Pralatrexate-Resistant Cell Lines Show Intact Internalization Via RFC1 but Collateral Sensitivity to Nucleoside Analogs
Authors: Oiwa Kana , Hosono Naoko , Nishi Rie , Ueda Takanori , Scott Luigi , O'Connor Owen A. , Yamauchi Takahiro 
Published: October 2019

Abstract
Background
Pralatrexate (PDX) is a novel dihydrofolate reductase (DHFR) inhibitor which is designed to have a high affinity for reduced folate carrier (RFC1), DHFR, and folylpolyglutamate synthetase (FPGS), resulting in greater internalization and accumulation in tumor cells than methotrexate. PDX has been approved by the U.S. FDA and many international health care ministries for the treatment of relapsed and refractory peripheral T-cell lymphoma (R/R PTCL). A recent case match control analysis has confirmed a survival advantage for patients enrolled on PROPEL compared to a well-matched population of patients receiving standard of care. Recently, PDX has demonstrated compelling activity in combination with romidepsin in R/R PTCL patients, where is role is likely to expand. In this study we explored the mechanisms of resistance to PDX and approaches to overcome them, including combination strategies using PDX-resistant (PDX-R) leukemic cell lines.
Methods
T-lymphoblastic leukemia cell lines, CCRF-CEM (CEM), MOLT-4 (MOLT4), and MOLT-3 (MOLT3), were used. To establish PDX-R cell lines, the cells were initially incubated with low-concentrations of PDX, and then concentrations of the drug were gradually increased. After acquisition of PDX-R, the single-cell separation was performed by a limiting dilution. Microarray analysis was performed in CEM, MOLT4 and each PDX-R counterpart.
Results
The 50% inhibitory concentration (IC50) values of PDX against CEM, MOLT4 and MOLT3 were 0.6 nM, 2.4 nM and 1.4 nM, respectively, while the PDX-R cell lines exhibited IC50 values of 20 nM, 80 nM and 40 nM, respectively. After 48-hour treatment with PDX, annexin-V positivity was significantly reduced in PDX-R cells (in CEM, MOLT4 and MOLT3 was 78.7%, 77.7%, 97.4% vs 4.4%, 5.9%, 6.6%, respectively). The expression of RFC1 was not altered in PDX-R cells, nor was any acquired mutation of RFC1 detected in cell lines. Intracellular uptake of [14C]-PDX was modestly decreased in CEM/PDX-R, while there was no difference of it in MOLT4/PDX-R, suggesting that the resistance mechanism was not due to the inability to internalize drug. Gene expression array revealed that the expression of DNA-methyltransferase 3 beta (DNMT3b) was significantly elevated in both CEM/PDX-R (12.8 times) and MOLT4/PDX-R (2.8 times) cells, whereas there was no significant difference in that of DHFR, FPGS and thymidylate synthase. When we explored the profile of cross-resistances in PDX-R cells, it was surprising that PDX-R cells were more sensitive to nucleoside analogs, including cytarabine (AraC) and forodesine (FDS). After 72 hours of FDS-treatment, induction of apoptosis was seen in both parental and PDX-R cells, suggesting that the mechanisms of PDX-R is not cross resistant. In addition, there was no cross-resistance to bortezomib or panobinostat in PDX-R cell lines.
Discussion
We established PDX-R cell lines which were around 30-fold resistant to PDX compared to their parental counterparts. The characteristics of PDX-R cells are as follows: (i) intact internalization of PDX, (ii) intact apoptotic mechanisms, (iii) no cross-resistance to BOR or LBH, (iv) collateral sensitive to nucleoside analogs, such as AraC and FDS. These data suggest that the mechanism of acquired resistance to PDX may be associate with not only cellular uptake of PDX, but also DNA methylation and/or nucleic-acid metabolism. These findings may be underscored by the fact that nucleoside analogs exhibited greater potency in the PDX-R cells in vitro. Next steps are focused on exploring the methylation status of these genes involved in PDX activity, and their merits of combining with hypomethylating agents.
Conclusions
We established PDX-R T-lymphoblastic leukemia cell lines which show collateral sensitivity to nucleoside analogs. The mechanisms of PDX resistance is not always cellular uptake of PDX, epigenetic alteration might contribute to the development PDX-resistance. Nevertheless, the new T-lymphoma-targeting agent, FDS can be effective in this setting. Our results may encourage further investigation to overcome resistance of PDX for patients with R/R PTCL.

Disclosures
O'Connor: ADC Therapeutics: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding.