Combinatorial epigenetic therapy in diffuse large B cell lymphoma pre-clinical models and patients

Abstract

Background: Refractory and/or relapsed diffuse large B cell lymphoma (RR-DLBCL) patients are incurable with conventional chemotherapy due to the aggressiveness and the chemorefractory state of these tumors. DNA hypermethylation and histone deacetylation are two major epigenetic modifications by which aggressive DLBCL maintain their oncogenic state. We have previously reported that DNA methyltransferase inhibitors (DNMTI) affect RR-DLBCL growth and improve chemosensitivity. Here, we hypothesized that the combination of DNMTI with histone deacetylase inhibitor (HDI) would be an active and feasible therapeutic strategy in RR-DLBCL. Thus, we evaluated the anti-lymphoma activity of the HDI vorinostat (VST) in combination with the DNMTI azacitidine (AZA) or decitabine (DAC) in pre-clinical models of RR-DLBCL, and we determined the feasibility of the combination by conducting a phase Ib trial in RR-DLBCL patients.

Results: Concurrent combination of DNMTI and HDI resulted in synergistic anti-lymphoma effect toward RR-DLBCL cells in vitro and in vivo, with no significant toxicity increase. In a phase Ib trial, a total of 18 patients with a median of three prior therapies were treated with four different dose levels of AZA and VST. The most common toxicities were hematological, followed by gastrointestinal and metabolic. The clinical benefit was low as only one subject had a partial response and three subjects had stable disease. Interestingly, two of the seven patients that received additional chemotherapy post-study achieved a complete response and three others had a significant clinical benefit. These observations suggested that the combination might have a delayed chemosensitization effect that we were able to confirm by using in vitro and in vivo models. These studies also demonstrated that the addition of VST does not improve the chemosensitizing effect of DAC alone.

Conclusions: Our data supports the strategy of epigenetic priming by employing DNMTI in RR-DLBCL patients in order to overcome resistance and improve their outcomes.

Keywords: Chemosensitization; Demethylating agents; Histone deacetylase inhibitors; Lymphoma; Phase Ib.

Fig. 1

Effect of the combination vorinostat with decitabine on DLBCL cells. a Scatter plots of the GI50 values of vorinostat (VST, left) and decitabine (DAC, right) in a panel of 27 DLBCL cell lines obtained at initial diagnosis or at relapse. b Isobologram for Toledo, Farage, OCI-Ly1, WSU-NHL, OCI-Ly7, and SU-DHL4, tested for the combination of DAC and VST. Circles represent each cell line with their relative position to the additive line indicating the result from the combination. Cells below the line indicate a synergistic effect. c DLBCL tumor growth curves (shown as area under the curve) in SU-DHL4 (left) and OCI-Ly7-xenografted (right) mice treated with vehicle, VST, DAC, or VST + DAC

Fig. 2

Chemosensitizing effect of the combination vorinostat with decitabine. a Treatment schedule performed in OCI-Ly1 cells receiving vorinostat (VST) and decitabine (DAC) followed by doxorubicin (Doxo) or mechlorethamine (Mechlo) (upper part), and mRNA and protein levels of p21 after 5 days of treatment (lower part). b Reduction in the growth inhibition 50 (GI50) values for mechlorethamine and doxorubicin after treatment with the VST and DAC combination. c Treatment schedule for mice receiving vehicle, VST, DAC, or VST + DAC, followed by doxorubicin (left part). On the right part, area under the curve (AUC) of tumor growth curves in OCI-Ly7-treated xenografts. d Representative images from OCI-Ly7 mice tumors after being treated, assayed for apoptosis by TUNEL. The percentages of stained area (mean ± the standard error) are shown at the bottom. The bar represents 100 μm