Abstract 4437: The relevance of aberrant FPGS splicing for ex vivo MTX resistance and clinical outcome in childhood acute lymphoblastic leukemia
Authors: Wojtuszkiewicz Anna , Assaraf Yehuda G. , Hoekstra Mirthe , Jansen Gerrit , Peters Godefridus J. , Sonneveld Edwin , Kaspers Gertjan L. , Cloos Jacqueline 
Published: August 2015

Abstract
Methotrexate (MTX) is one of the key components of current ALL treatment protocols. This antifolate inhibits de novo nucleotide biosynthesis, hindering DNA replication, hence leading to cell death. However, despite the overall good cure rates in ALL, drug resistance continues to hamper the efficacy of MTX, contributing to relapse of leukemia. Multiple molecular mechanisms underlying drug resistance have been characterized, however, their clinical relevance often remains unclear. We recently identified impaired FPGS splicing, in particular intron 8 partial retention (intron 8 PR), as a possible contributor to decreased FPGS activity in both MTX-resistant leukemia cell lines and primary ALL specimens.
In the current study we explored the association between FPGS splicing alterations and MTX resistance as well as the clinical outcome of 91 pediatric ALL patients. With respect to MTX resistance, we determined the levels of MTX polyglutamates in leukemic cells, as well as FPGS activity along with the expression of several enzymes and transporters involved in (anti)folate metabolism and transport, including FPGS, FPGH, RFC, DHFR and TS. The sensitivity of leukemic cells to MTX was determined using the thymidylate synthase inhibition assay (TSIA). Moreover, the sensitivity to other chemotherapeutics as measured in the MTT assay was determined for these patient specimens. The levels of FPGS splicing alterations were semi-quantified using PCR and fragment analysis. We found that intron 8 PR plays an important role in MTX resistance in the studied patient cohort. In a subset of patients with relatively low levels of MTX polyglutamates, high levels of intron 8 PR were associated with inferior EFS (HR = 4.2, P = 0.006) and OS (HR = 5.6, P = 0.003). The relation between intron 8 PR and OS remained significant in multivariate analysis, which included known prognostic factors, including WBC and age. Interestingly, in this subgroup of patients, intron 8 PR correlated with higher MTX resistance as determined by the TSIA assay (p = 0.015) and lower accumulation of long chain MTX polyglutamates (p = 0.027). Moreover, high levels of intron 8 PR were associated with resistance to the glucocorticoids dexamethasone and prednisone. Remarkably, we found that a MTX-resistant FPGS-deficient subline of CCRF-CEM - CEM/FPGS-, harbors a high level of intron 8 PR and displays high level of dexamethasone cross-resistance (over 350-fold).
Our findings suggest that intron 8 PR is not only relevant for MTX resistance but may also reflect a broader splicing defect affecting multiple genes, thereby resulting in multidrug resistance. These findings may have important implications for personalized treatment as well as circumvention of drug resistance in ALL patients.
Financial support by Kika (Children cancer-free)
Citation Format: Anna Wojtuszkiewicz, Yehuda G. Assaraf, Mirthe Hoekstra, Gerrit Jansen, Godefridus J. Peters, Edwin Sonneveld, Gertjan L. Kaspers, Jacqueline Cloos. The relevance of aberrant FPGS splicing for ex vivo MTX resistance and clinical outcome in childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4437. doi:10.1158/1538-7445.AM2015-4437