA-light patients, and at T6 for COBRA patients. Moreover, in COBRA-light patients, baseline expression of 8WT/8PR is significantly associated with remission at T3 or T6. This study is the first to show the impact of FPGS pre-mRNA splicing alterations in relation to unresponsiveness to MTX treatment in RA.
Muller, I., Lin, M., Lems, W., Wee, M. T., Cloos, J., Assaraf, Y., … Jonge, R. D. (2019). THU0063 PRE-MRNA SPLICING ALTERATIONS IN FOLYLPOLYGLUTAMATE SYNTHETASE IN BLOOD CELLS OF EARLY RHEUMATOID ARTHRITIS PATIENTS ARE ASSOCIATED WITH UNRESPONSIVENESS TO METHOTREXATE (pp. 299.1-299). BMJ. https://doi.org/10.1136/annrheumdis-2019-eular.3215