EHMT2 Inhibitor BIX-01294 Induces Endoplasmic Reticulum Stress Mediated Apoptosis and Autophagy in Diffuse Large B Cell Lymphoma Cells
Authors: Xu Linyan , Gao Xiang , Sang Wei , Yan Dongmei , Song Xuguang , Zhu Feng , Xu Kailin 
Published: November 2019

Diffuse large B cell lymphoma (DLBCL) is the most common type lymphoma and the standard therapy R-CHOP regimen has made most patients complete remission, however, 30-40% patients still remains be refractory or relapsed and have a dismal outcome, indicating standard cytotoxic therapy also has limits in these patients. Therefore, it is essential to identify novel therapeutic targets and agents to understand the depth molecular pathogenesis mechanism of DLBCL and overcome the relapsed/refractory. EHMT2 abnormally expression has been discovered in various kinds of malignant cells, and its higher expression may be concerned with poor prognostic of these cancers. BIX-01294 is a small molecule compound which specifically inhibits EHMT2 activity and induces demethylation of H3K9. However, the role of BIX-01294 and the involvement of EHMT2 in DLBCL is not well study now.
In the present study, we first examined the expression of EHMT2 and found EHMT2 were downregulated both in protein and mRNA levels. To determine the effect of BIX-01294 on DLBCL cells growth status, CCK-8 assay was preformed to detect the viability. BIX-01294 exhibited notable proliferation suppression in a dose-dependent manner in DLBCL cells, no matter the ABC type or GCB type cells. Then flow cytometry analysis was carried out to investigate the cell cycle distribution when treated with BIX-01294. The cells in G1 phase were increased in a dose-dependent fashion both in U2932 and SUDHL2 cells, and accompanied by the population in S phase was decreased. Furthermore, we preformed flow cytometric assay to elucidate apoptotic effect and found that BIX-01294 treatment induced U2932 and SUDHL2 apoptosis. By western blot and RT-qPCR, we also showed that the expression of anti-apoptotic protein c-FLIP was decreased and the level of DR4 and DR5 was upregulated. Conformably, BIX-01294 down-regulated anti-apoptotic protein Mcl-1 expression and up-regulated pro-apoptotic protein Bax level, indicating BIX-01294 activates exogenous and endogenous apoptotic signaling pathway in human DLBCL cells. We also showed both protein and mRNA levels of LC3B increased when cells treated with BIX-01294 in DLBCL cells, proves BIX-01294 also triggers autophagy.
To elucidate the mechanism of BIX-01294-induced apoptosis and autophagy in DLBCL cells, we studied the active stage of ER (endoplasmic reticulum) stress. We examined the expression of GRP78, CHOP, ATF3 and ATF4, which were regarded as important protein markers of ER stress and found that their expression were all enhanced in a dose-dependent fashion, indicate BIX-01294 activates ER stress. We then wondered whether ATF3 and ATF4 influenced apoptosis and autophagy induced by BIX-01294. We conducted shRNA to inhibit ATF3 or ATF4 expression and found inhibition of ATF3 or ATF4 upregulation decreased the expression of LC3B, indicating ATF3 and ATF4 were contributed to BIX-01294 induced autophagy. CCK8 assay showed that the viability was increased in ATF3 or ATF4 abrogated cells after exposure to BIX-01294. Consistently, the percentage of apoptosis was significantly decreased in ATF3 or ATF4 knockdown cells than control cells by Annexin-V staining flow cytometry. In our experiments, we also showed that suppressed ATF4 expression inhibited ATF3 and CHOP expression in DLBCL cells.
In conclusion, we showed that with the increased concentration of BIX-01294, the cell survival rate of DLBCL was obviously inhibited and cell cycle was arrested in G1 phase. BIX-01294 induced DLBCL cells apoptosis and autophagy. Furtherly, we explored one of the underlying mechanisms is through activating the ER stress pathway. We speculated that BIX-01294 treatment induces ATF4 upregulation, and then promotes ATF3 and CHOP expression, subsequently contributes to BIX-01294-mediated autophagy and apoptosis.

Disclosures
No relevant conflicts of interest to declare.