Research ArticleCancer

Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer

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Science Translational Medicine  02 Aug 2017:
Vol. 9, Issue 401, eaam7049
DOI: 10.1126/scitranslmed.aam7049
  • Fig. 1. MCL-1 and BCL-2 expression in PDX tumors and in vitro sensitivity to S63845 in cell lines and PDX models.

    (A) Box plots showing the relative expression (log2 RPKM) of BCL-2, MCL-1, BCL-XL, and BCL-W across breast tumor subtypes. BRCA1-mutant TNBC, n = 6; ER, n = 6; HER2, n = 8; and TNBC (BRCA1 wild type), n = 10. (B) Western blot analysis of ER and BH3 family member (MCL-1, BCL-2, BCL-XL, BAX, BAK, BIM, and BID) protein expression in PDX models (two independent tumors per PDX). Tubulin was used as a loading control. Arrowhead indicates MCL-1 band. B1, BRCA1-mutated. (C) Cell lines were treated at increasing concentrations of S63845 for 24 hours before assessment of viability using CellTiter-Glo. Means ± SEM for n ≥ 3 independent experiments are shown. (D) Western blot showing the expression of BCL-2 family members in breast cancer cell lines. HSP70 was used as loading control. (E) HER2-amplified and TNBC PDX tumor cells were cultured for 24 hours in mammosphere medium in the presence of ABT-737 (1 μM), ABT-199 (1 μM), WEHI-539 (1 μM), or S63845 (1 μM), and viability was determined compared to DMSO vehicle control. Means ± SEM are shown. The number of independent experiments is indicated. ND, not determined.

  • Fig. 2. Resistance to S63845-induced apoptosis through loss of BAK or elevated BCL-XL.

    (A) SK-BR-3 cells were infected with a genome-wide lentiviral sgRNA library and treated with S63845 (1 μM) or DMSO control, and then gDNA of surviving cells was isolated to identify the sgRNAs by NGS. The pooled analysis from six independent infections, displaying normalized values for S63845 or DMSO control, is shown. Solid red bar represents the regression line. The sgBAK1 hits are shown as blue dots. See table S5 for top up-regulated sgRNAs in the S63845-treated pools. (B) SK-BR-3 cells infected with CRISPR-Cas9 guides targeting proapoptotic proteins were treated with increasing concentrations of S63845 for 24 hours before assessment of viability using CellTiter-Glo. Ev, empty vector. (C) SK-BR-3 cells infected with CRISPR-Cas9 guides targeting BIM, BID, and PUMA were treated with increasing concentrations of S63845, as described above. (D) The MDA-MB-468 cell line was treated with increasing concentrations of S63845 in the presence of ABT-199, WEHI-539, or ABT-737 (500 nM) for 24 hours before assessment of viability using CellTiter-Glo. For (B) to (D), means ± SEM for three independent experiments are shown. (E) PDX tumor cells were cultured for 24 hours in mammosphere medium with ABT-199, ABT-737, WEHI-539, and S63845 (500 nM and 1 μM) or combination treatment with S63845 and other BH3 mimetics (both at 500 nM) before assessment of viability using CellTiter-Glo. Results are presented as percentages of untreated cells. Bars represent means ± SEM for at least five independent experiments per PDX. The tumor subtype for each PDX is shown in parentheses.

  • Fig. 3. Synergistic effect of S63845 with lapatinib, trastuzumab, or docetaxel.

    (A) SK-BR-3 cells were treated with lapatinib (500 nM), trastuzumab (100 μg/ml), and docetaxel (2 nM) or left untreated in the presence of S63845 (30 nM), with or without Q-VD-OPh (QVD; 10 μM), for 72 hours before viability analysis with propidium iodide staining. Results are presented as a percentage of untreated cells and represent three to five independent experiments. Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.001. (B and C) SK-BR-3 cells were treated with increasing concentrations of S63845 and docetaxel (B), or HER2-targeted therapies lapatinib (left panel) for 72 hours or trastuzumab (right panel) for 96 hours (C), and then subjected to viability assays using CellTiter-Glo followed by BLISS score analysis. BLISS synergy values are >0.0 on the vertical axis. (D) Western blot analysis of lysates from (A) showing expression of MCL-1, BCL-XL, BAK, BAX, BIM, BID, AKT, P-AKT, ERK, P-ERK, and cleaved caspase 3 (CC3). Tubulin was used as a loading control.

  • Fig. 4. Improved tumor response to docetaxel in TNBC and trastuzumab in HER2-amplified PDX models with the addition of S63845.

    Kaplan-Meier survival curves (left panels) and tumor volume curves (right panels) for PDX models. (A) TNBC PDX 110 from a BRCA1 mutation carrier (n = 10 to 12 mice per arm) and (B) TNBC PDX 838 (n = 10 to 11 mice per arm). Mice were treated with vehicle alone (black line), docetaxel (10 mg/kg intraperitoneally on days 1 and 22) plus vehicle for S63845 (blue line), S63845 (25 mg/kg intravenously once weekly for 6 weeks, on days 2, 9, 16, 23, 30, and 37) plus vehicle for docetaxel (green line), or combined docetaxel and S63845 (red line). (C) HER2-amplified PDX 231 (n = 6 to 8 per arm). Mice were treated with vehicle (black line), trastuzumab (30 mg/kg loading dose on day 1 and then 15 mg/kg twice weekly for 6 weeks starting on day 4) plus vehicle for S63845 (blue line), S63845 (25 mg/kg once weekly for 6 weeks on days 2, 9, 16, 23, 30, and 37) plus vehicle for trastuzumab (green line), or combined trastuzumab and S63845 (red line). For tumor volume curves, black bars indicate the total duration of the treatment. Mice, which remained otherwise healthy, were sacrificed when tumor size reached the experimental ethical end point (>600 mm3). Means ± SEM are shown. Log-rank (Mantel-Cox) P value is shown for combination therapy versus docetaxel or trastuzumab alone. Tumor growth curves for individual mice from PDX models 110, 838, and 231 are shown in fig. S10.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/401/eaam7049/DC1

    Materials and Methods

    Fig. S1. Expression of BCL-2 family members and sensitivity to S63845 or A-1210477.

    Fig. S2. RNA-seq analysis of BCL-2 family members in PDX models.

    Fig. S3. Expression of BCL-XL, BCL-2, BCL-W, and MCL-1 in METABRIC and TCGA databases.

    Fig. S4. Characterization of ER, PR, HER2, and BCL-2 family member protein expression in PDX models by immunohistochemistry.

    Fig. S5. Deep sequencing of genome-wide lentiviral sgRNA libraries, knockdown of BH3 proteins, and S63845-mediated disruption of MCL-1 complexes containing BH3-only proteins.

    Fig. S6. Generation and analysis of S63845-resistant cell lines.

    Fig. S7. Exploring molecular mechanisms of resistance to S63845.

    Fig. S8. Effect of concomitant treatment of SK-BR-3 cells with a BH3 mimetic and trastuzumab, lapatinib, or docetaxel.

    Fig. S9. Role of BIM in the synergistic effect of S63845.

    Fig. S10. Individual tumor growth curves in TNBC and HER2-amplified PDXs after combination treatment with S63845 and docetaxel or trastuzumab.

    Fig. S11. Effect of combination therapy on tumor cell death.

    Fig. S12. Effect of combination therapy on mouse weight, biochemistry, and blood counts.

    Table S1. Clinical, histopathological, and molecular features of primary breast tumors.

    Table S2. BCL-2 family mRNA expression in PDX models.

    Table S3. Statistical analysis of gene expression between different molecular subtypes of breast cancer in PDX models.

    Table S4. Statistical analysis of MCL-1, BCL-2, BCL-XL, and BCL-W gene expression between different molecular subtypes of breast cancer in METABRIC and TCGA data sets.

    Table S5. Normalized sgRNA counts up-regulated in S63845-treated cells compared to DMSO control.

    Table S6. Primers used for sequencing CRISPR clones.

    Table S7. Sequencing analysis of CRISPR clones for BAK, BAX, BMF, and NOXA.

    References (5366)

  • Supplementary Material for:

    Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer

    Delphine Merino, James R. Whittle, François Vaillant, Antonin Serrano, Jia-Nan Gong, Goknur Giner, Ana Leticia Maragno, Maïa Chanrion, Emilie Schneider, Bhupinder Pal, Xiang Li, Grant Dewson, Julius Gräsel, Kevin Liu, Najoua Lalaoui, David Segal, Marco J. Herold, David C. S. Huang, Gordon K. Smyth, Olivier Geneste, Guillaume Lessene, Jane E. Visvader,* Geoffrey J. Lindeman*

    *Corresponding author. Email: visvader{at}wehi.edu.au (J.E.V.); lindeman{at}wehi.edu.au (G.J.L.)

    Published 2 August 2017, Sci. Transl. Med. 9, eaam7049 (2017)
    DOI: 10.1126/scitranslmed.aam7049

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of BCL-2 family members and sensitivity to S63845 or A-1210477.
    • Fig. S2. RNA-seq analysis of BCL-2 family members in PDX models.
    • Fig. S3. Expression of BCL-XL, BCL-2, BCL-W, and MCL-1 in METABRIC and TCGA databases.
    • Fig. S4. Characterization of ER, PR, HER2, and BCL-2 family member protein expression in PDX models by immunohistochemistry.
    • Fig. S5. Deep sequencing of genome-wide lentiviral sgRNA libraries, knockdown of BH3 proteins, and S63845-mediated disruption of MCL-1 complexes containing BH3-only proteins.
    • Fig. S6. Generation and analysis of S63845-resistant cell lines.
    • Fig. S7. Exploring molecular mechanisms of resistance to S63845.
    • Fig. S8. Effect of concomitant treatment of SK-BR-3 cells with a BH3 mimetic and trastuzumab, lapatinib, or docetaxel.
    • Fig. S9. Role of BIM in the synergistic effect of S63845.
    • Fig. S10. Individual tumor growth curves in TNBC and HER2-amplified PDXs after combination treatment with S63845 and docetaxel or trastuzumab.
    • Fig. S11. Effect of combination therapy on tumor cell death.
    • Fig. S12. Effect of combination therapy on mouse weight, biochemistry, and blood counts.
    • Table S1. Clinical, histopathological, and molecular features of primary breast tumors.
    • References (5366)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). BCL-2 family mRNA expression in PDX models.
    • Table S3 (Microsoft Excel format). Statistical analysis of gene expression between different molecular subtypes of breast cancer in PDX models.
    • Table S4 (Microsoft Excel format). Statistical analysis of MCL-1, BCL-2, BCL-XL, and BCL-W gene expression between different molecular subtypes of breast cancer in METABRIC and TCGA data sets.
    • Table S5 (Microsoft Excel format). Normalized sgRNA counts up-regulated in S63845-treated cells compared to DMSO control.
    • Table S6 (Microsoft Excel format). Primers used for sequencing CRISPR clones.
    • Table S7 (Microsoft Excel format). Sequencing analysis of CRISPR clones for BAK, BAX, BMF, and NOXA.

    [Download Tables S2 to S7]

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