Selective targeting of BCL6 induces oncogene addiction switching to BCL2 in B-cell lymphoma

Abstract

The BCL6 oncogene plays a crucial role in sustaining diffuse large B-cell lymphomas (DLBCL) through transcriptional repression of key checkpoint genes. BCL6-targeted therapy kills lymphoma cells by releasing these checkpoints. However BCL6 also directly represses several DLBCL oncogenes such as BCL2 and BCL-XL that promote lymphoma survival. Herein we show that DLBCL cells that survive BCL6-targeted therapy induce a phenomenon of "oncogene-addiction switching" by reactivating BCL2-family dependent anti-apoptotic pathways. Thus, most DLBCL cells require concomitant inhibition of BCL6 and BCL2-family members for effective lymphoma killing. Moreover, in DLBCL cells initially resistant to BH3 mimetic drugs, BCL6 inhibition induces a newly developed reliance on anti-apoptotic BCL2-family members for survival that translates in acquired susceptibility to BH3 mimetic drugs ABT-737 and obatoclax. In germinal center B cell-like (GCB)-DLBCL cells, the proteasome inhibitor bortezomib and the NEDD inhibitor MLN4924 post-transcriptionally activated the BH3-only sensitizer NOXA thus counteracting the oncogenic switch to BCL2 induced by BCL6-targeting. Hence our study indicates that BCL6 inhibition induces an on-target feedback mechanism based on the activation of anti-apoptotic BH3 members. This oncogene-addition switching mechanism was harnessed to develop rational combinatorial therapies for GCB-DLBCL.

Keywords: BCL2; BCL6; lymphoma; resistance; targeting.

Figure 1. BCL6 knockdown induces BCL2 and BCL-XL upregulation in DLBCL

A. BCL6 immunoblot in OCI-Ly1 cells transfected with siRNA for BCL6 (siBCL6-1) or control (siNT). BCL6 chromatin immunoprecipitation (ChIP) for target genes BCL2 and BCL-XL and negative control in OCI-Ly1 cells transfected with siRNAs. Data is shown as percent of input. B. transcript changes (fold to RPL13A) in BCL2 and BCL-XL in OCI-Ly1 cells transfected with siBCL6-1 or siBCL6-2 for 24 h compared to siNT. C. Cell viability of OCI-Ly1 cells transfected with siBCL6-1 or siNT for 72 h and treated with the BCL2 and BCL-XL inhibitor ABT-737 vs. D.M.S.O. (Vehicle). D. effect of the BCL6 inhibitor RI-BPI on mRNA levels of BCL2 and BCLXL (to GAPDH) at 12 and 24 h. E. RI-BPI growth inhibitory concentration 50% (GI₅₀) in a panel of 22 DLBCL cell lines. The red line divides cell lines into sensitive or BCL6-dependent (top part) from resistant (bottom part). Color scale represents GI₅₀ values from more sensitive (light blue) to less sensitive (dark grey). GCB-DLBCL BCL6-dependent cell lines in bold. F. Baseline levels of anti-apoptotic (orange shadow) and pro-apoptotic (blue shadow) BCL2-family members in RI-BPI sensitive (i.e. BCL6-dependent) and resistant groups of DLBCL cells. ***p < 0.001 and **p < 0.05. All other differences are not statistically significant.

Figure 2. BCL2 inhibitors increase the anti-lymphoma effect of RI-BPI in double-sensitive DLBCLs

A. GI₅₀ for ABT-737 and obatoclax (Y-axes) plotted against GI₅₀ for RI-BPI (X-axes) in GCB-DLBCL cells. Dotted lines segregate resistant and sensitive cell lines. B. Effect of RI-BPI on pro-survival BCL2 family members in the double-sensitive GCB-DLBCL SU-DHL6, SC-1 and DoHH2 cells treated for 6 and 12 hours compared to their respective controls (full line). Dotted line represents 2-fold expression increase. C. RI-BPI effect on protein expression of BCL2, MCL1 and BCL-XL in SU-DHL6 cells. D. Isobologram analysis for ABT-737 (top) and obatoclax (bottom) in four double-sensitive GCB-DLBCL cell lines. Values bellow diagonal represent synergistic combinations.

Figure 3. RI-BPI changes the dynamic of the BCL2-family components and sensitizes cells to BH3 mimetic drugs

A. Effect of RI-BPI on pro-survival BCL2-family members in the RI-BPI sensitive GCB-DLBCL OCI-Ly1 and OCI-Ly7 cells treated for 6 and 12 hours compared to their respective controls (full line, 0 h). Dotted line represents 2-fold expression increase. B. Effect on BCL2, BCL-XL and MCL1 protein levels in OCI-Ly1 and OCI-Ly7 cells after 48 h of exposure to RI-BPI. Densitometry values are shown at the bottom normalized to their respective controls. C. Fold drug reduction (expressed as dose reduction index) for ABT-737 (left) and obatoclax (right) in four RI-BPI sensitive and BH3-mimetic inhibitor resistant cell lines pre-treated for 48 h with RI-BPI. Dose reduction indexes smaller than 1 represent favorable combinations. D. Effect on BCL2, BCL-XL and MCL1 protein levels in SU-DHL8 cells after 48 h of exposure to RI-BPI. Densitometry values (RI-BPI/vehicle to actin) are shown on the right.

Figure 4. Proteasome and NAE inhibitors increased the anti-lymphoma effect of RI-BPI

A. Effect of RI-BPI on pro-apoptotic BCL2-family members in the RI-BPI sensitive GCB-DLBCL SU-DHL6, OCI-Ly1, OCI-Ly7, SC-1 and DoHH2 cells treated for 6 and 12 hours compared to their respective controls (full line, 0 h). Dotted line represents 2-fold expression increase. B. Effect on BIM protein levels at 48 h after exposure to RI-BPI. C. Effect on NOXA protein levels at 24 h after exposure to bortezomib (Bo) and MLN4924 (ML) vs. vehicle (Veh) in SU-DHL6 and OCI-Ly1 cell lines. D. GI₅₀ for bortezomib and MLN4924 (Y-axes) plotted against GI₅₀ for RI-BPI (X-axes) in BCL6-dependent GCB-DLBCL cells. Dotted lines segregated resistant and sensitive cell lines. E. Fold drug reduction (expressed as dose reduction index) for MLN4924 and bortezomib in 8 BCL6-dependent GCB-DLBCL cell lines pre-treated with RI-BPI. Dose reduction indexes greater than 1 represent favorable combinations.

Figure 5. Targeting or pro-survival pathways increased the effect of RI-BPI in vivo

A. Flowchart of GCB-DLBCL mice xenografting, treatment and end-point evaluation. B. Tumor volume represented by the area under the curve (AUC) of xenograft growth from day 1 to day 10 of treatment in OCI-Ly1 mice treated with the compounds shown in A. T-test significant p values of combinatorial regimens are shown (pair-wise comparisons vs. RI-BPI). C. Representative hematoxylin and eosin microphotographs of bone marrow and liver tissues from C57BL/6 mice treated with RI-BPI, bortezomib or their combination as in A. The green bar (vehicle) represents 100 micrometers D. Representative TUNEL histochemistry microphotographs from lymphoma tissues from the mice shown in B. The green bar (vehicle) represents 50 micrometers. E. Quantification of apoptotic cells (% of positive TUNEL nuclei over total nuclei) in OCI-Ly1 mice xenografted lymphoma tissues. Only statistically significant p values are depicted (T-test). F. Cartoon representation of the proposed oncogene switching mechanism and combinatorial targeting of BCL6 and BCL2 pathways in GCB-DLBCL.