Acute lymphoblastic leukemia (ALL) remains the most common cancer diagnosis in children, accounting for 21% of all pediatric cancers from birth to 19 years. Antifolate therapy, specifically treatment with methotrexate, is central in ALL therapy. Genetic polymorphisms, such as those for MTHFR and SLC01B1 can contribute to how methotrexate is metabolized and can alter side effect profiles, but they do not fully explain why transformed cells may become resistant to methotrexate. Epigenetic abnormalities, including dysregulation of DNA methylation, is frequent in all types of cancers including ALL, often leading to silencing of tumor suppressor genes. Epigenetic silencing could potentially influence a cell's sensitivity to antifolate therapy.
PCFT is a low pH folate transporter and the regulation of PCFT gene expression includes DNA methylation. Previous studies have demonstrated that treatment with a hypomethylating agent results in reexpression of the gene. PCFT methylation, however, has not yet been studied in primary cancer tissues. Decreased or absent levels of PCFT could result in decreased methotrexate uptake and provide a mechanism of resistance.
Genomic DNA and RNA were isolated from primary pediatric ALL samples as well as ALL cell lines. Methylation status was evaluated using methylation specific PCR (MSP). Expression was measured using qRT-PCR. Cell lines were treated with the hypomethylating agent 5-Aza-2-deoxycytidine (5-Aza-2dC). After treatment with 5-Aza-2dC, RNA was isolated for expression. Cells were also evaluated for methotrexate entry into the cell using fluorescein-tagged methotrexate.
We found that 100% of ALL cell lines (n=5) were methylated at PCFT. In primary patient samples, among an initial cohort, 23/33 samples (70%) had methylation at PCFT by MSP. By subtype of ALL, methylation of PCFT was found in 14/21 (67%) of B-cell ALL and 9/12 (75%) of T-cell ALL. This cohort did not have RNA available or outcome data. In a second pediatric ALL cohort of newly diagnosed patients, 23/40 (57%) were methylated at the PCFT promoter. Among these samples, 19/33 B-cell ALL, and 4/7 T-cell ALL were methylated at PCFT. The mean diagnostic age of the patients with PCFT promoter methylation was lower (7.13 years) than those that were unmethylated (8.71 years). Within this cohort with between 4.5 and 15 years of time elapsed since diagnosis, only 3 patients experienced recurrent or refractory disease. Of these patients, 1 had methylation of PCFT.
Expression analysis by qRT-PCR demonstrated that 2 cell lines had no expression of PCFT (Tanoue and Nalm6) while 1 cell line expressed low level PCFT despite having promoter methylation (HB 11;19). Among the primary ALL samples, the unmethylated group had a higher mean expression of PCFT (1/ΔCt = 0.068) than the methylated group (1/ΔCt = 0.0635; p=0.045), suggesting that methylation of PCFT leads to decreased expression. Treatment of the ALL cell lines with 5-Aza-2dC did induces expression of PCFT in Nalm6 and Tanoue, but did not increase expression in HB 11;19 where expression was already present. After treatment with 5-Aza-2dC, Nalm6 and Tanoue also increased the intake of methotrexate by 2.25 and 1.7 times respectively, while in HB 11;19, the methotrexate intake decreased by a factor of 0.73.
PCFT is frequently methylated in both B-cell and T-cell pediatric ALL. DNA methylation was associated with decreased expression in primary samples. Hypomethylating agents can reverse this gene silencing resulting in higher methotrexate entry into the cell, providing a potential strategy for overcoming methotrexate resistance.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.