Inhibition of Autophagy by Chloroquine Sensitises Lymphoma Cells to ABT-737-Induced Apoptosis

Abstract 1625

Diffuse large B-cell lymphoma (DLBCL) is characterised by overexpression of the anti-apoptotic protein Bcl-2. It has been recently observed that Bcl-2 also inhibits autophagy by binding and sequestering Beclin-1, an essential autophagy protein, but it is unclear whether Bcl-2 inhibits both apoptosis and autophagy in DLBCL cells. We aimed to determine the dual role of Bcl-2 in both apoptosis and autophagy in Bcl-2 positive cell lines (Su-DHL4 and CRL) and Bcl-2 negative cell lines (Su-DHL8 and Su-DHL10) using the BH3 mimetic compound ABT-737.

The sensitivity of Bcl-2 positive and Bcl-2 negative cell lines to ABT-737-mediated mitochondrial depolarization (ΔΨm^LOW) and cell death (DAPI positive) was assessed by flow cytometry. Treatment of the Bcl-2 positive cell lines Su-DHL4 and CRL with ABT-737 significantly increased (p<0.01) the percentage of both ΔΨm^LOW cells, indicating mitochondrial damage as well as DAPI positive cells indicating cell death. Treatment with ABT-737 increased Bax activation and PARP cleavage in Bcl-2 positive cells, indicating that as expected, ABT-737-induced cell death is via apoptosis. ABT-737-induced cell death was not detected in Bcl-2 negative cell lines Su-DHL8 and Su-DHL10, demonstrating that, as expected, the sensitivity of DLBCL cell lines to ABT-737-induced apoptosis is Bcl-2 dependent.

Treatment of Bcl-2 positive cells with ABT-737 also resulted in a decreased cellular co-localisation of Bcl-2 and Beclin-1 as detected by immunofluorescent staining. Degradation of p62 and LC3-II, selective substrates of autophagy, was detected by Western blotting in Bcl-2 positive but not in Bcl-2 negative cell lines after treatment with ABT-737 for 15 hours. LC3-I is a diffuse cytoplasmic protein which upon activation of autophagy becomes cleaved and lipidated to LC3-II which becomes punctate within cells. Punctuate LC3-II is a widely used marker of active autophagy. ABT-737-induced autophagosome formation was determined at an earlier time point (3 hours after ABT-737 treatment) using immune-fluorescent microscopy. ABT-737 induced increased numbers of larger punctate LC3-II in Bcl-2 positive Su-DHL4 and CRL cell lines but not in Bcl-2 negative cells, indicating that inhibition of Bcl-2 induces autophagy in Bcl-2 positive cells.

We then determined whether autophagy affects ABT-737-induced apoptosis by blocking autophagy using an autophagy inhibitor chloroquine (CQ). Co-treatment with ABT-737 and CQ resulted in an increase in the percentage of ΔΨm^LOW cells, DAPI positive cells and PARP cleavage compared to cells treated with ABT-737 alone in Bcl-2 positive cell lines. Combined, these results indicate that inhibition of autophagy by chloroquine further sensitises Bcl-2 positive cells to ABT-737-induced apoptosis.

In summary, our results indicate that Bcl-2 inhibits autophagy in lymphoma cells by sequestering Beclin-1. Disruption of this interaction by ABT-737 induces autophagy which in turn inhibits apoptosis. Inhibition of autophagy results in increased sensitivity of Bcl-2 positive cells to ABT-737-induced apoptosis, suggesting a role for autophagy inhibitors in lymphoma treatment.