Abstract
Pralatrexate (Folotyn; PLX) and belinostat (Beleodaq; BLS) are registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and are being considered for other lymphomas. In this study we investigated whether BLS had the ability to potentiate the cytotoxicity of PLX. A panel of lymphoma cell lines was used for the combination studies: the B-cell SUDHL-4, SUDHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. Uptake of PLX was mediated by the reduced folate carrier (RFC). PLX showed a 6-fold better RFC substrate affinity compared to methotrexate, and 2-fold better than levoleucovorin (l-LV). Sensitivity expressed as the concentration that resulted in 50% growth inhibition (IC50) after 72 hr exposure to PLX varied from 2.8 to 20 nM and for BLS from 72 to 233 nM, independent of the background of the cell lines. The interaction between BLS and PLX was studied using the median-drug effect analysis. At a fixed molar ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: around 1.0). In three selected cell lines (SUDHL-4, SUDHL-5, and HT) sequential exposure (24 h pretreatment with BLS, followed by 48 h to PLX + BLS), did not improve interaction (CI: 0.9–1.4). As an alternative approach a non-fixed ratio was used by exposing SUDHL-4, SUDHL-5, and HT cells to IC25 concentrations of either BLS or PLX in combination with the other drug. Exposure to IC25 of PLX did not decrease the IC50 for BLS (CI from 0.6–1.2), but exposure to IC25 of BLS markedly increased PLX sensitivity (low CIs from 0.40 to 0.66). Mechanistic studies focused on induction of apoptosis, and showed cleavage of predominantly caspase-9 in HT and SUDHL-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 PLX and BLS showed additivity in various lymphoma cell lines, with a schedule-dependent synergism in B-cell lymphoma. Based on these data, proficient inhibition of HDAC activity by BLS holds promise in sensitization of tumor cells to PLX.
Highlights
Peripheral T-cell Lymphoma (PTCL) accounts for over 6,000 to 9,000 cases in the United States annually and worldwide this type of cancer represents 10 to 15% of all non-Hodgkin’s lymphoma (Rudiger et al, 2002; Laribi et al, 2018)
Since PLX and BLS are registered for the treatment of PTCL (O’Connor et al, 2011; Gooptu et al, 2015), we investigated the sensitivity of several other lymphoma cell lines, including B-cell, to these drugs, in comparison to the CEM cells used for transport studies (Table 2)
Most other combinations were additive. Both PLX and BLS have been tested earlier in various combinations, such as the combinations of PLX with gemcitabine in NonHodgkin Lymphoma models, in which sequential addition was five times more effective in inducing apoptosis compared with simultaneous exposure (Tonner et al, 2006)
Summary
Peripheral T-cell Lymphoma (PTCL) accounts for over 6,000 to 9,000 cases in the United States annually and worldwide this type of cancer represents 10 to 15% of all non-Hodgkin’s lymphoma (Rudiger et al, 2002; Laribi et al, 2018). Inhibition of DHFR by PLX results in depletion of purines and dTMP, leading to an imbalance of deoxynucleotides, with a depletion of deoxythymidine triphosphate (dTTP) and an increase in deoxyuridine triphosphate (dUTP) resulting in DNA strand breaks and in inhibition of DNA synthesis (Marchi et al, 2010). Based on these properties, PLX has been tested in several other hematological and solid tumors as a single agent and in various combinations (Marchi et al, 2010, 2013; Serova et al, 2011; Dovzhanskiy et al, 2012). Antifolates can be considered as epigenetic drugs since they affect cellular methylation reactions, due to inhibition of one-carbon metabolism (Frigerio et al, 2019)
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