Abstract
Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.
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Data availability
Source data for all figures are provided with the paper online. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
Code availability
Details and code for the power analyses can be found in the GitHub repository (https://github.com/celldeath/power_analysis).
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Acknowledgements
The authors thank K. B. Margulies and K. Bedi of the University of Pennsylvania for generously providing human heart samples; M. Graham for analysis of pharmacokinetic data; B. Bartholdy for RNA-sequencing analysis; V. Hadad for statistical analysis for in vivo studies; M. Zheng, H. Guzik and A. Vohra for technical assistance with mouse studies, imaging and zebrafish studies. This work was supported by grants from the National Institutes of Health (R01HL128071, R01HL130861, R01HL138475 and R01CA170911 to R.N.K.; R01CA178394 to E.G.; R01HL146691 and R00DK107895 to G.S.; R01CA100324 to J.S.C.; P30CA013330 to the Albert Einstein Cancer Center; T32CA200561 to L.R.S.; 1S10OD019961 Shared Instrumentation Grant to the Einstein Analytical Imaging Facility); American Heart Association (15CSA26240000 and 18SRG34280018 to R.N.K. and E.G.; and 15PRE25080032 Predoctoral Fellowship to D.A.); Harrington Scholar-Innovator Award to R.N.K.; Fondation Leducq (RA15CVD04 to R.N.K. and E.G.); Department of Defense (PR151134P1 to R.N.K); New York State Stem Cell Science (NYSTEM C029154 to the Einstein Stem Cell Isolation and Xenotransplantation Facility); Canadian Institutes for Health Research Foundation Grant to L.A.K.; Breast Cancer Research Foundation to R.B.H.; and John S. LaDue Memorial Fellowship at Harvard Medical School to A.A. R.N.K. was supported by the Dr. Gerald and Myra Dorros Chair in Cardiovascular Disease. Overall support was provided by the Wilf Family Cardiovascular Research Institute.
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Contributions
R.N.K. and E.G. conceived the research study. D.A. and R.N.K. designed the molecular, cellular and in vivo studies, which were executed by D.A., R.P., X.F.J., M.Y., F.G.L., J.J.C., J.L., Y.C., V.M., M.S., G.S. and L.A.K. E.G. and T.P.G. designed the MST studies, which were executed by T.P.G. E.G., R.N.K., A.L. and D.A. designed the BH3 profiling studies, which were executed by A.L. and D.A. V.P. performed echocardiography. R.N.K., R.B.H. and D.A. designed the LM2 xenograft studies, which were executed by D.A. and H.L. R.N.K, M.H.O., J.S.C. and D.A. designed the breast cancer patient-derived xenograft studies, which were executed by G.S.K., L.R.S. and D.A. R.N.K., U.S., E.G. and D.A. designed the leukemia transplant studies, which were executed by D.A., S.R.N. and K.M. A.A. and R.T.P. designed the zebrafish studies, which were executed by A.A., D.A., E.G. and R.N.K wrote the manuscript with contributions by M.Y. All authors read and approved the final manuscript.
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R.N.K., E.G., D.A., T.P.G. and L.A.K. are inventors on a patent application PCT/US2018/021644 submitted by Albert Einstein College of Medicine that covers compounds, compositions and methods for BAX inhibition for the treatment of diseases and disorders.
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Extended data
Extended Data Fig. 1 BAI1 binding to BAX as assessed by microscale thermophoresis (MST).
Relates to Fig. 2b. a, c, Examples of MST which measures the thermophoretic movement induced by infrared laser activation of labeled BAX as reflected by relative fluorescence as a function of time. Cold start region indicated by blue box. Warm region indicated by pink box. Each curve denotes a different concentration of BAI1. BAX was labeled with 1 (C1, red curves) or 2 (C2, blue curves) dye molecules/BAX molecule yielding similar results. Representative of 3 independent experiments. b, d, Binding curves determined using the temperature jump region of the curve (pink box) normalized to the cold start region (blue box).
Extended Data Fig. 2 BAI1 does not affect baseline or doxorubicin-induced Ca2+ handling or IRE1α activation.
a, Cytosolic Ca2+ concentrations assessed with Fluo-4 at baseline and following 1 hr treatment with doxorubicin (DOX) in the absence or presence of 1 hr pretreatment with BAI1 1 µM. Box plots depict IQR, horizontal lines show the median, and whiskers represent the minimum and maximum values. n=15, 16 (control); n=18, 19 (DOX 1 µM); n=17, 20 (DOX 5 µM); n=24, 23 (DOX 10 µM) cells. Representative of 2 independent experiments. One-way ANOVA, **** P<0.0001. b, Time course of mitochondrial Ca2+ concentrations assessed with Rhod-2 at baseline and following 1 hr treatment with DOX in the absence or presence of pretreatment with BAI1 1 µM. n=28 (DOX 1 µM), n=35 (DOX 5 µM), n=32 (DOX 10 µM) cells. Representative of 2 independent experiments. Data presented as mean ± s.e.m. c, Western blot for total and phosphorylated IRE1α in MEFs treated with DOX 10 μM for 12 hr with or without 1 hr pretreatment with BAI1 1 μM. Thapsigargin (TG) 1 μM served as a positive control. Unprocessed images of blots are provided as source data. Representative of 3 independent experiments.
Extended Data Fig. 3 BAI1 prevents doxorubicin-induced cardiomyopathy in zebrafish.
a, Zebrafish 30 hr post-fertilization treated with DMSO (control), doxorubicin (DOX), or DOX plus BAI1. Hearts indicated by arrows. Representative images of 3 independent experiments. b, BAI1 prevents doxorubicin-induced cardiomyopathy in a dose-dependent manner. Prevention indicates the absence of all 3 of the following manifestations assessed 40 hr post-treatment: decreased cardiac contraction, pericardial edema, and decreased tail blood flow. n=3 independent experiments. One-way ANOVA, * P=0.0456, ** P=0.0011. Data presented as mean ± s.e.m.
Extended Data Fig. 4 Pharmacokinetic analysis of BAI1.
a, Plasma BAI1 concentrations as a function of time following 1 mg/kg intravenous injection into adult male mice. Concentration determined by LC-MS/MS. n=3 males/time point. Data presented as mean ± s.e.m. b, Pharmacokinetic parameters estimated by non-compartmental analysis of the plasma BAI1 concentration-time curve. Kel: elimination rate constant; t1/2: half-life; Cmax: maximum plasma concentration; SE_Cmax: standard error of Cmax; Tmax: time to reach Cmax; C0: concentration at t=0; AUClast: area under the curve from t=0 to the time of the last quantifiable concentration; SE_AUClast: standard error of AUClast; AUCinf: AUC from t=0 to infinity; AUCextrap: extrapolated AUC from last to infinity, expressed as percentage of AUCinf; Vz: volume of distribution following administration; Cl: total body clearance following administration, calculated from dose/AUC; AUMCinf: area under the first moment curve from t=0 to infinity; MRTinf: mean residence time, calculated by dividing the AUMCinf by the AUC; Vss: steady-state volume of distribution, calculated from Cl · MRTinf.
Extended Data Fig. 5 BAI1 prevents cardiomyopathy in long-term doxorubicin model (females).
a, Schematic of low-dose, long-term doxorubicin (DOX)-induced cardiomyopathy mouse model. Mice were 12 weeks old at the start of experiment. b, Echocardiographic analysis of systolic function including fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and LVEDD-LVESD prior to initiation of treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=15 females. Mean values are shown on the graphs. c, Echocardiographic parameters 4 weeks following the 2-week course of treatment. Saline, n=10; BAI1, n=10; DOX, n=13; DOX+BAI1, n=15 females. Mean values are shown on the graphs. One-way ANOVA, FS: ** P=0.0045, ## P=0.0013; LVESD: * P=0.0368; LVEDD-LVESD: ** P=0.0013, ## P=0.0029. d, TUNEL of cardiac sections and quantification to assess apoptosis. Saline, n=10; BAI1, n=10; DOX, n=12; DOX+BAI1, n=11 females. One-way ANOVA, **** P<0.0001. e, Cleaved caspase-3 immunohistochemistry of cardiac sections and quantification to assess apoptosis. Saline, n=5; BAI1, n=5; DOX, n=9; DOX+BAI1, n=11 females. One-way ANOVA, ** P=0.0078, *** P=0.0003. f, Immunofluorescence for loss of nuclear HMGB1 in cardiac sections and quantification to assess necrosis. Saline, n=4; BAI1, n=5; DOX, n=4; DOX+BAI1, n=5 females. One-way ANOVA, **** P<0.0001. All data presented as mean ± s.e.m. One-way ANOVA, ns P>0.05. The data from the Saline, DOX, and DOX+BAI1 groups for females here are also displayed in Fig. 5b–d along with the corresponding male data in which BAI1 alone was not tested. All four groups are displayed together here so that they can be directly compared within one sex.
Extended Data Fig. 6 BAI1 prevents cardiomyopathy in acute doxorubicin model.
a, Schematic of high-dose, acute doxorubicin (DOX)-induced cardiomyopathy mouse model (females). Mice were 8 weeks old at the start of experiment. b, Echocardiographic analysis of systolic function including fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and LVEDD-LVESD prior to initiation of treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=15 females. Mean values are shown on the graphs. c, Echocardiographic parameters 5 days following the single treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=14 females. Mean values are shown on the graphs. One-way ANOVA, FS: **** P<0.0001; LVEDD: * P=0.0139, ** P=0.0014; LVESD: ** P=0.0043; LVEDD-LVESD: **** P<0.0001. d, Schematic of high-dose, acute DOX-induced cardiomyopathy mouse model (males). Mice were 8 weeks old at the start of experiment. e, Echocardiographic parameters 5 days following the single treatment. Saline, n=8; DOX, n=9; DOX+BAI1, n=12 males. Mean values are shown on the graphs. One-way ANOVA, FS: ** P=0.0011, ## P=0.0025; LVEDD-LVESD: ** P=0.0015, ## P=0.0039. f, TUNEL of cardiac sections and quantification to assess apoptosis. n=3 males/group. One-way ANOVA, ** P=0.0077, ## P=0.0032. g, Immunofluorescence for loss of nuclear HMGB1 in cardiac sections and quantification to assess necrosis. n=3 males/group. One-way ANOVA, * P=0.0235, ** P=0.0031. All data presented as mean ± s.e.m. One-way ANOVA, ns P>0.05.
Extended Data Fig. 7 BAI1 alone does not affect body or organ weights.
Treatment with BAI1 alone in the long-term model as illustrated in the schematic in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. n=10 mice/group. All data presented as mean ± s.e.m. P-values from two-tailed Student’s t-test shown on the graphs.
Extended Data Fig. 8 BAI1 alone does not affect histology of multiple tissues.
Treatment with BAI1 alone in the long-term model as illustrated in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. Hematoxylin and eosin staining of mouse tissues. Representative micrographs from n=5 mice/group. Scale bars: a, 1000 μm; b, 50 μm.
Extended Data Fig. 9 BAI1 alone does not affect hematological parameters.
Treatment with BAI1 alone in the long-term model as illustrated in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. All values are within the normal range for mice. n=10 mice/group. All data presented as mean ± s.e.m. P-values from two-tailed Student’s t-test shown on the graphs.
Extended Data Fig. 10 BAI1 does not attenuate doxorubicin-induced killing of human cancer cells.
a, b, BAI1 alone does not affect cell viability of various cancer cell lines. Cellular viability measured by maintenance of plasma membrane integrity (CytoTox-Glo). c, d, BAI1 does not interfere with the ability of doxorubicin to kill these cells. For panels c and d, untreated cells (no doxorubicin and no BAI1) assigned a viability of 100% (not shown). All panels, n=3 independent experiments. One-way ANOVA, compared with BAI1 0 µM, ** P=0.0091. All data presented as mean ± s.e.m. For comparison, BAI1 at nanomolar concentrations inhibit cardiomyocyte death (Fig. 4).
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Amgalan, D., Garner, T.P., Pekson, R. et al. A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy. Nat Cancer 1, 315–328 (2020). https://doi.org/10.1038/s43018-020-0039-1
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DOI: https://doi.org/10.1038/s43018-020-0039-1
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