Article Figures & Data
Figures
- Figure 1.
In vitro venetoclax response in representative human patient-derived DLBCL (26) and MCL (10) cell lines. A, The effect of 72 hours of venetoclax treatment on the viability of human patient-derived MCL and DLBCL cell lines relative to cells treated with dimethylsulfoxide. Waterfall graph shows the IC50 value of venetoclax for each cell line. B, Comparison of venetoclax IC50 values between non-GCB (n = 9) and GCB (n = 17) DLBCL subtypes (first panel); between DLBCL (n = 26) and MCL (n = 10) cell lines (second panel); and between BCL2-positive (n = 26) and BCL2-negative (n = 10) DLBCL and MCL cell lines (third panel). Comparison of venetoclax IC50 values between MYC/BCL2 DHL/DEL (n = 11) and non-DHL/DEL (n = 15) DLBCL cells (fourth panel). C, BCL-2 protein expression in DLBCL and MCL cell lines was measured by RPPA analysis. Gray bars indicate venetoclax sensitivity and black bars indicate venetoclax resistance. The asterisk (*) indicates cell lines that express BCL-2 protein but were resistant to venetoclax. D, RPPA analysis was assessed in 26 representative DLBCL cell lines, and the expression level of BCL-2 and its family members was plotted against the corresponding venetoclax IC50 for each cell line. Spearman rank correlation coefficient and P values were determined based on the above analysis. P values less than 0.05 indicate significant correlations. Solid line indicates the linear regression line, and the dotted line depicts the 95% confidence interval.
- Figure 2.
Venetoclax response in fresh primary human aggressive B-cell lymphoma patient samples and in vivo efficacy of venetoclax in MCL and DLBCL PDX models. A, Cell viability of freshly isolated tumor cells from 9 MCL and 4 DLBCL primary samples after treatment with increasing concentrations of venetoclax for 48 hours. B, BCL-2 protein expression in primary MCL and DLBCL samples analyzed by Western blotting. Actin served as a loading control. R, resistant (IC50 > 100 nmol/L); S, sensitive (IC50 < 100 nmol/L). C, PT-2 PDX mice were randomly divided into two groups (n = 5) and once human β2M was detectable, mice were administered venetoclax 50 mg/kg or vehicle control by daily oral gavage for 3 weeks. Mouse serum was collected, and the circulating human β2M levels in the mouse serum were used to monitor tumor burden (top). The body weight was calculated at the endpoint of treatment (bottom). D, PT-6 MCL PDX mice were randomized into groups, and once the tumor reached to 3 mm3 in size, the mice were administered either venetoclax 50 mg/kg or vehicle control by daily oral gavage for 3 weeks. Tumor burden was evaluated by measuring tumor volume to determine therapeutic efficacy (top; P = 0.028, venetoclax vs. vehicle control, n = 5). The body weight was calculated from day 0 to endpoint of treatment (bottom). E, The DLBCL PDX mice (PT-12) were randomized into groups, and once the tumor reached to 3 mm3 in size, the mice were treated with venetoclax 50 mg/kg or vehicle control by daily oral gavage for 3 weeks (top; P = 0.04, venetoclax vs. vehicle control, n = 5). The body weight was calculated from day 0 to endpoint of treatment (bottom). F, The body weight changed from day 0 to endpoint of treatment. I, H&E staining and anti-human BCL-2 IHC staining of tumor masses from 3 PDXs after mice were humanely euthanized at the endpoint.
- Figure 3.
Mechanisms underlying venetoclax activity in aggressive B-cell lymphomas. A, The Venn diagrams show common upregulated and downregulated proteins in venetoclax-treated RC, U-2932 and HF cells. B, Representative heatmaps showing the common up- and downregulated proteins in venetoclax-treated RC, U-2932 and HF cells. C, Table showing the venetoclax up- and downregulated proteins in different cellular processes. D, Representative DLBCL cell lines with high BCL-2 [(RC and U-2932) or low BCL-2 (HT)] were treated with venetoclax in a dose- (0–10 nmol/L) and time- (24 and 48 hours) dependent manner and then assessed for apoptosis by annexin V staining. DMSO treatment serves as a control. E, RC, U-2932, HT, and a primary DLBCL sample cells were treated with venetoclax (Ven) in a dose-dependent manner. Whole cell extracts were subjected to Western blot analysis for expression of cleaved caspase-3 (c-casp-3), PARP, γH2AX, and actin (loading control). The PI3K inhibitor KA2237 (KA) was used as a control agent to induce apoptosis in HT cells.
- Figure 4.
Venetoclax treatment activates the AKT compensatory pathway and novel agents targeting PI3K or AKT synergize with venetoclax in aggressive B-cell lymphomas. A, Representative venetoclax-sensitive (RC and Mino) cell lines were treated with venetoclax in a 1:1 ratio drug combinations with PI3K inhibitors (ACP-319, idelalisib, and KA2237) or AKT inhibitor (MK-2206) in concentration-dependent manner for 72 hours, and cell viability was assessed. The highest starting concentration for each drug was 20 μmol/L. The following are the drug combinations: 100 nmol/L: 20 μmol/L; 50 nmol/L:10 μmol/L; 25 nmol/L:5 μmol/L; 12.5 nmol/L:2.5 μmol/L; 6.25 nmol/L:1.25 μmol/L; 3.1 nmol/L:0.61 μmol/L; 1.5 nmol/L:0.3 μmol/L. Data from two independent experiments performed in triplicate are shown. B, RC and Mino cells were treated with venetoclax (5 nmol/L) alone, PI3K inhibitors or AKT inhibitor (10 μmol/L) alone, or combination of both drugs for 24 hours. Protein extracts from the cells were subjected to Western blot analysis to detect AKT, pAKT, cleaved caspase-3, cleaved PARP, and actin (protein loading control). C, Apoptotic cells were also detected by annexin V/propidium iodide staining of the above experiments. Data shown represent means ± SD from three independent experiments. D, Two representative DLBCL primary cells (PT-12 and PT-13) were treated with venetoclax in 1:1 ratio drug combinations with the dual, selective PI3K–p110β/δ inhibitor, KA2237, in concentration-dependent manner for 72 hours, and cell viability was assessed. The highest starting concentration for KA2237 was 20 μmol/L. E, RC, Mino, BJAB, and HT cells were transiently transfected with control or three different validated AKT siRNAs. At 48 hours posttransfection, protein purified from the transfected cells was subjected to Western blotting for AKT and actin (loading control). A small portion of the transfected cells were treated with increasing concentrations of venetoclax for an additional 72 hours, and viability was assessed. Data are expressed as the mean ± SD of at least two independent experiments with triplicate samples.
- Figure 5.
Mechanism of acquired venetoclax resistance in lymphoma B cells. A, The effect of 72 hours of venetoclax treatment on the viability of parental (Mino-P, Rec-1-P, and RC-P) and venetoclax-resistant (VR; Mino-VR, Rec1-VR, and RC-VR) cell lines. B, Protein extracts from the parental lines and venetoclax-resistant cell lines were subjected to Western blot analysis to detect pAKT, AKT, PI3K, PTEN, and GAPDH (loading control). C, Parental and venetoclax-resistant cells were treated with PI3K inhibitors (KA2237 or idelalisib) for 24 hours. Protein extracts were subjected to Western blot analysis to detect for pAKT, AKT, and GAPDH (loading control). D, Viability assays were assessed to measure the growth inhibitory effect of the PI3K inhibitor KA2237 in parental and venetoclax-resistant cell lines.
- Figure 6.
Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K inhibition. A, Src-pY527, AKT-pS473, and AKT-pT308 protein levels were plotted against the corresponding venetoclax IC50 for each cell line. Spearman rank correlation coefficient and P values determined based on the above analysis. P values less than 0.05 indicate significant correlations. B, RPPA analysis was assessed in 26 representative DLBCL cell lines, and the expression level of PTEN, AKT, pAKT, p-mTOR, p70-S6K, PRAS40, and S6 was plotted against the corresponding KA2237 IC50 for each cell line. Spearman rank correlation coefficient and P values were determined based on the above analysis. P values less than 0.05 indicate significant correlations. C, Left, cells with high protein levels of BCL-2, BIM, and BAK are highly sensitive to venetoclax treatment, presumably through the release of BIM and BAK to cause BAX-mediated cytochrome c release, and subsequently apoptosis. Short- and long-term exposure of venetoclax to lymphoma cells inhibits PTEN, leading AKT pathway activation, for maintenance of cell survival, presumably through upregulation of BCLx/L/BAD complex. Right, cells with low or no BCL-2 protein expression are intrinsically resistant to venetoclax and tend to acquire high AKT pathway activation, as well as BCL-xL and BAD protein expression. PI3K or AKT inhibitors are amenable to overcome both acquired and intrinsic venetoclax resistance.
Tables
- Table 1.
DLBCL and MCL cell line characterization and venetoclax sensitivity
Subtype STR DNA fingerprinting p53 status BCL-2/MYC Venetoclax IC50 (nmol/L) KA2237 IC50 (μmol/L) DLBCL cell lines 1 HF GCB Yes wt +/+ <1 9.85 2 U2932 ABC (DHL) Yes ND DHL <1 49.6 3 RC GCB (DHL) yes ND DHL <1 0.68 4 OCI-LY10 ABC Yes wt ± 5 6.04 5 McA GCB Yes wt +/+ 5 11.78 6 LR ABC Yes wt +/+ 5 2.27 7 FN GCB Yes wt +/+ 5 14.85 8 OCI-LY19 GCB Yes wt +/+ 5 4.74 9 CJ GCB (DHL) Yes mut DHL 20 11.5 10 OCI-LY3 ABC Yes wt +/+ 25 8.34 11 MS GCB Yes mut ± 50 2.03 12 SUDHL-10 GCB Yes ND ± 50 1.45 13 Toledo GCB Yes wt ± 95 6.59 14 SUDHL-4 GCB Yes mut ± 100 0.47 15 HB ABC Yes mut −/+ 1,000 10.42 16 LVP-03 PM-LBCL yes ND −/− 1,000 NA 17 TMD-8 ABC Yes ND −/+ 1,000 4.31 18 DB GCB Yes mut +/+ 1,000 12.08 19 HT GCB Yes ND −/− 1,000 0.68 20 HBL-1 ABC Yes ND ± 2,000 4.31 21 MZ GCB Yes wt +/+ 2,500 15.26 22 LP ABC Yes mut −/− 5,000 5.77 23 DS GCB Yes mut −/− 5,000 28.17 24 Pfeiffer GCB Yes mut −/− 5,000 25.73 25 DBr GCB Yes wt −/− >10,000 22.05 26 BJAB GCB Yes wt −/+ >10,000 0.65 MCL cell lines 1 Z-138 Blastoid Yes wt +/+ <1 2.3 2 Mino Classic Yes mut +/+ <1 5 3 JVM-13 Classic Yes wt +/+ 2 4.5 4 REC-1 Blastoid Yes mut ± 2.5 1.0 5 Granta Classic Yes wt +/+ 5 2.9 6 Jeko-1 Classic Yes mut +/+ 5 1.3 7 DBsp53 Classic Yes wt ± 85 − 8 PF-1 Classic Yes wt +/+ 100 − 9 JMP-1 Blastoid Yes wt −/− 500 − 10 Maver-1 Classic Yes mut +/+ >10,000 4.1 NOTE: Cell viability assays were performed to identify the venetoclax IC50 values. BCL-2 and MYC protein expression was analyzed by reverse-phase protein array.
Abbreviations: ABC, activated B cell-like; DHL, double-hit lymphoma; GCB, germinal center-derived B cell; mut, mutant; ND, no data; PM-BCL, primary mediastinal large B-cell lymphoma; STR, short-tandem repeats; wt, wild-type.
Supplementary Data
- Table S1 - Up and downregulated proteins in Mino cells treated with 5 nM of venetoclax for 24 h, analyzed by RPPA analysis. Highlighted boxes show commonly altered proteins between Mino cells treated with venetoclax and DLBCL cell lines treated with venetoclax.
- Supplementary Figure Legends - Supplementary Figure Legends
- Figure S1 - (A) Immunoblotting of BCL-2 levels in representative MCL and DLBCL cell lines with actin as a loading control. Cell viability assays were performed using the Celltiter-Glo Luminescent Cell Viability Assay in representative GCB-DLBCL (B), non-GCB-DLBCL (C), and MCL (D) cell lines treated with increasing concentrations of venetoclax.
- Figure S2 - Cell viability assays were performed using the Celltiter-Glo Luminescent Cell Viability Assay in representative MCL primary cells (PT-2 and PT-6) treated with increasing concentrations of ibrutinib (A) or idelalisib (B).
- Figure S3 - Representative venetoclax-resistant (BJAB and HT) cell lines were treated with venetoclax at a 1:1 ratio drug combinations with PI3K inhibitors (idelalisib and KA2237) or an AKT inhibitor (MK-2206) in a concentration-dependent manner for 72 h, and cell viability was assessed. The highest starting concentration for each drug was 20 mM. The following are the drug combinations: 100 nM: 20 mM; 50 nM:10 mM; 25 nM:5 mM; 12.5 nM:2.5 mM; 6.25 nM:1.25 mM; 3.1 nM:0.61 mM; 1.5 nM:0.3 mM. Data from two independent experiments performed in triplicate are shown. (B) MAPK-pT202-Y204, MEK1-pS217-S221, and JNK-pT183-Y185 protein levels were plotted against the corresponding venetoclax IC50 for each cell line. Spearman's rank correlation coefficient, and p values determined of the above analysis. P values less than 0.05 indicate significant correlations.
- Figure S4 - Representative heatmap showing the up and downregulated proteins in the Mino parental (Mino-P) vs. Mino-venetoclax resistant (Mino-VR) cell lines.
Volume 24, Issue 16
