Identifying Targetable Vulnerabilities to Circumvent or Overcome Venetoclax Resistance in Diffuse Large B-Cell Lymphoma

Abstract

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.

Keywords: B-cell lymphoma; BCL2; IDH2; mitochondrial electron transport chain (ETC); venetoclax/ABT-199 resistance.

Figure 1

Generation and characterization of DLBCL cell lines with acquired venetoclax resistance. (A) Venetoclax dose–response curves (MTS, 48 h, relative to DMSO vehicle control, quadruplicates, mean ± SEM) for DLBCL cell lines with intrinsic venetoclax sensitivity (blue shades) or resistance (red shades). IC50 values in parentheses. (B) Western blots of the indicated proteins from the 10 DLBCL lines in A. Each β-actin blot is associated with the blots above it. Two different BCL2 antibodies were needed to detect BCL2 in SUDHL4 and SUDHL6. (C) Venetoclax dose–response curves of parental (Par, blue) and acquired venetoclax-resistant (Res, red) DLBCL lines (MTS, 48 h, relative to DMSO vehicle control, quadruplicates, mean ± SEM). IC50 values of each indicated. (D) Caspase-3/7 activity after 12 h of venetoclax (Ven) treatment or DMSO vehicle control of the lines from C (triplicates/quadruplicates, mean ± SD). Low and high doses of venetoclax are the IC50 and 10× the IC50 of the parental line, respectively. Representative histograms following high-dose venetoclax treatment or DMSO shown. * p < 2.30 × 10⁻⁵, compared to vehicle (DMSO) control. (E) Chromatograms of BCL2 sequencing of the SUDHL4 parental and acquired venetoclax-resistant lines. (F) Western blots of BCL2 family members of the three parental (P) and acquired venetoclax-resistant (R) DLBCL lines (BCL2 Ab1 used for SU4 and SU16; BCL2 Ab2 used for SU6); note that some exposures in (F) were longer than in (B) to detect lower-expressed proteins. Each β-actin blot is associated with at least one of the blots above it.

Figure 2

Combination treatment with BCL2 family member inhibitors is effective for DLBCL, follicular, and marginal zone lymphomas. (A) IC50s (μM) of acquired venetoclax-resistant (Res) and parental (Par) DLBCL lines and intrinsically venetoclax-resistant DLBCL lines following 48 h of treatment with navitoclax (Nav, B2XWi), A-1331852 (A852, BCLXi), or MIK665 (MIK, MCL1i). (B) ZIP synergy scores of venetoclax-sensitive and acquired venetoclax-resistant DLBCL lines (top) or intrinsically venetoclax-resistant (bottom) treated with venetoclax (Ven, BCL2i) + A852 (BCLXi) or MIK (MCL1i). See Supplementary Table S4 for synergy scores from other synergy methods. (C–F) Intracellular flow cytometry of four anti-apoptotic BCL2 family members and treatment results with venetoclax (Ven, V), navitoclax (Nav, N), A-1331852 (A852, A), MIK665 (MIK, M), or untreated (UT) in fresh patient samples of DLBCL (C), normal B-cells (D), follicular lymphoma (E), and marginal zone lymphoma (F). Representative histograms shown with median fluorescence intensity (MFI) after subtracting the isotype control MFI value. Following B-cell enrichment, cell survival (MTS, relative to DMSO vehicle control, triplicates, mean ± SD) was measured 6–12 h after treatment with the compounds indicated. For (C) * p < 0.01, (E) * p < 0.05 (top) and * p < 0.01 (bottom), and (F) * p < 0.05, comparing each concentration used in the combination treatment to the same concentration of each single agent.

Figure 3

Targetable oxidative phosphorylation vulnerability identified in venetoclax-resistant DLBCL lines and lymphoma patient samples. (A) Schematic of the workflow of RNA-seq analysis and drug/compound screens (images modified from BioRender.com) with the cell line comparisons indicated and the number of genesets identified (Venn diagram). The six overlapping Hallmark genesets are listed. (B,C) Cell death caused by ETC inhibitor or tigecycline-treated acquired venetoclax-resistant and parental DLBCL lines (B) or intrinsically venetoclax-resistant DLBCL lines (C) was measured with live/dead flow cytometry assay (72–96 h, triplicates, relative to DMSO vehicle control, mean ± SD). For (B) * p < 0.01 (SUDHL6) and * p < 0.05 (SUDHL16), comparing each resistant cell line to its parental counterpart at each concentration. For (C) * p < 0.0001, comparing treated cells at each concentration to untreated cells. (D–F) Treatment of DLBCL (D,E) and marginal zone (F) lymphoma patient samples with venetoclax (Ven, V), mubritinib (Mub, M), BAY-87-2243 (BAY, B), and/or IACS-010759 (IACS, IA). Cell viability assays ((D,F), 24 h, triplicates, relative to DMSO vehicle control, mean ± SD) and Caspase-3/7 activity assay ((E), 24 h, triplicates, fold-change relative to DMSO vehicle control, mean ± SD). (D–F), * p < 0.0001, comparing each combination treatment to both single agent treatments at the same concentrations.

Figure 4

IDH2 is upregulated in acquired venetoclax-resistant DLBCL cells, and its inhibition synergizes with venetoclax to overcome resistance. (A) Number of overlapping and non-overlapping genes identified from Hallmark oxidative phosphorylation geneset evaluation after the comparisons shown in Figure 3A were performed. Venn diagram (left) and significantly (FDR < 0.05 with ≥1.5-fold change) altered genes are in the heatmaps (right). (B–D) Combination treatment with venetoclax (Ven, V) + IDH2 inhibitor (AGI-6780, AGI, A) in acquired venetoclax-resistant DLBCL lines. Survival assays ((B), left, 48 h, quadruplicates, relative to DMSO vehicle control, mean ± SEM) and 3D ZIP synergy plots ((B), right), live/dead flow cytometry analysis ((C), left, 48 h, triplicates, relative to DMSO vehicle control, mean ± SEM) and 3D ZIP synergy plots ((C), right), and Caspase-3/7 activity measured ((D), 48 h, triplicates, mean ± SD). Untreated (UT). (E–G) Treatment of DLBCL (E,F) and marginal zone lymphoma (G) patient samples with venetoclax (Ven, V) and AGI-6780 (AGI, A). Cell viability assays for IDH2i ((E,G), 24 h, triplicates, relative to DMSO vehicle control, mean ± SD) and Caspase-3/7 activity ((F), 24 h, triplicates, fold-change relative to DMSO vehicle control, mean ± SD). For (D–G) * p < 0.0001, comparing each combination treatment to both single-agent treatments at the same concentrations.

Figure 5

Targetable critical pathways in venetoclax-sensitive and -resistant DLBCL revealed from drug/compound screening. (A) Median fold-change of all compounds in the categories indicated with ≥1.5-fold increased sensitivity in acquired venetoclax-resistant DLBCL lines compared to venetoclax-sensitive lines identified by drug screens. (B,C) IC50s (μM) of acquired venetoclax-resistant (Res) and parental (Par) DLBCL lines and intrinsically venetoclax-resistant DLBCL lines following 48 h of treatment with CDK7/9 (B) or BCR (C) inhibitors. Transcription inhibitors: CDK9-IN-2 (CDK9i), SNS-032 (CDK2/7/9i), THZ1 (CDK7i). B-cell receptor signaling inhibitors: copanlisib (Copa, PI3Ki), ibrutinib (Ibru, BTKi), and R406 (SYKi). (D,E) MTS assays of enriched B-cells from fresh DLBCL (D,E), follicular (D,E), and marginal zone (D) lymphoma patient samples treated 24 h with venetoclax (Ven, V), THZ1 (T), CDK9 (CD), ibrutinib (Ib), R406 (R), and/or copanlisib (Copa, Co) (triplicates, relative to DMSO vehicle control, mean ± SD). For (D) * p < 0.05 (top), * p < 0.01 (middle), and * p < 0.0001 (bottom), and for (E) * p < 0.0001, comparing each combination treatment to both single-agent treatments at the same concentrations.