Abstract C69: Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro
Authors: Peters Godefridus J. , Van Gemert Frank H. , Kathmann I. , Cillessen Saskia A. , Jansen Gerrit , Reddy Guru 
Published: May 2016

Abstract
Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have promising activity in other lymphoma. FOL is a folate analog and a potent dihydrofolate reductase (DHFR) inhibitor, designed to accumulate in cancer cells via the reduced folate carrier (RFC) and retained via efficient polyglutamylation. DHFR inhibition leads to an imbalance of deoxynucleotides (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 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. In this study 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 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: the B-cell SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL (in competition with [3H]-methotrexate), showed a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin (l-LV). FOL had a very poor substrate binding affinity for FRα (&gt;100-fold lower than folic acid and &gt; 10 lower than l-LV) and a low affinity for PCFT (&gt;10-fold lower than folic acid and l-LV in [3H]-LV uptake competition experiments). Sensitivity (IC50 concentrations after 72 hr exposure) to FOL varied from 2.8 (Hut-78), 5.5 (SU-DHL4 and 5), 7.4 (HT) to 20 nM (Karpas-299 and Jeko-1) and for BEL from 100 (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. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: ±1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure (24 hr pretreatment with either BEL or FOL, followed by 48 hr to FOL + BEL), did not improve interaction (CI: 0.9-1.4). 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 of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 of BEL markedly increased FOL sensitivity (low CIs from 0.40-0.66). 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 additivity in various lymphoma cell lines, with a schedule-dependent synergism. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL.
Citation Format: Godefridus J. Peters, Frank H. Van Gemert, I. Kathmann, Saskia A. Cillessen, Gerrit Jansen, Guru Reddy. Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C69.