Date Published: February 23, 2018
Publisher: BioMed Central
Author(s): Naoki Shinojima, Kenji Fujimoto, Keishi Makino, Kohei Todaka, Kazumichi Yamada, Yoshiki Mikami, Kazutaka Oda, Kazumi Nakamura, Hirofumi Jono, Jun-ichi Kuratsu, Hideo Nakamura, Shigetoshi Yano, Akitake Mukasa.
The therapeutic response to high-dose methotrexate (HD-MTX) therapy for primary central nervous system lymphoma (PCNSL) varies. Polyglutamylation is a reversible protein modification with a high occurrence rate in tumor cells. MTX incorporated into cells is polyglutamylated and strongly binds to dihydrofolate reductase without competitive inhibition by leucovorin (LV). Tumor cells with high polyglutamylation levels are selectively killed, whereas normal cells with lower polyglutamylation are rescued by LV. We hypothesized that the extent of polyglutamylation in tumor cells determines treatment resistance. Here, we investigated the therapeutic response of PCNSL to HD-MTX therapy with LV rescue based on polyglutamylation status. Among 113 consecutive PCNSL patients who underwent HD-MTX therapy in our department between 2001 and 2014, polyglutamylation was evaluated by immunostaining in 82 cases, with relationships between polyglutamylation and therapeutic response retrospectively examined. Human malignant lymphoma lines were used for in vitro experiments, and folpolyglutamate synthetase (FPGS), which induces polyglutamylation, was knocked down with short-hairpin RNA, and a stable cell line with a low rate of polyglutamylation was established. Cell viability after MTX treatment with LV rescue was evaluated using sodium butyrate (NaBu), a histone-deacetylase inhibitor that induces polyglutamylation by elevating FPGS expression. The complete response rate was significantly higher in the group with polyglutamylation than in the non-polyglutamylation group [58.1% (25/43) and 33.3% (13/39), respectively] (p < 0.05), and progression-free survival was also significantly increased in the group with polyglutamylation (p < 0.01). In vitro, the relief effect of LV after MTX administration was significantly enhanced after FPGS knockdown in al cell lines, whereas enhancement of FPGS expression by NaBu treatment significantly reduced this relief effect. These findings suggested that polyglutamylation could be a predictor of therapeutic response to HD-MTX therapy with LV rescue in PCNSL. Combination therapy with HD-MTX and polyglutamylation-inducing agents might represent a promising strategy for PCNSL treatment.
The standard treatment for primary central nervous system lymphoma (PCNSL) is high-dose methotrexate (HD-MTX)-based chemo-radiotherapies with leucovorin (LV) rescue [10, 16, 27]. The median overall survival of patients with PCNSL who undergo HD-MTX-based therapies is ~ 40 months [16, 27]; however, the therapeutic response to HD-MTX therapies varies in patients with PCNSL, with some cases showing poor therapeutic response or recurrence . The established prognostic factors for therapeutic response to HD-MTX and for survival in PCNSL are age and performance status [2, 9], whereas no predictors have been identified for molecules supposedly targeted in this therapy. Polyglutamylation is a reversible post-translational modification of proteins that is thought to be involved in the stabilization of proteins, such as microtubules . In contrast to normal cells, tumor cells show frequent occurrence of polyglutamylation . Once the MTX transported into the tumor cells is polyglutamylated, it is retained and strongly binds to dihydrofolate reductase (DHFR) in a process that is not subject to competitive inhibition by LV, resulting in long-lasting inhibition of thymidylate synthase [14, 22, 24]. Therefore, MTX treatment can selectively kill cancer cells in which polyglutamylation has occurred, whereas normal cells with lower levels of polyglutamylation are rescued with LV . In this context, we hypothesized that the therapeutic response to HD-MTX therapy with LV rescue is dependent upon the extent of polyglutamylation in PCNSL. This study investigated whether the extent of polyglutamylation could predict the response to HD-MTX therapy in patients with PCNSL. To the best of our knowledge, this is the first study revealing that polyglutamylation could be a significant predictor of the therapeutic response to HD-MTX therapy with LV rescue in PCNSL.
HD-MTX-based therapy with LV rescue is established as the standard treatment method for patients with PCNSL [10, 16, 27]. The therapeutic response to HD-MTX therapy varies, and no predictors for therapeutic response or survival have been identified in patients with PCNSL. MTX treatment with LV rescue can selectively kill tumor cells harboring high levels of polyglutamylation, whereas healthy cells with lower levels of polyglutamylation are rescued by LV . We hypothesized that the therapeutic response to HD-MTX therapy with LV rescue depends upon the extent of polyglutamylation in PCNSL. We found that polyglutamylation is indeed a significant predictor of the response to HD-MTX therapy in patients with PCNSL. Additionally, our in vitro studies confirmed that the therapeutic response to HD-MTX treatment with LV rescue was dependent upon the extent of polyglutamylation in lymphoma cell lines, which was consistent with our clinical results. To the best of our knowledge, there have been no studies evaluating the correlation between therapeutic response and polyglutamylation in PCNSL. In other types of malignant neoplasms, high levels of MTX polyglutamylation (for example, in pediatric ALL, such as pediatric B cell lineage ALL) are correlated with therapeutic response, with pediatric B cell lineage ALL showing higher cure rates as compared with adult ALL and T cell lineage ALL . The mechanism of resistance to MTX in leukemia possibly involves decreased uptake of the drug or lack of drug retention due mainly to low levels of polyglutamylation, increased polyglutamate breakdown, or increased DHFR activity [5, 6, 14]. Decreased expression of the reduced folate carrier, a transport protein, was associated with impaired MTX transport and was observed in relapsed acute lymphocytic leukemia after treatment with MTX therapy . Low levels of DHFR gene amplification might also be an important cause of MTX resistance in ALL . Such mechanisms of resistance to MTX aside from impaired polyglutamylation might explain why CR to MTX therapy was observed in only 60% of the patients with PCNSL who showed polyglutamylation in the present study. In contrast, one third of patients in the non-polyglutamylation group had CR. AUCMTX might be a predictor for therapeutic response , and here, we found higher AUCMTX values in patients with CR as compared with those with no CR. Therefore, the combination of polyglutamylation status and AUCMTX might be useful for prediction of MTX therapeutic response.
Our findings suggest that polyglutamylation levels could represent a predictor of therapeutic response to HD-MTX therapy with LV rescue in PCNSL. Furthermore, combination therapy with HD-MTX and agents inducing polyglutamylation might be useful for treating PCNSL.