A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity

Abstract

B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.

Conflict of interest statement

Competing interests: S.K., X.Z, Y.H., P.Z., G.Z., and D.Z. are inventors of two pending patent applications for use of BCL-XL PROTACs as senolytic and antitumor agents. R.H., G.Z., and D.Z. are co-founders of and have equity in Dialectic Therapeutics, which develops BCL-XL PROTACs to treat cancer.

Figures

Extended Data Fig. 1. Expression of anti-apoptotic BCL-2 members and VHL in cancer, and the synthetic schemes of DT2216 and DT2216NC

a, A representative of three immunoblot analyses of VHL in three different human tumor cell lines and platelets from three different individuals (indicated as units 1–3). b, Immunoblot analyses of the basal protein levels of BCL-XL, BCL-2, MCL-1 and VHL in different solid tumor cells. Data are a representative of two independent experiments. c,BCL2L1 and VHL mRNA expression (Log2 transformed) and mutational status were analyzed using TCGA PanCancer Atlas studies via cBioPortal. d, Synthetic schemes of DT2216, DT2216NC and VHL-L. Reagents and conditions: (i) (1) N-methylmorpholine, isobutyl chloroformate, THF, −25 °C then −15 °C; (2) NaBH4, H2O; (ii) Bu3P, diphenyl disulfide, toluene, 80 °C; (iii) DIBAL-H, toluene, −78 °C (iv) compound 5, NaBH(OAc)3, TEA, DCM; (v) TFA, DCM; (vi) TEA, acetonitrile, reflux; (vii) compound 10, EDCI, DMAP, DCM; (viii) Zn, HOAc, THF; (ix) HATU, TEA, DCM; (x) (1) LiOH monohydrate, MeOH, H2O; (2) compound 12, HATU, TEA, DCM; (xi) Ac2O, TEA, DCM.

Extended Data Fig. 2. The BCL-XL degradation by DT2216 is rapid and long lasting

a, Immunoblot analysis of BCL-XL expression in MOLT-4 cells after they were treated with DT2216 for various durations as indicated. A representative immunoblot is presented on the top panel. Densitometric analysis of BCL-XL expression is presented on the bottom panel as mean of two independent experiments. Each symbol represents data (% of 0 h) from an individual experiment. b, Analysis of BCL-XL expression by immunoblot in MOLT-4 cells treated with DT2216 for 16 h followed by drug withdrawal and then cultured without DT2216 for 0 to 48 h as indicated. A representative immunoblot is on the top panel. Densitometric analysis of BCL-XL expression is presented on the bottom panel as the mean of two independent experiments. Each symbol represents data (% of Veh) from an individual experiment. c, Caspase-3 activity in MOLT-4 cells was measured 24 h after they were treated with 1 μM of DT2216 or ABT263. Data are presented as mean (n = 2 technical replicates) of a representative experiment. Each symbol represents data (% of Veh) from an individual replicate. Similar results were obtained in an additional independent experiment. d, Representative immunoblot to confirm CRISPR/Cas9-mediated double knockout of Bax and Bak in H146 cells. The experiment was repeated independently one more time with similar results.

Extended Data Fig. 3. ABT263 or VHL-L blocks the formation of ternary complex of BCL-XL, DT2216 and VHL

a, Recombinant His-tagged BCL-XL protein (100 nM) was incubated with the VHL-Elongin B/C complex (50 nM) with 0.039 μM of DT2216. The ternary complex formation was abrogated in the presence of ABT263 (1 μM) or VHL-L (10 μM). Data are expressed as mean (n = 2 technical replicates). Each symbol represents data from an individual replicate. b, The negative-control of DT2216 (DT2216NC) cannot form ternary complex with BCL-XL and the VHL-complex. Recombinant His-tagged BCL-XL protein (100 nM) was incubated with the VHL-Elongin B/C complex (50 nM) with increasing concentrations of DT2216 or DT2216NC. Data are expressed as mean (n = 2 technical replicates).

Extended Data Fig. 4. DT2216 binds to both BCL-XL and BCL-2 but cannot degrade BCL-2

a, The binding affinities of DT2216 and ABT263 towards BCL-XL, BCL-2 and BCL-W were measured by AlphaScreen and are represented in terms of inhibition constant (Ki). The data are average of two independent experiments each performed in duplicates. b, c, Immunoblot analyses of BCL-2 and BCL-W are shown after the RS4 cells were treated with indicated concentrations of DT2216 for 16 h (upper panels), or with 1 μM of DT2216 for indicated durations (lower panels). d, Cell viability of BCL-2-dependent RS4 cells after treatment with increasing concentrations of DT2216, ABT199 or ABT263 for 72 h. Data are presented as mean ± SD from six replicate cell cultures in one experiment. Similar results were obtained in two additional independent experiments for DT2216. The EC50 of DT2216 is the average of three independent experiments. e, Cell viability of MCL-1-dependent H929 cells after treatment with increasing concentrations of DT2216 or S63845 for 72 h. Data are presented as mean ± SD from six replicate cell cultures in one experiment. f, Schematic representation of proteomic assay shown in fig. 3d. g, Representative immunoblot analyses are shown after the cells were treated with indicated concentrations of PZ-15227 for 16 h (top panel) or with 0.1 μM of PZ-15227 for indicated time points (bottom panel). Similar results were obtained in one more independent experiment. h, CETSA assay for BCL-XL and BCL-2. MOLT-4 (for BCL-XL) or RS4 (for BCL-2) cells were treated with DMSO, or 1 μM of DT2216 for 6 h. Raw band intensities are mean of two independent experiments.

Extended Data Fig. 5. Drug metabolism and PK/PD profile of DT2216

a, DT2216 stability in mouse blood and plasma. b, DT2216 liver microsomal stability. c, Plasma concentrations of DT2216 after a single administration of 2 mpk (i.v. injection), 20 mpk (i.p. injection) or 20 mpk (p.o. administration) are presented as Mean ± SD (n = 3 mice/group). These studies were done by BioDuro (San Diego, CA, USA), a global contract research organization, through a contract. d, MRM chromatograms of (A) DT2216 in drug-free brain homogenate, (B) Internal standard in spiked drug-free tumor homogenate (40 ng/mL), (C) DT2216 in spiked tumor homogenate (5 ng/mL; LLOQ), (D) DT2216 in tumor sample taken from vehicle dosed mouse at 24 h, (E) DT2216 in tumor sample taken at 24 h after 15 mpk/i.p. administration. e, Representative immunoblot analysis of BCL-XL in tumors at different durations after DT2216 (DT, 15 mpk/i.p.) administration (n = 2 mice in Veh and DT2216 groups at each time points). Similar results were obtained in two more immunoblot experiments. Mpk, mg/kg.

Extended Data Fig. 6. DT2216 induces BCL-XL degradation and apoptosis in MOLT-4 T-ALL xenografts and suppresses their growth in a dose-dependent manner

a, b, T-ALL xenograft tumors were harvested two days after female CB-17 SCID mice received one i.p. injection of DT2216 at 7.5 mpk and 15 mpk. A single immunoblot analysis of BCL-XL, BCL-2, MCL-1, cleaved and full length caspase-3 and PARP1 in the tumors is shown. c, Changes in tumor volume over time after the start of treatment with vehicle (Veh), or DT2216 (7.5 or 15 mpk/q7d/i.p.). All the data presented are mean ± SEM (n = 8 mice in Veh, 8 mice in DT2216 7.5 mpk, and 7 mice in DT2216 15 mpk). The data from DT2216 15 mpk group are also presented in Extended Data Fig. 8f in which the tumor size in these mice were continuously monitored till day 55 post treatment.

Extended Data Fig. 7. Images of MOLT-4 tumor-bearing mice and excised tumors

The images shown (quantification data are shown in Fig. 4f,g) were captured at the end of experiment when the mice were treated with Veh, DT2216 (15 mpk/q7d/i.p.) or ABT263 (50 mpk/qd/p.o.). The tumor-bearing mice (shown in the top panel) and harvested tumors (shown in the bottom panel) are not placed in an identical order.

Extended Data Fig. 8. DT2216 induces regression of MOLT-4 xenografts without causing thrombocytopenia

a, Illustration of the experimental design of MOLT-4 T-ALL xenograft mouse model. b, Representative of two independent immunoblot analyses of BCL-XL, cleaved and full-length caspase-3 and PARP1 in MOLT-4 T-ALL xenografts harvested two days after tumor-bearing mice were treated with a single injection of vehicle (Veh) or DT2216 (15 mpk/i.p.). c, Blood platelets (PLT), white blood cells (WBC), lymphocytes (LYM) and granulocytes (GRA) were numerated one day after first treatment with vehicle (Veh), DT2216 (15 mpk/i.p.) or ABT263 (15 mpk/i.p.) as shown in a. a and b represents statistical significance vs. Veh and DT2216, respectively, determined by one-way ANOVA and Tukey’s multiple comparison test. d, Body weight changes in mice after the start of treatment with vehicle (Veh), DT2216 or ABT263 as shown in a. e, Numeration of PLT one day after the 6th dose of DT2216 (15 mpk/q7d/i.p. or 15 mpk/q4d/i.p.). Data are presented as mean ± SEM (n = 2 mice in Veh, 7 mice in DT2216 q7d, and 6 mice in DT2216 q4d). f, Changes in tumor volume over time after the start of treatment with vehicle (Veh), DT2216 or ABT263 as shown in a. Data presented in c, d and f are mean ± SEM (n = 6 mice for Veh group and 7 mice each for DT2216 and ABT263 groups). Each symbol in c and e represents data from an individual animal.

Extended Data Fig. 9. Images of excised H146 SCLC tumors

The tumor images shown for the quantification data presented in Fig. 5g. Mice were treated as shown in Fig. 5c.

Extended Data Fig. 10. Anti-leukemic activity of DT2216 alone and in combination with chemotherapy in T-ALL PDX models

a, 8 weeks old female NSG mice (n = 20 mice) were injected with 1 × 106 D115 cells 24 h after 0.25 Gy irradiation. Upon engraftment, mice were randomized to receive vehicle, DT2216 (15 mpk/i.p./q4d for 3 weeks), chemotherapy [Chemo (vincristine 0.15 mpk + dexamethasone 5 mpk + L-asparaginase 1000 U/kg, i.p., q7d for 3 weeks)], or the combination of DT2216 with chemotherapy. Disease burden was followed by engraftment in bone marrow by checking hCD45% in bone marrow aspiration samples through flow cytometry. Data are presented as mean ± SEM (n = 5 mice in each group). Each symbol represents data from an individual animal, and the middle horizontal line represents mean. b, Mice survival was followed and statistical significance was determined by Log-rank test (n = 5 mice in each group). c, 8 weeks old female NSG mice (n = 20 mice) were injected with 1 × 106 332X-luci cells 24 h after 0.25 Gy irradiation. Upon engraftment, mice were randomized to receive vehicle, DT2216, chemotherapy, or the combination of DT2216 with chemotherapy as mentioned in a. Disease burden was followed by checking hCD45% in peripheral blood samples (retro orbital) through flow cytometry. Data are presented as mean ± SEM (n = 5 mice in each group). Each symbol represents data from an individual animal, and the middle horizontal line represents mean. d, Mice survival was followed and statistical significance was determined by Log-rank test (n = 5 mice in each group).

Figure 1.. DT2216, a BCL-XL PROTAC, selectively induces BCL-XL degradation and apoptosis in BCL-XL-dependent MOLT-4 T-ALL cells but not in platelets.

a, Chemical structures of DT2216 and its negative-control DT2216NC showing a BCL-2/-XL ligand linked to a VHL ligand via an optimized linker. DT2216NC has the inactive VHL ligand that does not bind to VHL. b, c, DT2216 selectively degrades BCL-XL in MOLT-4 cells but not in platelets after treatment with increasing concentrations of DT2216 as indicated for 16 h. A representative immunoblot is presented on the top panel. Densitometric analyses of BCL-XL expression are presented on the bottom panel as mean (n = 2 and 3 independent experiments for MOLT-4 and platelets, respectively). DC50, the drug concentration causing 50% protein degradation; Dmax, the maximum level of degradation. d, Viability of MOLT-4 cells and human platelets were determined after they were incubated with increasing concentrations of DT2216 and ABT263 for 72 h. The data are presented as mean ± SD from six and three replicate cell cultures in a representative experiment for MOLT-4 and platelets, respectively. Similar results were also observed in two additional independent experiments. For platelet viability assay, each experiment used platelets from one individual donor. EC50 values are the average of three independent experiments. e, A representative of two independent immunoblot analyses of cleaved and full-length caspase-3 and PARP1 in MOLT-4 cells 24 h after they were treated with vehicle (Veh), DT2216, or ABT263. f, Cell viability of MOLT-4 cells was determined after the cells were pretreated with the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM) and then treated with DT2216 for 72 h at indicated concentrations. Data are presented as mean ± SD from six replicate cell cultures in a representative experiment. Each symbol represents data from an individual replicate. Similar results were also observed in one additional independent experiment. g, h, Viability of non-targeting sg-RNA-transfected (sgCTRL) and Bax/Bak double knockout (KO; represented as sgBAX+BAK) H146 cells was determined after they were incubated with increasing concentrations of DT2216 or ABT263 for 72 h. Data are presented as mean ± SD from three replicate cell cultures in a representative experiment. Similar results were also observed in one additional independent experiment. i, j, MOLT-4 cells and human platelets were treated with either DT2216 (DT, 1 μM) or ABT263 (1μM) for 6 h. MOLT-4 cells were pretreated with MG132 (MG, 1 μM) for 1 h in order to block BCL-XL degradation. Immunoblots after immunoprecipitation with BCL-XL and in whole cell lysates (Input) are shown from a single experiment. β-actin was used as an equal loading control in all immunoblot analyses shown in Fig. 1b, c, e, i and j. The uncropped immunoblot images related to this figure are provided in separate source data file.

Figure 2.. DT2216 degrades BCL-XL in a VHL- and proteasome-dependent manner.

a, ABT263 and/or VHL-L cannot induce BCL-XL degradation in MOLT4 cells. An immunoblot analysis of BCL-XL in MOLT-4 cells is shown after the cells were treated with ABT263 or VHL-L or the combination of both for 16 h. b, Pretreatment with ABT263 blocks the BCL-XL degradation by DT2216. An immunoblot analysis of BCL-XL in MOLT-4 cells after they were either left untreated or pretreated with ABT263 for 1 h and then treated with or without DT2216 as indicated for 16 h before being assayed. c, Pretreatment with VHL-L blocks the BCL-XL degradation by DT2216. An immunoblot analysis of BCL-XL in MOLT-4 cells is shown. The cells were either left untreated or pretreated with VHL-L for 1 h and then treated with or without DT2216 as indicated for 16 h before being assayed. d, An immunoblot analysis of BCL-XL in VHL-null 786-O cells treated with vehicle (Veh) or increasing concentrations of DT2216 for 16 h. e, An immunoblot analysis of BCL-XL in MOLT-4 cells is shown. The cells were treated with 1 μM of DT2216 and its negative-control DT2216NC for 16 h before being assayed. f, Proteasome inhibition blocks the BCL-XL degradation by DT2216. A representative of two immunoblot analyses of BCL-XL in MOLT-4 cells after they were either left untreated or pretreated with the proteasome inhibitor MG132 for 1 h, and then treated with or without DT2216 for 16 h. g, Cell viability of MOLT-4 cells treated with or without ABT263 in the presence or absence of VHL-L for 72 h. h, Cell viability of MOLT-4 cells after they were either left untreated or pretreated with VHL-L for 1 h, and then treated with DT2216 for 72 h. The data presented in g and h are mean ± SD from three replicate cell cultures in one representative experiment. Each symbol represents data from an individual replicate. Similar results were obtained in an additional independent experiment. i, Cell viability of MOLT-4 cells after treatment with increasing concentrations of DT2216 or its negative-control DT2216NC for 72 h. The data represent mean ± SD from six replicate cell cultures in one representative experiment. Each symbol represents data from an individual replicate. Similar results were obtained in one additional independent experiment. β-actin was used as an equal loading control in immunoblot analyses shown in Fig. 2a–f. The uncropped immunoblot images related to this figure are provided in separate source data file.

Figure 3.. DT2216 is a BCL-XL-specific PROTAC and induces BCL-XL degradation through K87 ubiquitination.

a, A representative of two immunoblot analyses of BCL-XL, BCL-2, MCL-1 and VHL in distinct tumor cell lines. b, Representative immunoblot analysis of BCL-XL, BCL-2 and MCL-1 in H146 SCLC after they were treated with 0.1 μM DT2216 for 48 h and in RS4 B-ALL cells and EJM and H929 multiple myeloma cells after they were treated with 1 μM DT2216 for 16 h. Similar results were obtained in one additional independent experiment. c, Proteomic analysis showing specificity of DT2216 on BCL-XL degradation in comparison with its negative-control DT2216NC in WI-38 cells. d, Ternary complex formation of BCL-XL or BCL-2 with DT2216 and VHL determined by AlphaLISA assay. Data are expressed as mean of a single experiment (n = 2 technical replicates). Similar results were obtained in two more independent assays performed with BCL-XL. e, pH stability of ternary complex formed by DT2216 and VHL complex and BCL-XL or BCL-2 as measured by AlphaLISA assay. Data are expressed as mean (n = 2 technical replicates). Similar results were obtained in one additional independent experiment. f, NanoBRET ternary complex formation of BCL-XL and BCL-2. Ternary complex formation was determined in 293T cells after they transiently expressed HiBit-BCL-XL, LgBit, and HaloTag-VHL or HiBit-BCL-2, LgBit, and HaloTag-VHL and then treated with a serial dilution of DT2216. Data are expressed as mean ± SEM of three independent experiments. g, Representative immunoblot of HA, Flag, β-actin following Flag immunoprecipitation of protein extracts from 293T cells cotransfected as indicated with Flag-BCL-XL and HA-Ub or Flag-BCL-2 and HA-Ub plasmids, then the cells were treated with or without DT2216 (1 μM) and MG132 (10 μM) as indicated for 4 h. Data are representative of three independent experiments. h, Crystal structure of BCL-XL with ABT263 (BCL-2/XL ligand). The lysines are colored in blue. i, A representative immunoblot analysis of BCL-XL showing that DT2216 induces wild-type (WT) BCL-XL degradation in a proteasome-dependent manner. Flag-BCL-XL-WT plasmid was transfected into 293T cells for 40 h and then the cells were treated with or without DT2216 (1 μM) and MG132 (10 μM) as indicated for 6 h. Similar results were obtained in two more independent experiments. j, Representative immunoblot analysis of BCL-XL showing that DT2216 induces BCL-XL degradation dependent on K87 ubiquitination. For the analysis, Flag-BCL-XL-WT, K87R and K157R (lysine to arginine), K87H (lysine to histidine), K-ko (all the lysines in BCL-XL were mutated to arginines) and K87-only (all the lysines in BCL-XL were mutated to arginines except K87) mutant plasmids were transfected into 293T cells for 40 h, and then the cells were treated with or without 1 μM DT2216 for 6 h. Similar results were obtained in two more independent experiments. k, K87 is the only ubiquitination site triggered by DT2216. 293T cells were co-transfected with Flag-BCL-XL and HA-Ub vectors. Extracts were immunoprecipitated with anti-Flag Affinity Resin, followed by trypsin and AspN digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated EVIPMAAVkQALR peptide (ubiquitinated K87 residue) of BCL-XL. Parent ion corresponding to this peptide has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been labeled. The results were obtained from a single experiment. β-actin was used as equal loading control in immunoblotting experiments shown in Fig. 3a, b, h, i and j. The uncropped immunoblot images related to this figure are provided in separate source data file.

Figure 4.. DT2216 is more potent against MOLT-4 T-ALL xenografts and less toxic to platelets than ABT263 in mice.

a, Concentration of DT2216 in MOLT-4 tumors after a single DT2216 administration (15 mpk/i.p.). Data represents average of two mice in a group at each time point. b, Densitometric analysis of BCL-XL protein levels in tumors (mean, n = 2 mice in a group at each time point) at different durations after a single Vehicle (Veh) or DT2216 administration (15 mpk/i.p.). Each symbol represents data (% of Veh) from an individual animal. The representative immunoblots are shown in Extended Data Fig. 5e. c, Numeration of platelets (PLT) 0.25, 1, 2, 3, 4, 5, 7 and 10 days after a single i.p. injection with DT2216 or p.o. dosing with ABT263 at indicated doses. Data are represented as mean ± SEM (n = 3 mice in each group). Each symbol represents data from an individual animal. d, Numeration of PLT after the mice were continuously treated with DT2216 or ABT263 as indicated. Data are represented as mean ± SEM (n = 3 mice in each group till day 12.25; n = 7 mice in Veh, 7 mice in DT2216, and 8 mice in ABT263 at day 17.25; n = 7 mice in Veh, 6 mice in DT2216, and 8 mice in ABT263 at day 24.25). Each symbol represents data from an individual animal. e, f, Body weight and tumor volume changes in mice after the start of treatment with vehicle (Veh), DT2216 (15 mpk/q7d/i.p.) or ABT263 (50 mpk/qd/p.o.). Data presented are mean ± SEM (n = 7 mice in Veh, 7 mice in DT2216 and 8 mice in ABT263 at the start of treatment). Statistical significance was determined by two-sided unpaired Student’s t-test. g, MOLT-4 tumor bearing mice were sacrificed 25 days after treatment initiation (four days and one day after last dose of DT2216 and ABT263, respectively). Tumor weights at the end of study are shown. Data are presented as mean ± SEM (n = 7 mice in Veh, 6 mice in DT2216 and 8 mice in ABT263). Each symbol represents data from an individual animal and the middle horizontal line represents mean. Statistical significance was determined by two-sided unpaired Student’s t-test. h, Immunoblot analysis of BCL-XL, BCL-2 and MCL-1 in MOLT-4 tumors (n = 3 mice in each group). Mpk, mg/kg; q7d, once a week treatment; qd, daily treatment. The uncropped immunoblot images related to this figure are provided in separate source data file.

Figure 5.. Synergy of DT2216 with other BCL-2 family protein inhibitors.

a, Percentage of viable H146 SCLC cells after 72 h treatment with increasing concentrations of DT2216 (DT) and ABT199 (199, a BCL-2 specific inhibitor) alone or the combination of these two at equimolar concentrations (1:1) as indicated is presented on the left panel. EC50 values for each of these treatments, the combination index (CI) at EC75 and EC90 values, and the average CI are presented in the table on the right panel. Data are presented as mean ± SD from six replicate cell cultures in one representative experiment. Similar results were obtained in one additional independent experiment. b, Percentage of viable EJM multiple myeloma cells after 72 h treatment with increasing concentrations of DT2216 (DT) and S63845 (S, a MCL-1 specific inhibitor) alone or the combination of these two at equimolar concentrations (1:1) is presented on the left panel. EC50 values for each of these treatments, CI at EC75 and EC90 values, and the average CI are presented in the table on the right panel. Data are presented as mean ± SD from six replicate cell cultures in one representative experiment. Similar results were obtained in one additional independent experiment. c, Illustration of the experimental design of H146 SCLC xenograft model. d, Body weight changes in H146 SCLC tumor bearing mice after the start of treatment with vehicle (Veh), DT2216, ABT263 or ABT199 alone or the combination of DT2216 (DT) and ABT199 (199) as shown in c. Data are presented as mean ± SEM (n = 6 mice per group at the start of treatment). e, Blood platelets (PLT) were numerated one day after first treatment with all the agents (left panel), and one day after last treatment with DT2216 or ABT263 and 22nd dose of ABT199 (right panel) as shown in c. Data are presented as mean ± SEM (n = 5 mice each for DT+199 in left panel and Veh in right panel, n = 6 mice each in other groups). Each symbol represents data from an individual animal. Statistical significance was determined by two-sided unpaired Student’s t-test. f, Changes in tumor volume over time after the start of treatment as shown in c. Data are presented as mean ± SEM (n = 6 mice per group at the start of treatment). a (P = 0.0381 ABT263 vs. Veh, P = 0.0400 ABT199 vs. Veh); b ( P = 0.0069 vs. Veh, P = 0.0152 vs. ABT263, P = 0.0044 vs. ABT199); and c (P = 0.0022 vs. Veh, P = 0.0001 vs. DT2216, P = 0.0003 vs. ABT263, P <0.0001 vs. ABT199) determined by two-sided unpaired Student’s t-test at post-treatment day-27. g, The average wet weight of excised tumors from each group. Data are presented as mean ± SEM (n = 5 mice in Veh, and 6 mice each in other groups). Each symbol represents data from an individual animal. a (P = 0.0524 vs. Veh); b (P = 0.0086 vs. Veh, P = 0.0116 vs. ABT263, P = 0.0072 vs. ABT199); and c (P = 0.002 vs. Veh, P <0.0001 vs. DT2216, P = 0.0006 vs. ABT263, P <0.0001 vs. ABT199) determined by two-sided unpaired Student’s t-test. h, A representative of two immunoblot analyses of BCL-XL, BCL-2 and MCL-1 in the H146 SCLC tumors excised at the end of experiment (n = 6 mice in each group). β-actin was used as a loading control. The uncropped immunoblot images related to this figure are provided in separate source data file.

Figure 6.. Synergy of DT2216 with chemotherapy.

a, Percentage of viable MDA-MB-231 triple negative breast cancer cells after 72 h treatment with increasing concentrations of DT2216, ABT263, docetaxel (DTX), doxorubicin (DOX), or vincristine (VIN) alone or the combination of DT2216 (DT) or ABT263 (ABT) with one of these chemotherapeutic agents. Data are presented as mean ± SD from three replicate cell cultures in one representative experiment. Similar results were obtained in two additional independent experiments for DTX. b, Percentage of viable VHL-null 786-O renal cell carcinoma cells after 72 h treatment with docetaxel (DTX), DT2216, or ABT263 alone, or the combination of DT2216 (DT) or ABT263 (ABT) with DTX as indicated. Data are presented as mean ± SD from three replicate cell cultures of a single experiment. For the combination treatment, cells were treated with equimolar ratio of two drugs (i.e. 1:1) except the combination of DTX with DT2216 or ABT263 in MDA-MB-231 cells where the ratios of DTX and DT2216 or ABT263 were 1:10. c, Illustration of the experimental design of MDA-MB-231 breast cancer xenograft model. d, e, Body weight and tumor volume changes in MDA-MB-231 tumor bearing mice after the start of treatment with vehicle (Veh), DTX, DT2216, or the combination of these two as shown in c. Data are presented as mean ± SEM (n = 10 mice in each group at the start of treatment). Statistical significance was determined by two-sided unpaired Student’s t-test at post-treatment day-26. f, Illustration of the experimental design of CUL76 T-ALL patient-derived xenograft (PDX) model. g, Percentage of CUL76 T-ALL cells in mouse blood collected at various times after the initiation of the treatments as shown in f. Data are presented as mean ± SEM (n = 5 mice in each group at the start of treatment). Each symbol represents data from an individual animal, and the middle horizontal line represents mean. h, Kaplan-Meier survival curve showing the survival of mice after CUL76 T-ALL engraftment. Medium survival time within each treatment group is presented along with statistical analysis results (n = 5 mice in each group at the start of treatment). Statistical significance was determined by Log-rank (Mantel-Cox) test.

Additional References (for methods)