The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models

Journal name:
Nature
Volume:
538,
Pages:
477–482
Date published:
DOI:
doi:10.1038/nature19830
Received
Accepted
Published online

Abstract

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

At a glance

Figures

  1. S63845 binds to the BH3-binding groove of MCL1 and kills tumour cell lines by inducing BAX/BAK-dependent apoptosis.
    Figure 1: S63845 binds to the BH3-binding groove of MCL1 and kills tumour cell lines by inducing BAX/BAK-dependent apoptosis.

    a, Structure of the MCL1 inhibitor, S63845 with binding affinities (nM) for human MCL1, BCL-2 and BCL-XL (see source data). FP, Fluorescence polarization and SPR, surface plasmon resonance.b, Structure of S63845 in complex with MCL1. MCL1 is shown as a surface representation (C and H atoms in grey, O in red, N in blue and S in yellow), the ligand as a stick representation (C and H atoms in green, O in red, N in blue, Cl in green, F in blue–grey and S in yellow). The P2 and P4 hydrophobic pockets and the conserved Arg263 are shown. c, Cellular activity of S63845 and other BH3 mimetics in H929, H146 and RS4;11 human tumour-derived cell lines. Mean ± s.e.m. of n = 3– 8 biological replicates are shown (see source data). d, Western blot analysis of MCL1 and BCL-XL protein levels in HCT-116 cells treated with S63845 or A-1210477. e, Apoptosis induction in H929 cells treated with the MCL-1 inhibitor, S63845, with or without further addition of the caspase inhibitor, QVD-OPh. Mean and individual data points from n = 3 biological replicates are shown (see source data). f, BAX and BAK dependency for S63845 induced killing in H929 cells, as indicated by propidium iodide exclusion assay. Individual data points shown from n = 2 (sgEV, single guide RNA empty vector) or n = 3 (Bak/,Bax/) biological replicates (see source data). Western blot showing protein expression levels of BAK and BAX. For gel source images, see Supplementary Fig. 1.

  2. S63845 is effective against haematological cancer-derived cell lines in vitro and in vivo.
    Figure 2: S63845 is effective against haematological cancer-derived cell lines in vitro and in vivo.

    a, Viability of 25 multiple-myeloma-derived cell lines treated with S63845 (sensitive cell lines in red, IC50 < 0.1 μM; moderately sensitive cell lines in blue, 0.1 μM < IC50 < 1 μM,; insensitive cell lines in green, IC50 > 1 μM). Cell lines are grouped according to their characteristic cytogenetic abnormalities. The p53 status is indicated (*, mutated; #, wild type; †, unknown). Data from n = 2–5 biological replicates and mean ± s.d. are shown (see source data). b, Anti-tumour effect of S63845 in AMO1 multiple myeloma xenograft model. Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. c, Viability of lymphoma- or chronic-myeloid-leukaemia (CML)-derived cell lines treated with S63845 (sensitive cell lines in red, IC50 < 0.1 μM; moderately sensitive cell lines in blue, 0.1 μM < IC50 < 1 μM; insensitive cell lines in green, IC50 > 1 μM). Mean IC50 and individual data points from n = 2 biological replicates are shown (see source data). d, Viability of Eμ-Myc mouse lymphoma cell lines treated with S63845. Mean IC50 and individual data points from n = 3 biological replicates are shown (see source data). e, Survival curves of C57BL/6–LY5.1 mice transplanted with Eμ-Myc lymphoma cells treated with S63845 or vehicle. Statistical analysis was performed by Mantel–Cox test.

  3. S63845 is effective against AML samples in vitro and in vivo, but does not readily kill normal human haematopoietic progenitor cells.
    Figure 3: S63845 is effective against AML samples in vitro and in vivo, but does not readily kill normal human haematopoietic progenitor cells.

    a, Viability of AML-derived cell lines treated with S63845 (sensitive cell lines in red, IC50 < 0.1 μM; moderately sensitive in blue, 0.1 μM < IC50 < 1 μM). Mean IC50 and individual data points from n = 2 biological replicates are shown (see source data). b, Impact of BCL-XL mRNA expression levels on the sensitivity of 24 haematological cancer-derived cell lines to S63845 (IC50 values from Figs. 2c and 3a). DLBCL, diffuse large B cell lymphoma; FPKM, fragments per kilobase per million mapped fragments. The FPKM value is log2(FPKM + 0.1) to account for values of zero. BCL-XL mRNA expression levels (RNA-seq) were extracted from the CCLE database (see source data). The Pearson correlation coefficient and P value are indicated. pIC50 = −log10(IC50) in mol l−1. c, Anti-tumour activity of S63845 in the MV4-11 AML xenograft model. Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. d, Comparison of S63845 activity against primary AML samples relative to healthy CD34+ donor cells. The viability of primary AML cells and normal CD34+ cells (from four healthy human donors, red dashed lines) normalized to vehicle control after 48-h exposure to S63845 (in μM) are shown (see source data). The mean viability of vehicle-treated primary AML cells after 48-h exposure was 85% (range, 70–97%).

  4. Activity of S63845 against solid-tumour-derived cell lines in vitro with or without other targeted agents.
    Figure 4: Activity of S63845 against solid-tumour-derived cell lines in vitro with or without other targeted agents.

    Cell lines were treated with S63845 with or without the MEK1/2 inhibitor, trametenib (SK-MEL-2), the HER-2 inhibitor, lapatinib (BT-474), the B-RAF inhibitor, PLX-4032 (A2058) or the EGFR inhibitor, tarceva (PC-9). Data are normalized to baseline cell survival at the time of initial drug treatment (day 0). Mean and individual data points from three biological replicates are shown (see source data).

  5. S63845 is well tolerated in mice at doses that efficiently kill tumour cells.
    Figure 5: S63845 is well tolerated in mice at doses that efficiently kill tumour cells.

    Cell counts of major lymphoid and myeloid subsets in the bone marrow (BM), spleen and thymus of C57BL/6–LY5.1+ mice at day 9 following treatment with 25 mg per kg body weight S63845 for 5 days (days 1–5); n = 6 vehicle and n = 6 drug treated mice (see source data). Students unpaired t-test was carried out and significant changes are shown. LSKs, lineage marker negative, SCA1+, cKIT+ haematopoietic stem cell population; SPs, CD4+, CD8 or CD4, CD8+ single positive thymocytes; DN, CD4CD8 double negative thymocyte progenitors; mono, monocytes; macro, macrophages; slg+, surface immunoglobulin positive.

  6. Biophysical characterization of the binding of MCL1 inhibitors to human and mouse MCL1 and serum effect on their cellular potency.
    Extended Data Fig. 1: Biophysical characterization of the binding of MCL1 inhibitors to human and mouse MCL1 and serum effect on their cellular potency.

    a, Affinity comparison of binding of S63845 to human or mouse MCL1 (n = 4 and 3 biological replicates for human and mouse, respectively; see source data). b, Binding affinity data of S63845 and A-1210477 for MCL1 (fluorescence polarization (FP) and SPR), BCL-2 (fluorescence polarization) and BCL-XL (n = 2 for fluorescence polarization and n = 3–4 for SPR, see source data). c, SPR sensograms, KD and kinetic parameters for the binding of S63845 and A-1210477 to human MCL1 (n = 4 and 3 biological replicates for S63845 and A-1210477, respectively). d, Impact of serum concentration (FBS, fetal bovine serum) on the biological activity of the MCL1 inhibitors, S63845 and A-1210477, in H929 cells (n = 3 biological replicates, see source data).

  7. Targeting MCL1 genetically in H929 cells induces caspase-mediated cell death.
    Extended Data Fig. 2: Targeting MCL1 genetically in H929 cells induces caspase-mediated cell death.

    a, H929 cells rely on MCL1 for their survival. The viability of H929 cells was determined with CellTiter-Glo assays 72 h after addition of doxocycline to induce expression of sgRNAs to target BCL-2, BCL-XL, BCL-W, MCL1 or BFL1 using CRISPR/Cas9 technology30. Two sgRNAs were tested for each gene: sg#1 (no fill pattern) and sg#2 (slash pattern). Mean and individual data points of n = 2 biological replicates performed in triplicate are shown (see source data). b, Targeting MCL1 by CRISPR/Cas9 causes rapid cell death. The viability (CellTiter-Glo assays) of H929 cells stably expressing Cas9 and containing an inducible expression construct for a sgRNA to target MCL1 was determined 0–96 h after addition of doxocycline. Mean and individual data points of at least n = 2 biological repicates performed in triplicate are shown (see source data). c, Caspases mediate the killing of H929 cells when MCL1 is genetically targeted. The viability (propidium iodide uptake determined by flow cytometry) of H929 cells after switching on the expression of the indicated sgRNAs by the addition of doxocycline (+DOX) was measured after culture in the presence (+) or absence (−) of the broad-spectrum caspase inhibitor, QVD-OPh (25 μM). Data (mean ± s.d.) are derived from one experiment performed in triplicate (see source data). EV, empty vector.

  8. Impact of treatment with S63845 on the interaction of MCL1 with pro-apoptotic BCL-2 family members and on the level and stability of MCL1.
    Extended Data Fig. 3: Impact of treatment with S63845 on the interaction of MCL1 with pro-apoptotic BCL-2 family members and on the level and stability of MCL1.

    a, HeLa cells transduced with Flag–BCL-XL, Flag–BCL-2 or Flag–MCL1 expression constructs were treated for 4 h with increasing concentrations of S63845, before immunopreciptation using anti-FLAG antibody. Immunoprecipitates and total inputs were analysed by immunoblotting for the FLAG-tagged proteins as well as the associated BAK and BAX proteins. For gel source images, see Supplementary Fig. 1. b, HCT-116 cells were incubated for 16 h with increasing concentrations of the proteasome inhibitor, MG132, as a control, before assessing the levels of MCL1, BCL-XL and actin (protein loading control) by immunoblotting. c, HCT-116 cells were pre-incubated for 1 h with DMSO or 1 μM S63845 and then treated with the translation inhibitor, emetin (20 μg ml−1), for 0.5, 1, 2 or 4 h. Determination of MCL1 protein levels was performed by immunoblotting (actin served as a loading control). d, Densitometry of MCL1 immunoblot (Extended Data Fig. 3c, long exposure for DMSO-treated cells and short exposure for S63845-treated cells) and MCL1 half-life estimation (21 min for DMSO-treated cells and 79 min for S63845-treated cells). e, HCT-116 cells were incubated for 16 h with 300 nM S63845, or transfected with 3× Flag–MCL1 expressing plasmid (400 ng, for 48 h) before assessing MCL1 mRNA level by reverse transcriptase–PCR quantification (see source data).

  9. S63845 induces apoptosis of sensitive tumour derived cell lines.
    Extended Data Fig. 4: S63845 induces apoptosis of sensitive tumour derived cell lines.

    a, PARP cleavage in H929 cells after treatment for 6 h with the MCL1 inhibitors, S63845 or A-1210477. Mean and individual data points from three biological replicates are shown (see source data). b, Treatment with S63845 causes mitochondrial release of cytochrome c in sensitive cells. H929 cells were incubated for 4 h with increasing concentrations of S63845 before assessing cytochrome c abundance in the cytosolic fraction by immunoblotting. LDH was used as a protein loading control. For gel source images, see Supplementary Fig. 1. c, Treatment with MCL1 inhibitors causes PARP cleavage in sensitive cells. AMO1 and MV4-11 cells were treated for 6 h with increasing concentrations of the MCL1 inhibitors, S63845 or A-1210477. Cleaved PARP (a marker of apoptotic cell death) was detected by a Meso Scale eletrochemiluminescence assay. Means and individual data points from three biological replicates are shown (see source data).

  10. Correlation between the sensitivity of multiple-myeloma-derived cell lines to S63845 and killing by the MCL1-selective ligand BIM2A. BAX/BAK dependency for S63845-induced killing in KMS-12-PE and AMO1 myeloma cells. Expression of pro-survival BCL-2 family proteins across a panel of multiple-myeloma-derived lines.
    Extended Data Fig. 5: Correlation between the sensitivity of multiple-myeloma-derived cell lines to S63845 and killing by the MCL1-selective ligand BIM2A. BAX/BAK dependency for S63845-induced killing in KMS-12-PE and AMO1 myeloma cells. Expression of pro-survival BCL-2 family proteins across a panel of multiple-myeloma-derived lines.

    a, Correlation between the sensitivity of multiple myeloma (MM)-derived cell lines to S63845 and killing by enforced expression of the MCL1-selective ligand BIM2A. The sensitivity of 25 multiple-myeloma-derived cell lines to S63845 was plotted against the viability of the same cells infected with a lentivirus that expresses the MCL1-selective ligand BIM2A (ref. 33) (r = 0.55) or with a lentivirus expressing the BCL-2, BCL-XL and BCL-W binding ligand BIM–BAD (ref. 34) (r = −0.22) (data from Extended Fig. 5b, see source data). The r and P values were determined by Pearson correlation analysis. b, Table of IC50 values and per cent viability as defined in Extended Data Fig. 5a measured using the CellTiter-Glo assay. The IC50 values were calculated from 2–5 biological replicates (see source data, mean ± s.d.). As a comparison, the dependence of these multiple myeloma cell lines on MCL1 or BCL-2/BCL-XL/BCL-W was validated by a genetic approach. The survival of multiple myeloma cell lines 24 h after enforced expression of BIM2A (which inhibits MCL1) or BIM–BAD (which inhibits BCL-2, BCL-XL and BCL-W) was determined by CellTiter-Glo assay and data were normalized to the survival observed when the inert BIM variant (BIM4E) was expressed. These BIM variants have been previously described13, 34; the data with the BIM variants, shown for comparison, are extracted from a larger data set17. The multiple myeloma cell lines are grouped based on their characteristic cytogenetic abnormality. c, BAX/BAK-dependent killing of KMS-12-PE and AMO1 myeloma cells by S63845. KMS-12-PE or AMO1 myeloma cells were engineered to lack BAX and BAK using CRISPR/Cas9 technology1 (see Methods). Cells expressing the sgRNA empty vector (sgEV) were also generated to serve as negative controls. These cells were treated with S63845 and their viability was determined after 48 h by the propidium iodide exclusion assay. Data shown are mean viability ± s.d. derived from three (KMS-12-PE) or two (AMO1) biological replicates (see source data). d, Expression of MCL1 and related pro-survival BCL-2 proteins across a selected panel of multiple-myeloma-derived lines (shown in Fig. 2a). The levels of MCL1, BCL-2 and BCL-XL were compared between the six multiple-myeloma-derived cell lines most sensitive to S63845 (left, IC50 < 10 nM; data extracted from Extended Data Table 1) and six least sensitive ones (right, IC50 > 100 nM). For gel source images, see Supplementary Fig. 1.

  11. Anti-tumour efficacy and effect of dose scheduling of S63845 in H929 and AMO1 multiple myeloma xenograft models. The in vitro activity of S63845 in human Burkitt-lymphoma-derived cell lines and in vivo activity of S63845 on individual Eμ-Myc lymphoma cell lines.
    Extended Data Fig. 6: Anti-tumour efficacy and effect of dose scheduling of S63845 in H929 and AMO1 multiple myeloma xenograft models. The in vitro activity of S63845 in human Burkitt-lymphoma-derived cell lines and in vivo activity of S63845 on individual Eμ-Myc lymphoma cell lines.

    a, Anti-tumour effect of S63845 in H929 multiple myeloma xenograft models. Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. Tumour volumes of 8 individual mice per treatment group (shown in Fig. 2b) are shown. b, SCID mice were inoculated with 5 × 106 AMO1 cells and randomized nine days after grafting. After randomization, mice were either not treated (black line) or treated by i.v. administration of S63845, every day for 5 consecutive days at 25 mg per kg body weight (red line). For tumour source data, see Supplementary Fig. 2. c, Comparison of S63845 anti-tumour efficacy in the AMO1 model after administration at 25 mg per kg body weight either twice a week for two weeks (red diamonds) or once a week for two weeks (orange triangles). Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. d, Body weight evaluation of mice that were either left untreated (black squares) or treated with S63845 at 25 mg per kg body weight twice a week for two weeks (red diamonds) or once a week for two weeks (orange triangles). Mean ± s.e.m. body weights of eight animals per treatment group are shown. For source data, see Supplementary Fig. 2. e, Sensitivity of human Burkitt-lymphoma (BL)-derived cell lines to S63845. IC50 values of Burkitt-lymphoma-derived cell lines were determined following 24 h in vitro exposure to increasing doses of S63845. Average IC50 values were determined from three biological replicates (each carried out in triplicate, see source data); s.d. are shown. f, Representative haematoxylin and eosin (H&E)-stained sections of spleens and bone marrow from mice transplanted with Eμ-Myc lymphoma cells 4 days after the end of treatment with either vehicle or S63845 from Fig. 2e. Scale bars represent 553 μm for the ×5 objective, 272 μm for the ×10 objective, 133 μm for the ×20 objective and 68 μm for the ×40 objective. g, Response of different Eμ-Myc lymphoma-derived cell lines to in vivo treatment with S63845. Survival curves of C57BL/6–Ly5.1+ mice transplanted with each of the six independent Eμ-Myc lymphoma-derived cell lines that were treated on days 5–9 after transplant with either vehicle (black line) or 25 mg per kg body weight S63845 (red line), by tail vein injection (see source data). A composite of the data from all of these Eμ-Myc lymphoma-derived cell lines is presented in Fig. 2e.

  12. Spearman correlations distribution in haematological cell lines and AML data on patient samples.
    Extended Data Fig. 7: Spearman correlations distribution in haematological cell lines and AML data on patient samples.

    a, Waterfall plots on Spearman correlations between human transcriptome (RNA-seq gene FPKM from CCLE database) and S63845 pIC50 (pIC50 = −log10(IC50)) measured on a panel of 24 haematological cancer-derived cell lines. Raw P values were adjusted for multiple testing using Benjamini-Hochberg correction. Only correlations with false discovery rate (FDR) < 0.05 were retained for the analysis. Spearman correlations were ranked and plotted as a distribution. The threshold was set at r = 0.4, which corresponded to half of the maximum correlation. b, S63845 activity against primary AML samples relative to comparable drugs. Cell viability was assessed by flow cytometric enumeration of Sytox Blue-negative cells after gating on blast cells. The concentration of each drug (in μM) causing 50% lethality for each sample (LC50) is shown. Cytogenetic risk for each sample is defined as intermediate (I) or adverse (A). NA indicates not available. Cell viability (%) after 48 h of treatment with vehicle is also shown. c, AML cell colony-forming assays showing clonogenic capacity of CD34+ sorted cells from two G-CSF mobilized normal, healthy donors (red lines and filled circles) and primary samples from 3–6 patients with AML (black symbols and lines) after 14–21 days exposure to S63845, ara-C or idarubicin, normalized to vehicle control. Error bars show ± s.d. of colony counts from duplicate plates (see source data).

  13. S63845 activity in solid tumour-derived cell lines, correlation with BCL-XL mRNA expression and impact of treatment with the MEK1/2 inhibitor, trametenib, the HER-2 inhibitor, lapatinib, the B-RAF inhibitor, PLX-4032, or the EGFR inhibitor, tarceva, on the levels of p-ERK and the pro-apoptotic protein BIM.
    Extended Data Fig. 8: S63845 activity in solid tumour-derived cell lines, correlation with BCL-XL mRNA expression and impact of treatment with the MEK1/2 inhibitor, trametenib, the HER-2 inhibitor, lapatinib, the B-RAF inhibitor, PLX-4032, or the EGFR inhibitor, tarceva, on the levels of p-ERK and the pro-apoptotic protein BIM.

    a, Viability of NSCLC-, melanoma- and breast tumour-derived cell lines treated with S63845. Sensitive cell lines (IC50 < 0.1 μM) are indicated in red, moderately sensitive cell lines (0.1 μM < IC50 < 1 μM) in blue and insensitive cell lines (IC50 > 1 μM) in green. Mean IC50 and individual data points from n = 2 biological replicates are shown (see source data). b, The impact of the BCL-XL mRNA expression levels on the sensitivity of a panel of 110 solid tumour-derived cell lines to S63845 (see source data). BCL-XL mRNA expression levels (RNA-seq) were extracted from the CCLE database. The Pearson correlation coefficient and P value are shown. c, Waterfall plots on Spearman correlations between human transcriptome (RNA-seq Gene FPKM from CCLE database) and S63845 pIC50 measured on a panel of 110 solid tumour cell lines. Raw P values were adjusted for multiple testing using Benjamini-Hochberg correction. Only correlations with FDR < 0.05 were retained for analysis. Spearman correlations were ranked and plotted as a distribution. The threshold was set at r = 0.4, which corresponded to half of the maximum correlation. d, Tumour cells were incubated with the indicated inhibitors for 16 h, before assessing the protein levels of BIM, ERK, phosphorylated (that is, activated) ERK (P-ERK) and actin (used as a protein loading control) by immunoblotting. For gel source images, see Supplementary Fig. 1. e, SK-MEL-28 cells were treated with S63845 with or without further addition of the MEK1/2 inhibitor, trametenib. Data are normalized to baseline cell survival at the time of initial drug treatment (day 0). Mean and individual data points from three biological replicates are shown (see source data).

  14. Tolerability of S63845 in mice and impact of treatment on healthy tissues at doses that prevent tumour expansion.
    Extended Data Fig. 9: Tolerability of S63845 in mice and impact of treatment on healthy tissues at doses that prevent tumour expansion.

    a, Impact of treatment with S63845 at doses that prevent tumour expansion on healthy tissues in mice. Healthy (not tumour-burdened) C57BL/6–Ly5.1+ mice were treated for 5 consecutive days with either vehicle or 25 mg per kg body weight S63845, and analysed 3 days after treatment had finished. Student’s unpaired t-test was carried out to compare the organ weights of spleen, thymus, lymph nodes, liver and kidney between vehicle and drug-treated mice. The organ weights were not significantly altered in response to treatment with S63845 (see source data). b, Representative haematoxylin and eosin-stained sections of the heart, liver, muscle and kidneys taken from vehicle- or S63845-treated mice at day 9 following treatment for 5 days (days 1–5). Scale bars represent 112 μm for the ×20 objective and 57 μm for the ×40 objective. c, Impact of treatment with increasing doses of S63845 on C57BL/6 mice. Kaplan–Meier survival curves are shown for mice treated with vehicle or with 25, 40, 50 or 60 mg per kg body weight S63845 for 5 consecutive days (see source data). There were 3 female and 3 male 7–8-week-old mice in each treatment group (except for the 40 mg per kg group where n = 5 because one mouse from this treatment group had to be killed owing to an injury incurred during drug administration). Using a Mantel–Cox test only the 60 mg per kg body weight dose showed significant changes relative to treatment with vehicle, ***P = 0.0006. d, Blood cell count analysis of the mice described in b (see source data). The mice were analysed as they were killed, or for the mice surviving the entire treatment, 3 days after the 5 day-treatment had been completed. Unpaired two-tailed t-test was performed and compared to vehicle. For PLT counts, compared to vehicle, **P = 0.0027 for the 25 mg kg dose, ***P = 0.0005 for the 40 mg per kg dose and *P = 0.0245 for the 50 mg per kg dose of S63845. For the RBC counts, compared to vehicle, *P = 0.0312 for the 50 mg per kg dose and *P = 0.0242 for the 60 mg per kg dose of S63845.

Tables

  1. Data collection and refinement statistics
    Extended Data Table 1: Data collection and refinement statistics

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Author information

Affiliations

  1. Servier Research Institute of Medicinal Chemistry, Budapest 1031, Hungary

    • András Kotschy,
    • Zoltán Szlavik,
    • Márton Csekei,
    • Attila Paczal,
    • Zoltán B. Szabo,
    • Szabolcs Sipos,
    • Gábor Radics,
    • Agnes Proszenyak,
    • Balázs Balint,
    • Levente Ondi &
    • Gábor Blasko
  2. Vernalis (R&D) Ltd., Cambridge CB21 6GB, UK

    • James Murray,
    • James Davidson,
    • Alan Robertson,
    • Allan Surgenor,
    • Pawel Dokurno,
    • Ijen Chen,
    • Natalia Matassova,
    • Julia Smith,
    • Christopher Pedder &
    • Christopher Graham
  3. Institut de Recherches Servier Oncology R&D Unit, Croissy Sur Seine 78290, France

    • Ana Leticia Maragno,
    • Gaëtane Le Toumelin-Braizat,
    • Maïa Chanrion,
    • Alain Bruno,
    • Aurélie Studeny,
    • Gaëlle Lysiak-Auvity,
    • Anne-Marie Girard,
    • Fabienne Gravé,
    • Ghislaine Guasconi,
    • Nicolas Cauquil,
    • Frédéric Colland,
    • John A. Hickman &
    • Olivier Geneste
  4. The Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia

    • Gemma L. Kelly,
    • Jia-Nan Gong,
    • David Segal,
    • Chris D. Riffkin,
    • Laura C. A. Galbraith,
    • Brandon J. Aubrey,
    • Margs S. Brennan,
    • Marco J. Herold,
    • Catherine Chang,
    • Andrew W. Roberts,
    • David C. S. Huang,
    • Andreas Strasser &
    • Guillaume Lessene
  5. Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia

    • Gemma L. Kelly,
    • Jia-Nan Gong,
    • David Segal,
    • Chris D. Riffkin,
    • Laura C. A. Galbraith,
    • Brandon J. Aubrey,
    • Margs S. Brennan,
    • Marco J. Herold,
    • Catherine Chang,
    • Andrew W. Roberts,
    • David C. S. Huang,
    • Andreas Strasser &
    • Guillaume Lessene
  6. Australian Centre for Blood Diseases, Monash University, Melbourne 3004, Australia

    • Donia M. Moujalled,
    • Giovanna Pomilio &
    • Andrew H. Wei
  7. Department of Clinical Haematology and Bone Marrow Transplantation, The Royal Melbourne Hospital, Victorian Comprehensive Cancer Centre, Melbourne 3050, Australia

    • Brandon J. Aubrey &
    • Andrew W. Roberts
  8. Institut de Recherches Servier, Biomarker Research Division, Croissy Sur Seine 78290, France.

    • Fabien Melchiore,
    • Nolwen Guigal-Stephan &
    • Brian Lockhart
  9. Faculty of Medicine, The University of Melbourne, Melbourne 3010, Australia.

    • Andrew W. Roberts
  10. Department of Clinical Haematology, The Alfred Hospital, Melbourne 3004, Australia.

    • Andrew H. Wei
  11. Department of Pharmacology and Pharmaceutics, The University of Melbourne, Melbourne 3010, Australia

    • Guillaume Lessene

Contributions

A.K., Z.S., J.D., M.Cs., A.Pa., Z.B.S., S.S., G.R., A.Pr., B.B., L.O., G.B. and C.G. supervised and performed the chemistry. A.K., Z.B.S., J.M., J.D. and I.C. performed the drug design and molecular modelling. A.L.M., G.L.T.-B., G.L.K., J.-N.G., D.M.M., A.Stu., D.S., C.D.R., G.P., C.C., G.G. and N.C. performed cell based experiments. G.L.K., G.L.-A., A.-M.G., F.G., M.S.B., L.C.A.G. and M.J.H. performed the in vivo experiments. B.J.A. contributed to the histology analysis. J.M., A.R., A.Su., P.D., N.M., J.S. and C.P. produced recombinant proteins, performed biochemical assays and crystallographic studies. M.Ch., G.L.K., A.B. and M.J.H. designed the in vivo experiments. F.M., N.G.-S. and B.L. designed and performed the bioinformatic analysis. A.-L.M., J.M., J.D., G.L.K., F.C., J.A.H., A.W.R., D.C.S.H., A.H.W., A.Str., G.L. and O.G. supervised the studies, designed the experiments and interpreted the results. A.Str., G.L., D.C.S.H. and O.G. wrote the manuscript with the assistance of A.L.M., G.L.K. and the other authors.

Competing financial interests

G.L.T.-B., A.L.M., G.G., A.Stu., N.C., M.Ch., A.B., G.L.-A., A.-M.G., F.G., O.G., J.A.H., B.L., N.G.-S., F.C., F.M., A.K., Z.Szl., M.Cs., A.Pa., Z.Sza., S.S., G.R., L.O., A.Pr., B.B. and G.B. are employees of Servier. N.M., C.P., J.S., I.C., C.G., J.D., A.R., A.Su., P.D. and J.M. are employees of Vernalis (R&D) Ltd., G.L., A.Str., G.L.K., M.J.H., J.G., D.S., C.D.R., A.W.R., C.C., M.S.B. and D.C.S.H. are employees of the Walter and Eliza Hall Institute of Medical Research, which receives research funding and milestone payments in relation to venetoclax (ABT-199). G.L., A.Str., G.L.K., M.J.H., J.-N.G., D.S., C.D.R., A.W.R., M.S.B. and D.C.S.H. receive research funding from Servier. A.H.W. serves on the advisory board for Servier and receives research funding from Servier.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks S. Fletcher, F. Stegmeier, G. Wagner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Biophysical characterization of the binding of MCL1 inhibitors to human and mouse MCL1 and serum effect on their cellular potency. (298 KB)

    a, Affinity comparison of binding of S63845 to human or mouse MCL1 (n = 4 and 3 biological replicates for human and mouse, respectively; see source data). b, Binding affinity data of S63845 and A-1210477 for MCL1 (fluorescence polarization (FP) and SPR), BCL-2 (fluorescence polarization) and BCL-XL (n = 2 for fluorescence polarization and n = 3–4 for SPR, see source data). c, SPR sensograms, KD and kinetic parameters for the binding of S63845 and A-1210477 to human MCL1 (n = 4 and 3 biological replicates for S63845 and A-1210477, respectively). d, Impact of serum concentration (FBS, fetal bovine serum) on the biological activity of the MCL1 inhibitors, S63845 and A-1210477, in H929 cells (n = 3 biological replicates, see source data).

  2. Extended Data Figure 2: Targeting MCL1 genetically in H929 cells induces caspase-mediated cell death. (157 KB)

    a, H929 cells rely on MCL1 for their survival. The viability of H929 cells was determined with CellTiter-Glo assays 72 h after addition of doxocycline to induce expression of sgRNAs to target BCL-2, BCL-XL, BCL-W, MCL1 or BFL1 using CRISPR/Cas9 technology30. Two sgRNAs were tested for each gene: sg#1 (no fill pattern) and sg#2 (slash pattern). Mean and individual data points of n = 2 biological replicates performed in triplicate are shown (see source data). b, Targeting MCL1 by CRISPR/Cas9 causes rapid cell death. The viability (CellTiter-Glo assays) of H929 cells stably expressing Cas9 and containing an inducible expression construct for a sgRNA to target MCL1 was determined 0–96 h after addition of doxocycline. Mean and individual data points of at least n = 2 biological repicates performed in triplicate are shown (see source data). c, Caspases mediate the killing of H929 cells when MCL1 is genetically targeted. The viability (propidium iodide uptake determined by flow cytometry) of H929 cells after switching on the expression of the indicated sgRNAs by the addition of doxocycline (+DOX) was measured after culture in the presence (+) or absence (−) of the broad-spectrum caspase inhibitor, QVD-OPh (25 μM). Data (mean ± s.d.) are derived from one experiment performed in triplicate (see source data). EV, empty vector.

  3. Extended Data Figure 3: Impact of treatment with S63845 on the interaction of MCL1 with pro-apoptotic BCL-2 family members and on the level and stability of MCL1. (641 KB)

    a, HeLa cells transduced with Flag–BCL-XL, Flag–BCL-2 or Flag–MCL1 expression constructs were treated for 4 h with increasing concentrations of S63845, before immunopreciptation using anti-FLAG antibody. Immunoprecipitates and total inputs were analysed by immunoblotting for the FLAG-tagged proteins as well as the associated BAK and BAX proteins. For gel source images, see Supplementary Fig. 1. b, HCT-116 cells were incubated for 16 h with increasing concentrations of the proteasome inhibitor, MG132, as a control, before assessing the levels of MCL1, BCL-XL and actin (protein loading control) by immunoblotting. c, HCT-116 cells were pre-incubated for 1 h with DMSO or 1 μM S63845 and then treated with the translation inhibitor, emetin (20 μg ml−1), for 0.5, 1, 2 or 4 h. Determination of MCL1 protein levels was performed by immunoblotting (actin served as a loading control). d, Densitometry of MCL1 immunoblot (Extended Data Fig. 3c, long exposure for DMSO-treated cells and short exposure for S63845-treated cells) and MCL1 half-life estimation (21 min for DMSO-treated cells and 79 min for S63845-treated cells). e, HCT-116 cells were incubated for 16 h with 300 nM S63845, or transfected with 3× Flag–MCL1 expressing plasmid (400 ng, for 48 h) before assessing MCL1 mRNA level by reverse transcriptase–PCR quantification (see source data).

  4. Extended Data Figure 4: S63845 induces apoptosis of sensitive tumour derived cell lines. (398 KB)

    a, PARP cleavage in H929 cells after treatment for 6 h with the MCL1 inhibitors, S63845 or A-1210477. Mean and individual data points from three biological replicates are shown (see source data). b, Treatment with S63845 causes mitochondrial release of cytochrome c in sensitive cells. H929 cells were incubated for 4 h with increasing concentrations of S63845 before assessing cytochrome c abundance in the cytosolic fraction by immunoblotting. LDH was used as a protein loading control. For gel source images, see Supplementary Fig. 1. c, Treatment with MCL1 inhibitors causes PARP cleavage in sensitive cells. AMO1 and MV4-11 cells were treated for 6 h with increasing concentrations of the MCL1 inhibitors, S63845 or A-1210477. Cleaved PARP (a marker of apoptotic cell death) was detected by a Meso Scale eletrochemiluminescence assay. Means and individual data points from three biological replicates are shown (see source data).

  5. Extended Data Figure 5: Correlation between the sensitivity of multiple-myeloma-derived cell lines to S63845 and killing by the MCL1-selective ligand BIM2A. BAX/BAK dependency for S63845-induced killing in KMS-12-PE and AMO1 myeloma cells. Expression of pro-survival BCL-2 family proteins across a panel of multiple-myeloma-derived lines. (895 KB)

    a, Correlation between the sensitivity of multiple myeloma (MM)-derived cell lines to S63845 and killing by enforced expression of the MCL1-selective ligand BIM2A. The sensitivity of 25 multiple-myeloma-derived cell lines to S63845 was plotted against the viability of the same cells infected with a lentivirus that expresses the MCL1-selective ligand BIM2A (ref. 33) (r = 0.55) or with a lentivirus expressing the BCL-2, BCL-XL and BCL-W binding ligand BIM–BAD (ref. 34) (r = −0.22) (data from Extended Fig. 5b, see source data). The r and P values were determined by Pearson correlation analysis. b, Table of IC50 values and per cent viability as defined in Extended Data Fig. 5a measured using the CellTiter-Glo assay. The IC50 values were calculated from 2–5 biological replicates (see source data, mean ± s.d.). As a comparison, the dependence of these multiple myeloma cell lines on MCL1 or BCL-2/BCL-XL/BCL-W was validated by a genetic approach. The survival of multiple myeloma cell lines 24 h after enforced expression of BIM2A (which inhibits MCL1) or BIM–BAD (which inhibits BCL-2, BCL-XL and BCL-W) was determined by CellTiter-Glo assay and data were normalized to the survival observed when the inert BIM variant (BIM4E) was expressed. These BIM variants have been previously described13, 34; the data with the BIM variants, shown for comparison, are extracted from a larger data set17. The multiple myeloma cell lines are grouped based on their characteristic cytogenetic abnormality. c, BAX/BAK-dependent killing of KMS-12-PE and AMO1 myeloma cells by S63845. KMS-12-PE or AMO1 myeloma cells were engineered to lack BAX and BAK using CRISPR/Cas9 technology1 (see Methods). Cells expressing the sgRNA empty vector (sgEV) were also generated to serve as negative controls. These cells were treated with S63845 and their viability was determined after 48 h by the propidium iodide exclusion assay. Data shown are mean viability ± s.d. derived from three (KMS-12-PE) or two (AMO1) biological replicates (see source data). d, Expression of MCL1 and related pro-survival BCL-2 proteins across a selected panel of multiple-myeloma-derived lines (shown in Fig. 2a). The levels of MCL1, BCL-2 and BCL-XL were compared between the six multiple-myeloma-derived cell lines most sensitive to S63845 (left, IC50 < 10 nM; data extracted from Extended Data Table 1) and six least sensitive ones (right, IC50 > 100 nM). For gel source images, see Supplementary Fig. 1.

  6. Extended Data Figure 6: Anti-tumour efficacy and effect of dose scheduling of S63845 in H929 and AMO1 multiple myeloma xenograft models. The in vitro activity of S63845 in human Burkitt-lymphoma-derived cell lines and in vivo activity of S63845 on individual Eμ-Myc lymphoma cell lines. (817 KB)

    a, Anti-tumour effect of S63845 in H929 multiple myeloma xenograft models. Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. Tumour volumes of 8 individual mice per treatment group (shown in Fig. 2b) are shown. b, SCID mice were inoculated with 5 × 106 AMO1 cells and randomized nine days after grafting. After randomization, mice were either not treated (black line) or treated by i.v. administration of S63845, every day for 5 consecutive days at 25 mg per kg body weight (red line). For tumour source data, see Supplementary Fig. 2. c, Comparison of S63845 anti-tumour efficacy in the AMO1 model after administration at 25 mg per kg body weight either twice a week for two weeks (red diamonds) or once a week for two weeks (orange triangles). Mean ± s.e.m. tumour volumes of eight animals per treatment group are shown (***P < 0.001 compared to vehicle). For tumour source data, see Supplementary Fig. 2. d, Body weight evaluation of mice that were either left untreated (black squares) or treated with S63845 at 25 mg per kg body weight twice a week for two weeks (red diamonds) or once a week for two weeks (orange triangles). Mean ± s.e.m. body weights of eight animals per treatment group are shown. For source data, see Supplementary Fig. 2. e, Sensitivity of human Burkitt-lymphoma (BL)-derived cell lines to S63845. IC50 values of Burkitt-lymphoma-derived cell lines were determined following 24 h in vitro exposure to increasing doses of S63845. Average IC50 values were determined from three biological replicates (each carried out in triplicate, see source data); s.d. are shown. f, Representative haematoxylin and eosin (H&E)-stained sections of spleens and bone marrow from mice transplanted with Eμ-Myc lymphoma cells 4 days after the end of treatment with either vehicle or S63845 from Fig. 2e. Scale bars represent 553 μm for the ×5 objective, 272 μm for the ×10 objective, 133 μm for the ×20 objective and 68 μm for the ×40 objective. g, Response of different Eμ-Myc lymphoma-derived cell lines to in vivo treatment with S63845. Survival curves of C57BL/6–Ly5.1+ mice transplanted with each of the six independent Eμ-Myc lymphoma-derived cell lines that were treated on days 5–9 after transplant with either vehicle (black line) or 25 mg per kg body weight S63845 (red line), by tail vein injection (see source data). A composite of the data from all of these Eμ-Myc lymphoma-derived cell lines is presented in Fig. 2e.

  7. Extended Data Figure 7: Spearman correlations distribution in haematological cell lines and AML data on patient samples. (685 KB)

    a, Waterfall plots on Spearman correlations between human transcriptome (RNA-seq gene FPKM from CCLE database) and S63845 pIC50 (pIC50 = −log10(IC50)) measured on a panel of 24 haematological cancer-derived cell lines. Raw P values were adjusted for multiple testing using Benjamini-Hochberg correction. Only correlations with false discovery rate (FDR) < 0.05 were retained for the analysis. Spearman correlations were ranked and plotted as a distribution. The threshold was set at r = 0.4, which corresponded to half of the maximum correlation. b, S63845 activity against primary AML samples relative to comparable drugs. Cell viability was assessed by flow cytometric enumeration of Sytox Blue-negative cells after gating on blast cells. The concentration of each drug (in μM) causing 50% lethality for each sample (LC50) is shown. Cytogenetic risk for each sample is defined as intermediate (I) or adverse (A). NA indicates not available. Cell viability (%) after 48 h of treatment with vehicle is also shown. c, AML cell colony-forming assays showing clonogenic capacity of CD34+ sorted cells from two G-CSF mobilized normal, healthy donors (red lines and filled circles) and primary samples from 3–6 patients with AML (black symbols and lines) after 14–21 days exposure to S63845, ara-C or idarubicin, normalized to vehicle control. Error bars show ± s.d. of colony counts from duplicate plates (see source data).

  8. Extended Data Figure 8: S63845 activity in solid tumour-derived cell lines, correlation with BCL-XL mRNA expression and impact of treatment with the MEK1/2 inhibitor, trametenib, the HER-2 inhibitor, lapatinib, the B-RAF inhibitor, PLX-4032, or the EGFR inhibitor, tarceva, on the levels of p-ERK and the pro-apoptotic protein BIM. (570 KB)

    a, Viability of NSCLC-, melanoma- and breast tumour-derived cell lines treated with S63845. Sensitive cell lines (IC50 < 0.1 μM) are indicated in red, moderately sensitive cell lines (0.1 μM < IC50 < 1 μM) in blue and insensitive cell lines (IC50 > 1 μM) in green. Mean IC50 and individual data points from n = 2 biological replicates are shown (see source data). b, The impact of the BCL-XL mRNA expression levels on the sensitivity of a panel of 110 solid tumour-derived cell lines to S63845 (see source data). BCL-XL mRNA expression levels (RNA-seq) were extracted from the CCLE database. The Pearson correlation coefficient and P value are shown. c, Waterfall plots on Spearman correlations between human transcriptome (RNA-seq Gene FPKM from CCLE database) and S63845 pIC50 measured on a panel of 110 solid tumour cell lines. Raw P values were adjusted for multiple testing using Benjamini-Hochberg correction. Only correlations with FDR < 0.05 were retained for analysis. Spearman correlations were ranked and plotted as a distribution. The threshold was set at r = 0.4, which corresponded to half of the maximum correlation. d, Tumour cells were incubated with the indicated inhibitors for 16 h, before assessing the protein levels of BIM, ERK, phosphorylated (that is, activated) ERK (P-ERK) and actin (used as a protein loading control) by immunoblotting. For gel source images, see Supplementary Fig. 1. e, SK-MEL-28 cells were treated with S63845 with or without further addition of the MEK1/2 inhibitor, trametenib. Data are normalized to baseline cell survival at the time of initial drug treatment (day 0). Mean and individual data points from three biological replicates are shown (see source data).

  9. Extended Data Figure 9: Tolerability of S63845 in mice and impact of treatment on healthy tissues at doses that prevent tumour expansion. (573 KB)

    a, Impact of treatment with S63845 at doses that prevent tumour expansion on healthy tissues in mice. Healthy (not tumour-burdened) C57BL/6–Ly5.1+ mice were treated for 5 consecutive days with either vehicle or 25 mg per kg body weight S63845, and analysed 3 days after treatment had finished. Student’s unpaired t-test was carried out to compare the organ weights of spleen, thymus, lymph nodes, liver and kidney between vehicle and drug-treated mice. The organ weights were not significantly altered in response to treatment with S63845 (see source data). b, Representative haematoxylin and eosin-stained sections of the heart, liver, muscle and kidneys taken from vehicle- or S63845-treated mice at day 9 following treatment for 5 days (days 1–5). Scale bars represent 112 μm for the ×20 objective and 57 μm for the ×40 objective. c, Impact of treatment with increasing doses of S63845 on C57BL/6 mice. Kaplan–Meier survival curves are shown for mice treated with vehicle or with 25, 40, 50 or 60 mg per kg body weight S63845 for 5 consecutive days (see source data). There were 3 female and 3 male 7–8-week-old mice in each treatment group (except for the 40 mg per kg group where n = 5 because one mouse from this treatment group had to be killed owing to an injury incurred during drug administration). Using a Mantel–Cox test only the 60 mg per kg body weight dose showed significant changes relative to treatment with vehicle, ***P = 0.0006. d, Blood cell count analysis of the mice described in b (see source data). The mice were analysed as they were killed, or for the mice surviving the entire treatment, 3 days after the 5 day-treatment had been completed. Unpaired two-tailed t-test was performed and compared to vehicle. For PLT counts, compared to vehicle, **P = 0.0027 for the 25 mg kg dose, ***P = 0.0005 for the 40 mg per kg dose and *P = 0.0245 for the 50 mg per kg dose of S63845. For the RBC counts, compared to vehicle, *P = 0.0312 for the 50 mg per kg dose and *P = 0.0242 for the 60 mg per kg dose of S63845.

Extended Data Tables

  1. Extended Data Table 1: Data collection and refinement statistics (208 KB)

Supplementary information

PDF files

  1. Supplementary Information (7.6 MB)

    This file contains Supplementary Figures 1-2, Supplementary Tables 1-2 and Supplementary Methods.

Additional data