Single agent and combination studies of pralatrexate and molecular correlates of sensitivity

Abstract

Background: Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents.

Methods: Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT-PCR.

Results: Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance.

Conclusion: Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome.

Figure 1

Pralatrexate cytotoxicity in a panel of human cancer cell lines. (A) Pralatrexate (PDX) time-course (IC₅₀ values, 1, 5, 24 and 72 h drug exposure) cytotoxicity in sensitive cell lines. (B) Comparative analysis of 72 h cytotoxicity of pralatrexate, methotrexate, pemetrexed, 5-FU and 5′-DFUR in a panel of cancer cell lines. The indicated values are calculated as follows: log (IC₅₀ individual cell line) – mean (log IC₅₀). Negative values indicate that the cell line is more sensitive than the average, whereas positive values indicate that the cell line is more resistant than the average.

Figure 2

Effects of pralatrexate (PDX) and methotrexate (MTX) (24 h exposure) on cell cycle distribution (A), apoptosis induction (Annexin V staining) (B) and activation of PARP, caspase 3 and caspase 9 (C) in DU145 prostate cancer cells. ^*Significant difference (P<0.05) comparing with control.

Figure 3

Relative mRNA expression of folate genes in pralatrexate sensitive and -resistant cell lines. (A) Relative expression of DHFR, FPGS, TS, GART, SLC25A32, SCL19A1/RFC-1 and ABCB1/MDR1 mRNA in sensitive- and resistant groups. (B) Correlation between pralatrexate sensitivity (IC₅₀ values) and mRNA expression of SCL19A1/RFC-1 and SLC25A32 folate transporters in nine pralatrexate-sensitive cell lines.

Figure 4

Characterization of pralatrexate-resistant cell lines. (A) Pralatrexate (top panel) and methotrexate (bottom panel) cytotoxicity in DU-PDX and HEP-PDX cell lines compared with their parental counterparts DU145 and HEP2. (B) Relative mRNA expression of folate genes in pralatrexate-resistant cell lines. (C) Western blot of DHFR protein in DU145 and HEP2 sensitive and DU-PDX, DU-MTX, HEP-PDX and HEP-MTX pralatrexate and methotrexate-resistant cell lines.