Histone Deacetylase Inhibitors Enhance Cell Death In Burkitt Lymphoma Cells By Modulating Expression Of The WNT/β-Catenin Pathway and Survivin

Burkitt Lymphoma (BL) is an aggressive non-Hodgkin B cell neoplasm primarily affecting children. While overall cure rates are high, prognosis for the 20% of patients with relapsed or refractory is dismal with a 16% survival rate at four years, indicating the need for novel therapeutic approaches. While epigenetic modulators have shown potential therapeutic benefit in other hematologic malignancies, their use in BL has been limited. Here, we evaluate whether histone deacetylase (HDAC) inhibitors could enhance cell death in chemotherapy resistant BL cells.

The BL cell lines Ramos, Raji, or previously generated/characterized immunochemotherapy resistant Raji BL cell lines (Raji 2R and Raji 4RH) (Barth et al., Br J Haematol, 2012) were treated for 48 hours with 4-hydroperoxycyclophosphamide (4-HC, the active metabolite of cyclophosphamide), cytarabine, prednisolone, HDAC inhibitors (suberoyanilide hydroxamic acid (SAHA, vorinostat), and romidepsin), or vehicle control. Cell viability was measured on a Via Cell, by Alamar Blue staining (Invitrogen), or by measuring Caspase-3 activation by flow cytometry. Levels of pro- (survivin, XIAP, MCL-1, livin, and BCL-XL) and anti- (Bax, Bak) apoptotic proteins were evaluated using standard western blot techniques. To evaluate the wiring of signaling networks in the basal state or in response to drug treatment, samples were fixed, permeabilized, and simultaneously stained for Caspase-3, phospho GSK-3 (pGSK3, the inactive form of this protein), active β-catenin, and survivin. Cells were processed on a BD FACSVerse and analyzed by gating on Caspase-3 negative (chemotherapy resistant) cells and monitoring the activity of the pGSK3, active β-catenin, and survivin using FlowJO (V9.6) software.

Relative to the BL cell line Ramos, Raji BL cells were resistant to all doses of conventional chemotherapy (cyclophosphamide, ara-c, and prednisolone) tested. Because hyper-activation of the WNT/b-catenin network due to epigenetic modulation has been implicated in chemotherapy resistance in other B-lineage pediatric malignancies (Hogan et al., Blood 2011), we treated cells with chemotherapy or vehicle and used phosphoflow cytometry to evaluate the expression of pGSK-3, active β-catenin, and its downstream target survivin. Relative to vehicle control, we found that exposure to cytotoxic chemotherapy resulted in rewiring of the cellular networks with increased levels of pGSK3, β-catenin, and survivin. Western blot analyses of the immunochemotherapy resistant Raji 2R and Raji 4RH cells revealed similar upregulation of anti-apoptotic proteins including survivin, downregulation of pro-apoptotic proteins Bak and Bax, and increased expression of pGSK3 compared to Raji cells. To test whether alterations in these signaling axes might enhance the survival of chemorefractory BL cells, we treated Raji cells with the HDAC inhibitor SAHA and found induction of apoptosis at nanomolar doses. Sensitivity to SAHA correlated with down-regulation of active β-catenin and survivin in a dose-dependent manner. To extend these results, we also tested whether immunochemotherapy resistant Raji 2R and Raji 4RH cells were sensitive to HDAC inhibition and found similar robust induction of cell death upon treatment with a panel of HDAC inhibitors.

Active β-catenin and survivin are upregulated in chemotherapy resistant BL cells. HDAC inhibition results in their rapid down regulation and enhanced apoptotic cell death. Further evaluation of inhibitors of the WNT/ β-catenin pathway and HDAC inhibitors is warranted in chemotherapy resistant BL and could have a role in treating refractory or relapsed BL patients.

No relevant conflicts of interest to declare.