Background
The treatment outcome of pediatric acute lymphoblastic leukemia (ALL) has greatly improved over the past seven decades with the current regimens resulting in a 5-year event-free survival (EFS) of around 80 % [
1‐
3]. This impressive improvement has been largely attributed to novel prognostic factors, including cytogenetic abnormalities such as
TEL-AML1 and
E2A-PBX1 gene fusions associated with good prognosis as well as
MLL gene rearrangements that confer unfavorable prognosis [
4,
5]. Yet, the high cure rates achieved with current treatment protocols are still paralleled by approximately 20 % risk of relapse, which is consequently associated with poor prognosis [
2,
3]. The emergence of relapse is largely attributable to drug resistance phenomena of leukemic cells. Thus, further advances in understanding the molecular basis underlying these drug resistance phenomena as well as accurate prediction of chemotherapy resistance prior to drug treatment may pave the way to overcoming chemoresistance.
Historically, one of the backbones of contemporary ALL treatment is the folate antimetabolite—methotrexate (MTX) [
6]. Folates are essential enzyme cofactors involved in one-carbon metabolism, which includes several cellular biosynthetic pathways including thymidylate and de novo purine biosynthesis, amino acid metabolism, and mitochondrial protein synthesis [
7]. Antifolates potently inhibit several folate-dependent enzymes engaged in nucleotide biosynthesis, which leads to cessation of DNA replication eventually resulting in cell death [
6]. High-dose (HD)-MTX is used as part of the central nervous system (CNS)-directed therapy (intrathecal MTX), and MTX is an essential component of the maintenance treatment [
1]. MTX is predominantly taken up into cells via the reduced folate carrier (RFC/SLC19A1), and in the case of HD-MTX treatment, also by passive diffusion across cellular membranes, at least to some extent [
8,
9]. Upon entry into the cytoplasm, MTX undergoes polyglutamylation—a unique metabolic conversion catalyzed by folylpolyglutamate synthetase (FPGS) [
9]. This polyglutamylation, which is based on the sequential addition of multiple glutamate residues to the γ-carboxyl group of both folates and MTX, ensures efficient intracellular retention as well as sustains and enhances target enzyme inhibition [
10‐
12]. The main targets of polyglutamylated MTX are dihydrofolate reductase (DHFR—also inhibited by MTX monoglutamates), thymidylate synthase (TS), and several enzymes involved in purine synthesis [
9]. On the other hand, a lysosomal glycoprotein—folylpolyglutamate hydrolase (FPGH)—can counteract polyglutamylation, thereby increasing the efflux of MTX by the efflux transporters of the ATP-binding cassette superfamily including for example ABCC1 and ABCG2 [
9,
13,
14]. Overall, the intracellular accumulation of MTX polyglutamates in leukemic cells proved to be an important determinant of the antileukemic activity of MTX in childhood ALL patients in vivo [
15‐
17]. At the same time, high concentration of long-chain but not total MTX polyglutamates was associated with inhibition of de novo purine synthesis [
15]. Consequently, a spectrum of alterations in MTX metabolism resulting in its decreased cellular accumulation has been identified to induce MTX resistance and compromise its curative effect. MTX resistance has been attributed to inactivating mutations or down-regulation affecting the
RFC gene as well as increased levels of DHFR and TS enzymes together with mutations that decrease their affinity for antifolates [
8,
9,
18‐
21]. In addition, different polymorphisms in
RFC,
TS, and
DHFR were previously reported, several of which were related to increased risk of relapse [
22‐
24]. The cytotoxic effect elicited by MTX is also largely influenced by FPGS activity. Consequently, loss of FPGS function is a well established mechanism of resistance to MTX and other polyglutamylation-dependent antifolates in leukemic cells [
17,
25‐
28]. Differential MTX sensitivity was shown to be associated with several cytogenetic abnormalities. Precursor B cell ALL displaying
TEL-AML1 or
E2A-PBX1 gene fusions were characterized by decreased levels of MTX polyglutamates as compared to precursor B cell ALL with normal karyotype [
16], while patients with hyperdiploid karyotype were highly sensitive to MTX [
29,
30]. Next, to its own cytotoxic effect, MTX is also important in the metabolism of other chemotherapeutics, such as mercaptopurine, used routinely in ALL treatment. MTX was shown to promote the conversion of mercaptopurine to one of its active metabolites—thioguanine nucleotides [
31,
32]—of which high concentration in leukemic cells was associated with increased EFS in leukemia patients [
33]. Therefore, it is imperative to characterize the extent of resistance to this important chemotherapeutic as well as the mechanisms underlying this phenomenon.
The aim of the current study was therefore to determine which parameters of MTX resistance are related to the long-term clinical outcome in childhood ALL patients treated with combination chemotherapy. Towards this goal, we have determined a range of in vitro parameters associated with MTX resistance in a large cohort of pediatric ALL patients and subsequently assessed their relation with treatment outcome as well as with clinical characteristics.
Discussion
In the current study, which used multiple parameters associated with MTX sensitivity, we demonstrate that the cellular level of MTX-induced cytotoxicity is an important determinant of long-term treatment outcome in childhood ALL. We showed that accumulation of long-chain MTX polyglutamates was the strongest MTX resistance-associated variable, as reflected by its significant association with both OS and EFS in the analyzed patient cohort. Our data support and further extend previous studies documenting better antileukemic effect of MTX as well as favorable 5-year EFS in childhood ALL patients, whose leukemic cells accumulated higher levels of MTX polyglutamates [
15,
29,
37]. On the other hand, a study of the Children’s Oncology Group (COG) suggested that the association between the concentration of MTX polyglutamates and EFS is abrogated by higher dose MTX therapy [
38]. However, this study involved a different mode of MTX administration and cut-off levels for high/low polyglutamate accumulation, which might have influenced the different outcome of the analysis. When analyzed in multivariate Cox regression with correction for established clinical factors (including WBC, age, CNS involvement, lineage, and DNA index) as covariates, the cellular level of long-chain MTX polyglutamates was not an independent prognostic factor. This indicates that despite the important role in ALL treatment response, MTX polyglutamates may not have additional predictive value to the already existing models. Surprisingly, low levels of
RFC were predictive of a better overall survival in both univariate and multivariate analysis, contradicting previous reports [
9,
39]. The relation between
RFC mRNA expression and overall survival was the strongest when corrected for the treatment protocol, suggesting that it might be linked to high doses of MTX used in some treatment protocols. When examined within each treatment protocol separately, this association was only observed in patients treated at DCOG ALL8 protocol, on which patients depending on the risk group were administered MTX at 2 g/m
2 or 5 g/m
2 [
40]. In contrast, on COALL protocol ALL-97, for which this relation was absent, MTX was administered at 1 g/m
2 [
2,
41]. This discrepancy may be explained by the limited number of patients treated with 1 g/m
2 in this cohort (6 patients for COALL-97 and 16 for DCOG ALL8). Decreased
RFC expression as well as mutations in the
RFC gene resulting in less efficient uptake of antifolates by tumor cells, have been associated with MTX resistance [
9,
39]. On the other hand, some of the mutations found in the
RFC gene were previously reported to increase the affinity of RFC to folates, resulting in increased intracellular accumulation and competition of folate with MTX [
42]. It is therefore possible that extremely high
RFC expression results in high concentration of folates in the cells, leading to decreased efficacy of MTX and worse treatment outcome. Unfortunately, the numbers of patients treated on the other protocols for which
RFC mRNA expression data were available were too low to allow this analysis.
Moreover, we show that the lineage as well as a number of chromosomal abnormalities are associated with distinct levels of MTX sensitivity. In agreement with previous analysis in a partially overlapping patient cohort as well as in other studies [
17,
34,
35], we observed here elevated accumulation of MTX polyglutamates together with decreased expression of
DHFR and
TS mRNA and consequently higher MTX sensitivity in precursor B cell ALL, as compared to T cell ALL. The difference in the accumulation of the long-chain MTX polyglutamates between these two distinct subtypes of ALL exceeded that obtained for the total MTX polyglutamates. This observation, together with the FPGS activity being significantly increased in the precursor B cell ALL, suggests that differences in MTX response between the precursor B cell and T cell leukemia are highly dependent on FPGS activity and consequent MTX polyglutamylation. Indeed, a previous study comparing lineage-based differences in MTX sensitivity in leukemia cell lines also pointed to the involvement of FPGS activity and DHFR levels [
43]. The importance of the cellular accumulation of MTX polyglutamates was further corroborated by its association with the TSIA, which was previously shown to be a good determinant of MTX sensitivity, as reflected by the correlation between the TSIA results and MTX cytotoxicity [
34]. This association was found in both the entire patient cohort as well as in precursor B cell ALL alone but not in T cell ALL when analyzed separately. The linear regression analysis did not show that the association of cellular MTX polyglutamate levels with the TSIA is actually different between precursor B cell and T cell leukemia. The apparent differences between the correlations may be due to a large uncertainty in the estimates for the correlations of the T cell ALL, which may have been caused by the small sample size for this subgroup. Taken together, our data indicate that despite the differences in concentration of MTX polyglutamates and MTX sensitivity profiles between precursor B cell and T cell ALL, the accumulation of MTX polyglutamates is likely an indicator of MTX resistance as determined by the TSIA in both of these ALL subtypes.
Intriguingly, in parallel to differences in both FPGS mRNA levels and activity observed between precursor B cell and T cell ALL, we found no correlation between
FPGS mRNA level and enzyme activity or the concentration of MTX polyglutamates. This suggests that the level of
FPGS mRNA might not directly translate to its enzymatic activity. A strong correlation was previously reported between
FPGS mRNA expression and activity in leukemic cell lines [
43]. In fact, our previous finding of the high propensity of human
FPGS gene in ALL specimens to undergo impaired splicing is consistent with the fact that transcript levels may not correlate at all with FPGS catalytic activity [
28,
44], Wojtuszkiewicz et al. unpublished observations. Moreover, decreased rates of
FPGS mRNA translation affecting its activity were previously detected in murine leukemic cell lines selected with another polyglutamylation-dependent antifolate—edatrexate [
45]. In addition, we have reported that aberrant
FPGS splicing is a potential contributing factor to the loss of FPGS function, as various
FPGS mRNA splicing alterations were detected in MTX-resistant leukemic cell lines devoid of FPGS activity as well as in adult ALL patient samples [
28].
MTX sensitivity in the patient cohort under study differed between several genetic subtypes of precursor B cell ALL. This included the hyperdiploid patients, displaying high MTX sensitivity associated with increased
RFC expression as well as
TEL-AML1 fusion and
MLL rearrangements displaying an increased MTX resistance. Elevated
RFC expression might be caused by an additional copy of
RFC gene-carrying chromosome 21, which is often observed in hyperdiploid ALL. Our results support previous findings showing that hyperdiploid patients accumulate higher levels of MTX polyglutamates associated with elevated
RFC expression [
29,
30]. Consequently, this subset of patients responded favorably to MTX-containing chemotherapy as compared to patients with a non-hyperdiploid karyotype [
37]. Although the remarkable sensitivity of hyperdiploid ALL cannot be entirely explained by high MTX sensitivity, the current results as well as observations of others [
29,
30,
37] suggest that it is an important biological feature contributing to sensitivity of this ALL subtype. Precursor B cell ALL with
TEL-AML1 fusion as well as with rearranged
MLL gene were associated with lower accumulation of MTX polyglutamates, which in the case of
TEL-AML1 was accompanied by increased MTX resistance as reflected by the TSIA. Precursor B cell ALL carrying either
TEL-AML1 or
E2A-PBX1 gene fusions was previously shown to accumulate decreased levels of MTX polyglutamates as compared to precursor B cell ALL which are devoid of these cytogenetic aberrations [
16]. This was associated with diminished expression of RFC in
E2A-PBX1 ALL and elevated expression of ABCG2—an MTX efflux transporter—in ALL patients with
TEL-AML1 fusion [
16]. Decreased RFC activity resulting from transcriptional silencing as well as mutations and allele loss was previously reported as the cause of antifolate resistance in several tumor types, including leukemia, breast cancer, and osteosarcoma [
19‐
21]. Similar overexpression of ABC transporters is an established mechanism of antifolate resistance [
9,
13,
46]. Moreover, another study suggests that both
TEL-AML1 and
E2A-PBX1 gene fusions downregulate FPGS expression by interacting with its promoter region [
47,
48]. Interestingly,
TEL-AML1 and
E2A-PBX1 are generally associated with a relatively good prognosis, as opposed to patients harboring rearranged
MLL gene [
4], which can be explained by the differences in sensitivity to other chemotherapeutics between these ALL subtypes [
5]. The numbers of ALL patients displaying these cytogenetic abnormalities for which MTX sensitivity-associated variables were measured was very limited in this study. Hence, we were not able to evaluate the role of particular trisomies (i.e., trisomy 21) across the patients with hyperdiploid karyotype or the analyzed cytogenetic abnormalities. Therefore, these associations should be further addressed in large future studies. However, the current data suggest that
TEL-AML1,
E2A-PBX1, and
MLL-rearranged ALL may benefit from courses with high-dose MTX, partly overcoming the polyglutamylation and transport defects [
16,
18].
One of the major limitations of the current study was the low numbers of patients for which particular parameters were recorded. This especially influenced analyses in various ALL groups, such as cytogenetic subtypes, where the patient numbers were extremely low. Moreover, patients included in this study were treated on diverse protocols with distinct MTX administration doses. This issue, however, was addressed in the statistical analysis by including the treatment protocol as a covariate. Finally, the lack of MTX-related toxicity data limited our insight into the associations of particular MTX resistance-related parameters and the clinical outcome of ALL patients. These issues should be carefully considered in future studies.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JC was the principal investigator and takes primary responsibility for the paper; GE, KS, and ES provided patient samples and clinical data; AW, NLW, PMV, and BD performed the research; JC, GJLK, GJ, GJP, and YGA designed the research; AW wrote the paper; JC, GJLK, GJ, GJP, GE, KS, ES, NLW, PMV, BD, YGA, and RP edited the paper. All authors read and approved the final manuscript.