Schedule-Dependent Synergy Between Belinostat and Pralatrexate in T-and B-Cell Lymphoma Cells in Vitro

Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) are two new agents that have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have also shown promising activity in other types of lymphoma. FOL is a folate analogue and a potent inhibitor of dihydrofolate reductase (DHFR), designed to accumulate in cancer cells preferentially via the reduced folate carrier (RFC) and retained therein via efficient polyglutamylation. Inhibition of DHFR leads to an imbalance of deoxynucleotides (e.g. depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that broadly inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. We investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether the toxicity of FOL can be controlled by levo-leucovorin (Fusilev), the natural stereoisomer of leucovorin, and whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL.

A panel of lymphoma cell lines was used for the combination studies including: the B-cell lymphoma cell lines SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell lymphoma cell lines Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL was determined in competition uptake experiments with [3H] Methotrexate (MTX), revealing a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin. FOL displayed very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than levo-leucovorin). FOL had a low substrate affinity for PCFT (>10-fold lower than folic acid and levo-leucovorin in [3H] leucovorin uptake competition experiments). Levo-leucovorin could completely protect toxicity by FOL, but had no effect on BEL toxicity. Sensitivity of lymphoma cell lines (IC50 concentrations after 72 hrs drug exposure) to FOL drug varied from 2.8 nM (Hut-78), 5.5 nM (SU-DHL4 and 5), 7.4 nM (HT) to 20 nM (Karpas-299 and Jeko-1) while IC50 values for BEL were in the range of 100 nM (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT).

The interaction between BEL and FOL was studied using the median-drug effect analysis with Calcusyn software. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all the lymphoma cell lines revealed an additive effect (CI: all around 1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure to the drugs (24 hr pretreatment with either BEL or FOL) followed by 48 hr to the combination, did not improve the results with CI values varying between 0.9 and 1.4. As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 concentrations of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 concentrations of BEL markedly increased the sensitivity to FOL as reflected by the low CIs varying from 0.40 to 0.66. Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest.

In conclusion, the combination of FOL and BEL showed additive activity in various lymphoma cell lines, while a schedule-dependent synergism was observed. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Furthermore, toxicity of FOL could be completely protected by levo-leucovorin.