HDAC Inhibition Effectively Induce Apoptosis in Diffuse Large B Cell Lymphoma with High Bfl-1

Venetoclax, a Bcl-2 specific inhibitor, shows potential benefit in certain patients with diffuse large B cell lymphoma (DLBCL) when combined with conventional cytotoxic chemotherapy. However, both de novo and acquired resistance to venetoclax is frequently observed in DLBCL and endogenous Bfl-1 expression can render DLBCL insensitive to venetoclax. Given the difficulties for direct inhibition of Bfl-1, we looked for the indirect inhibitory strategies for Bfl-1 at both transcriptional and post-translational levels. Accordingly, we decided to use a pan-histone deacetylase (HDAC) inhibitor to decrease a Bfl-1 transcriptional factor, Wilms' tumor-1 (Wt-1), while increasing Noxa, an inactivating Bfl-1 binding partner in DLBCL.

We screened the expression levels of Bcl-2 family in 6 DLBCL cell lines (SUDHL-2, OCI-LY-3, TMD-8, RC-K8, OCI-LY-1, and OCI-LY-19) and divided them into 2 groups based on Bfl-1 expression level (Bfl-1+/Bfl-1-). Bfl-1+ group cell lines (SUDHL-2, OCI-LY-3, TMD-8, RC-K8) exhibited reduced sensitivity to venetoclax compared to Bfl-1- group cell lines (OCI-LY-1, OCI-LY-19) as expected. In contrast, the cell lines in Bfl-1+ group were more sensitive to panobinostat, a representative pan-HDAC inhibitor, than Bfl-1- group cell lines. We could also validate the association between Bfl-1 expression and panobinostat response using 12 DLBCL cell lines data from Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC).

With western blot, we confirmed that as the concentration of panobinostat increases, Bfl-1 and Wt-1 were decreased and Noxa was increased as we hypothesized. In a time-course analysis, Bfl-1 was downregulated by panobinostat in a Wt-1, and Noxa-dependent manner. Additionally, we confirmed that the association of Bfl-1 with venetoclax and panobinostat was linked to apoptosis rate using Western blot and Flow cytometry.

Finally, to evaluate the impact of Bfl-1 on the vulnerability of cells to panobinostat, we compared the sensitivity to panobinostat upon shRNA-mediated Bfl-1 knockdown using SUDHL-2 cells (the strongest Bfl-1 expression level among DLBCL cell lines). Reduced sensitivity was observed in Bfl-1 knocked down cells comparing to Non-Infected (NI) and scrambled vector transduced cells. Apoptosis rate was decreased significantly in Bfl-1 knocked down cells as well. Therefore, we identified the sensitivity to panobinostat depends on the expression of Bfl-1 in DLBCL.

In summary, we identified the significance of Bfl-1 on HDAC inhibitor response and confirmed that Wt-1 and Noxa play important roles in that process. Our results provide a mechanistic rationale for utilizing HDAC inhibitors for DLBCL patients using Bfl-1 as a prediction marker. Accordingly, HDAC inhibitor can be therapeutic options for some DLBCL patients with high Bfl-1.