Targeting the Epigenome and the Cell Signaling As Novel Therapeutic Approaches for Splenic Marginal Zone Lymphoma

Background: DNA promoter methylation is a well-studied major regulatory machinery of gene expression. Aberrant DNA methylation is not only reported in many cancers but may have utility as a prognostic and/or diagnostic implications in lymphoma. Since pharmacological intervention may revert the aberrant methylation program, new therapeutic opportunities may be explored. We have previously published data that point to epigenetic aberrant reprogramming in the pathogenesis of splenic marginal zone lymphoma (SMZL) (Arribas, Rinaldi et al, Blood 2015). One quarter of SMZL cases are characterized by a high degree of DNA promoter methylation and this associates with inferior survival, a higher risk of histological transformation, IGHV1-02*04 allele usage, mutations of NOTCH2 gene, and 7q31-32 loss. Exposure of SMZL cell lines and primary cells to the demethylating agent decitabine is able to partially revert the aberrant methylation program. Here, we further explored the therapeutic implications of these early observations.

Materials and methods: SMZL cell lines (VL51, Karpas-1718, SSK41) were treated with decitabine with a short (48 h, 2µM) or long exposure (5 days, 100nM). For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously inoculated with 15x10⁶ SSK41 cells and divided in two groups (n= 8 each). Intraperitoneal treatment with decitabine started with tumors of 100-110 mm³ volume. For in vitro combinations, cell lines were exposed to increasing doses of agents alone or in combination for 72h and synergy was assessed with the Chou-Talalay combination index (CI): <0.3, very strong synergy 0.3-0.9, synergy; 0.9-1.1 additive effect; >1.1, antagonism/no benefit.

Results: SMZL cell lines, bearing the aberrant methylation program observed in clinical SMZL samples, showed an important reduction in cell growth upon exposure to decitabine (48 h, 2µM or 5 days, 100nM). In particular, the long exposure to low concentrations of decitabine induced G0/G1 cell cycle arrest and apoptosis in all the SMZL cell lines. We the validated the anti-tumor effect of decitabine in a SMZL xenograft model with the SSK41 cell line. Decitabine was given at the dose of 2 mg/kg on day 1 and day 3. The drug led to a complete eradication of the tumors in all the treated mice, that lasted for 15 days, while the mice of control developed tumors with an average volume of 1500 mm³.

Since the aberrant methylation program is associated with activation of NOTCH, EZH2, NF-κB and PI3K/AKT signaling pathway (Arribas, Rinaldi et al, Blood 2015), we have been assessing possible combinations schemes with additional targeted agents. The sequential combination of demethylating agent (decitabine 50 nM, 5 days) followed by a gamma-secretase inhibitor (PF-03084014, 200 nM for 6 days) resulted in much higher rate of cell death than the single agents. Also, combinations of gamma-secretase inhibitor given concomitantly to the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib and the EZH2 inhibitor DZNep were tested. All the combinations were active in the three cell lines, achieving strong synergy in two cell lines and synergy in the remaining one.

Conclusions: The demethylating agent decitabine showed in vitro and in vivo anti-tumor activity in SMZL, providing the rationale for possible clinical studies. Moreover, combinations of agents targeting pathways deregulated in SMZL showed a very promising in vitro synergism and are worth of further investigations.