Inhibition of Autophagy with 3-Methyladenine Synergistically Enhances Cellular Apoptosis Induced by the Histone Deacetylase Inhibitor PCI 24781 in Non-Hodgkin’s Lymphoma Cells
Authors: Davi Kevin A , Bhalla Savita , Prachand Sheila , Evens Andrew M , Gordon Leo I. 
Published: October 2019

Abstract
Background: Autophagy represents a potential mechanism of cellular survival in settings of external stressors, such as starvation and exposure to cytotoxic drugs. Driven by the formation of autophagosomes, which digest nonessential cellular components during stress, autogphagy may be pharmacologically inhibited by 3-methyladenine (3-MA), an inhibitor of autophagosome formation. Histone deacetylase inhibitors (HDACI) block cancer cell proliferation by mechanisms that involve epigenetic gene regulation leading to cell growth arrest, differentiation, and apoptosis. Previous investigators have demonstrated synergistic cellular killing by a combination of HDACI and autophagy inhibitors in leukemia cell lines (Carew et al., Blood. 2007).
Methods: We investigated whether a similar effect was possible in lymphoma. Specifically, we studied the combination of the pan-HDACI PCI-24781 and 3-MA in three lymphoma cell lines—Ramos (Burkitt’s lymphoma), Jeko, and Granta (both mantle cell lymphoma lines). Cells were cultured in RPMI (Invitrogen) and were incubated for 48 hours with 3-MA (1 mM and 2 mM), PCI-24781 (0.125 uM, 0.25 uM, 0.5 uM) and combinations of both agents. Apoptosis was determined by fluorescence-activated cell sorting (FACS) using AnnexinV-FITC/propidium iodide (AnnexinV+/PI+) staining. Western blots were performed to assess markers of apoptosis—beclin-1 and LC3B isoform I to II conversion.
Results: In all three cell lines, treatment with PCI-24781 resulted in a dose-dependent increase in apoptosis. Our previous studies have shown that the IC70 (dose to achieve 70% AnnexinV+/PI+) was 1uM for PCI-24781 in the Ramos cell line. When 3-MA was combined with 0.25 uM PCI-24781, apoptosis increased from 13% to 35%. Similarly, 3-MA increased apoptosis from 8% to 18% and from 30% to 50% when added to PCI 24781 0.125 uM and 0.5 uM, respectively. Similar, albeit less striking, trends were seen in Jeko and Granta cells. Synergy was determined in Ramos cells by the combination index (CI) using isobolograms (CalcuSyn software). Moderate synergy was seen at PCI concentrations of 0.25 and 0.5 uM and a 3-MA concentration of 2 mM, with a CI of approximately 0.7. Immunoblots were analyzed for markers of autophagy—beclin-1 and LC3B isoforms I and II. Combined 3-MA and PCI-24781 reduced Beclin-1 expression as well as the conversion of LC3B isoform I to II in cells treated with the PCI-24781and 3-MA combination, as compared to cells treated with PCI-24781 alone, suggesting that inhibition of autophagy is responsible for the synergistic increase in apoptosis. This reduction was most prominent at 6 hours, indicating that autophagy inhibition is a relatively early event in this model.
We conclude that inhibition of autophagy with 3-MA represents a novel approach to synergistically enhance cellular apoptosis induced by HDACI in lymphoma cells. Clinical studies taking advantage of this novel biology are warranted.