Research ArticleTOXICITY SCREENING

High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells

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Science Translational Medicine  15 Feb 2017:
Vol. 9, Issue 377, eaaf2584
DOI: 10.1126/scitranslmed.aaf2584
  • Fig. 1. hiPSC-CMs exhibit sarcomeric proteins and express human RTK families.

    (A) Diagram of study workflow. Somatic tissue samples were obtained from 13 individuals and reprogrammed into hiPSC colonies with either Sendai virus or lentivirus vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC (OKSM). hiPSCs were differentiated into hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. Purified cardiomyocytes were treated with TKIs and examined for alterations in cell viability, contractility, cellular signaling, and gene expression. (B) Confocal microscopic immunofluorescence images of differentiated hiPSC-CMs expressing the sarcomeric markers cardiac troponin T (TNNT2) and α-actinin (ACTN2). DAPI, 4′,6-diamidino-2-phenylindole. (C) Day 30 hiPSC-CMs from five healthy control lines expressing major RTKs including INSR, IGF1R, PDGFRA, and KDR. High TNNT2 and low PECAM1 expression indicates a pure hiPSC-CM population devoid of ECs. n = 3 biological replicates conducted for gene expression analysis in each hiPSC-CM line. Data are means ± SEM. RPKM, reads per kilobase per million.

  • Fig. 2. High-throughput analysis of TKI toxicity in purified hiPSC-CMs allows for the development of a TKI cardiac safety index.

    (A) Dose-response curves quantifying cytotoxicity after a 72-hour TKI treatment of five healthy control hiPSC-CM lines using a PrestoBlue viability assay. n = 5 biological replicates conducted per line. Data are means ± SEM. (B) Evaluation of hiPSC-CM contractility after a 72-hour TKI treatment with the IC200 Kinetic Imaging Cytometer. Average results from triplicate wells shown at each concentration. Red indicates decreased contraction rate, whereas green indicates increased contraction rate. (C) Values gathered from cytotoxicity and contractility analyses in hiPSC-CMs. Green shading indicates values associated with less cardiotoxicity. Red shading indicates values associated with higher cardiotoxicity. Cessation of beating is the concentration at which >50% of triplicate wells ceased beating. Effective concentration is the concentration at which a significant alteration in all listed contractility parameters was detected (see fig. S7 and Materials and Methods for details). Amplitude of effect is the degree to which all listed contractility parameters were altered at the effective concentration (see Materials and Methods for details). LD50 is the TKI concentration at which a 50% loss in viability is observed from viability assays, averaged across patient hiPSC-CM lines. Patient Cmax represents the maximum TKI blood plasma concentration experienced by patients reported in FDA literature. The cardiac safety index is a value from 0 to 1 that normalizes contractility and viability parameters to patient Cmax and combines these parameters to provide a relative metric for TKI cardiotoxicity. Highlighted drugs (surrounded by a red rectangle) have a safety index at or below 0.10, our threshold for highly cardiotoxic compounds. Clinically reported cardiotoxicities are alterations in patient cardiac function (see table S1). QT, QT interval prolongation; Hy, hypertension; LV, left ventricular ejection fraction decrease; HF, heart failure; MI, myocardial infarction; TdP, Torsades de pointes; SCD, sudden cardiac death; Brady, bradycardia; PE, pericardial effusion; Vas, vascular abnormalities; Afib, atrial fibrillation; **cardiovascular toxicity–associated boxed warning; #noncardiovascular toxicity–associated boxed warning.

  • Fig. 3. hiPSC-CMs exhibit alterations in intracellular calcium handling after a 2-hour treatment with known QT interval–prolonging TKIs.

    (A) Schematic illustrating TKI treatment regimen for hiPSC-CMs before calcium imaging. (B) Raw line scans of individual hiPSC-CM calcium transients after TKI treatment at indicated clinically relevant concentrations and calcium dye treatment over multiple beats. (C) Quantification of hiPSC-CM calcium imaging parameters after a 2-hour TKI treatment. n = 10 cells recorded for each condition. Data are presented as box-and-whisker plots showing the minimum, first quartile, median, mean, third quartile, and maximum of the data set. Student’s t test indicates significance compared to control (*P < 0.05 and **P < 0.01).

  • Fig. 4. hiPSC-CMs exhibit alterations in cellular electrophysiology after a 2-hour treatment with known QT interval–prolonging TKIs.

    (A) Schematic illustrating setup for acute and 2-hour TKI treatment before AP recording. (B) Representative AP tracings after acute TKI treatment for up to 10 min at clinically relevant concentrations in hiPSC-CMs. (C) Representative AP tracings after a 2-hour TKI treatment at clinically relevant concentrations in hiPSC-CMs. (D) Quantification of hiPSC-CM electrophysiological parameters after a 2-hour TKI treatment. Data are means ± SEM. *P < 0.05, compared to DMSO, Student’s t test. n = 10 cells recorded for each condition.

  • Fig. 5. Treatment with VEGFR2/PDGFR-inhibiting TKIs causes dose-dependent alterations in RTK signaling in hiPSC-CMs.

    Normalized quantification of RTK phosphorylation in purified hiPSC-CMs treated with 0 to 1 μM of the VEGFR2/PDGFR-inhibiting TKIs sorafenib, cabozantinib, ponatinib, axitinib, regorafenib, or sunitinib for 72 hours. Phosphorylation array blots are shown in fig. S12. n = 3 biological replicates conducted. Data are means ± SEM.

  • Fig. 6. Insulin and IGF1 activate cardioprotective signaling pathways and alleviate cytotoxicity in hiPSC-CMs.

    (A) Phosphorylation arrays demonstrating alterations in hiPSC-CM kinase activity after a 12-hour IGF1 or insulin treatment. n = 3 biological replicate phosphorylation arrays conducted. Data are means ± SEM. *P < 0.05, Student’s t test. We observed a significant increase in phosphorylation of the following protein amino acid residues after IGF1 treatment (P values listed): Akt1/2/3-S473 (0.01), Akt1/2/3-T308 (0.04), GSK3α/β-S21/S9 (0.03), p53-S15 (0.002), p53-S392 (0.02), p53-S46 (0.003), PRAS40-T246 (0.001), TOR-S2448 (0.0001), and WNK1-T60 (0.002). We observed a significant increase in phosphorylation of the following protein amino acid residues after insulin treatment (P values listed): Akt1/2/3-S473 (0.01), Akt1/2/3-T308 (0.04), GSK3α/β-S21/S9 (0.005), p53-S46 (0.04), PRAS40-T246 (0.007), TOR-S2448 (0.01), and WNK1-T60 (0.006). (B) Immunofluorescence of hiPSC-CMs treated with sorafenib, regorafenib, or ponatinib at increasing concentrations for 72 hours in the presence of IGF1 or insulin. Calcein-AM stains viable cells. (C) CellTiter-Glo quantification of hiPSC-CM viability with or without insulin/IGF1 cotreatment during TKI treatment. n = 5 biological replicates conducted. IGF and insulin treatment significantly rescued ponatinib toxicity (P = 0.004). Data are means ± SEM.

  • Fig. 7. Treatment with sorafenib, regorafenib, or ponatinib leads to hyperactivation of compensatory signaling through noncanonical VEGFRs.

    (A) Microarray heat map illustrating differentially expressed genes after a 72-hour sorafenib, regorafenib, and ponatinib treatment in hiPSC-CMs. Cells were treated with 1 μM TKI to avoid cytotoxicity at higher doses. Red indicates high gene expression, and blue indicates low gene expression. (B) Graph represents fold expression change (compared to control) of significantly altered genes after drug treatment. Significantly altered genes defined by P < 0.05 compared to untreated control. Multiple P-value comparisons made using one-way between-subject analysis of variance (ANOVA).

  • Fig. 8. Model for the activation of compensatory survival signaling in hiPSC-CMs in response to treatment with VEGFR2/PDGFR-inhibiting TKIs.

    (A) In hiPSC-CMs, the RTKs VEGFR2, PDGFRα, INSR, and IGF1R are upstream of prosurvival signaling pathways. (B) Our results suggest that VEGFR2/PDGFR-inhibiting TKIs up-regulate INSR and IGF1R signaling (phosphorylation) to compensate for the loss of VEGFR/PDGFR signaling (phosphorylation). This compensatory effect can augment cardiomyocyte survival during TKI treatment via introduction of exogenous insulin and IGF1 ligands. We observed an increase in the downstream gene expression of noncanonical VEGF-binding receptors and VEGFR pathway members, presumably to compensate for VEGFR2/PDGFR-inhibiting TKI-induced loss in upstream VEGFR signaling (phosphorylation).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/377/eaaf2584/DC1

    Materials and Methods

    Fig. S1. hiPSCs exhibit characteristic morphologies and markers of pluripotent stem cells.

    Fig. S2. Quantitative and qualitative cell viability assays illustrate sorafenib, regorafenib, and ponatinib cytotoxicity in hiPSC-CMs.

    Fig. S3. Quantitative cell viability assays on additional hiPSC-CM lines demonstrate VEGFR2/PDGFR-inhibiting TKI toxicity.

    Fig. S4. Quantitative cell viability assays in hiPSC-CMs and hiPSC-ECs derived from patients receiving TKI treatment.

    Fig. S5. Commercially available, healthy control hiPSC-CMs exhibit alterations in cellular contractility after a 72-hour TKI treatment.

    Fig. S6. Heat maps of high-throughput contractility analysis on commercially available, healthy control hiPSC-CMs treated with TKIs.

    Fig. S7. Extended calculations for TKI safety index after a 72-hour TKI treatment on commercially available, healthy control hiPSC-CMs.

    Fig. S8. hiPSC-CMs exhibit alterations in cellular contractility after a 72-hour treatment with known QT interval–prolonging TKIs.

    Fig. S9. hiPSC-ECs exhibit EC characteristics and demonstrate cytotoxicity in response to TKI treatment.

    Fig. S10. hiPSC-CFs exhibit properties of adult cardiac fibroblasts and demonstrate cytotoxicity in response to TKI treatment.

    Fig. S11. hiPSCs demonstrate a TKI cytotoxicity profile that is unique from those of hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs.

    Fig. S12. VEGFR2/PDGFR-inhibiting TKI treatment in hiPSC-CMs results in activation of compensatory insulin/IGF1 signaling.

    Fig. S13. IGF1 and insulin treatment activates cardioprotective Akt signaling in hiPSC-CMs.

    Fig. S14. IGF1 and insulin treatment rescues doxorubicin toxicity in hiPSC-CMs.

    Fig. S15. IGF1 and insulin treatment rescues ponatinib toxicity at early time points in hiPSC-CMs.

    Fig. S16. RNA-seq of hiPSC-CMs treated with the VEGFR2/PDGFR-inhibiting TKI sorafenib illustrates compensatory hyperactivation of VEGF signaling.

    Table S1. Small-molecule TKIs selected for high-throughput cardiotoxicity screen.

    Table S2. Adverse cardiac events associated with small-molecule TKIs selected for high-throughput cardiotoxicity screen.

    Movie S1. hiPSC-CMs before purification via glucose deprivation.

    References (3250)

  • Supplementary Material for:

    High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells

    Arun Sharma, Paul W. Burridge, Wesley L. McKeithan, Ricardo Serrano, Praveen Shukla, Nazish Sayed, Jared M. Churko, Tomoya Kitani, Haodi Wu, Alexandra Holmström, Elena Matsa, Yuan Zhang, Anusha Kumar, Alice C. Fan, Juan C. del Álamo, Sean M. Wu, Javid J. Moslehi, Mark Mercola, Joseph C. Wu*

    *Corresponding author. Email: joewu{at}stanford.edu

    Published 15 February 2017, Sci. Transl. Med. 9, eaaf2584 (2017)
    DOI: 10.1126/scitranslmed.aaf2584

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. hiPSCs exhibit characteristic morphologies and markers of pluripotent stem cells.
    • Fig. S2. Quantitative and qualitative cell viability assays illustrate sorafenib, regorafenib, and ponatinib cytotoxicity in hiPSC-CMs.
    • Fig. S3. Quantitative cell viability assays on additional hiPSC-CM lines demonstrate VEGFR2/PDGFR-inhibiting TKI toxicity.
    • Fig. S4. Quantitative cell viability assays in hiPSC-CMs and hiPSC-ECs derived from patients receiving TKI treatment.
    • Fig. S5. Commercially available, healthy control hiPSC-CMs exhibit alterations in cellular contractility after a 72-hour TKI treatment.
    • Fig. S6. Heat maps of high-throughput contractility analysis on commercially available, healthy control hiPSC-CMs treated with TKIs.
    • Fig. S7. Extended calculations for TKI safety index after a 72-hour TKI treatment on commercially available, healthy control hiPSC-CMs.
    • Fig. S8. hiPSC-CMs exhibit alterations in cellular contractility after a 72-hour treatment with known QT interval–prolonging TKIs.
    • Fig. S9. hiPSC-ECs exhibit EC characteristics and demonstrate cytotoxicity in response to TKI treatment.
    • Fig. S10. hiPSC-CFs exhibit properties of adult cardiac fibroblasts and demonstrate cytotoxicity in response to TKI treatment.
    • Fig. S11. hiPSCs demonstrate a TKI cytotoxicity profile that is unique from those of hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs.
    • Fig. S12. VEGFR2/PDGFR-inhibiting TKI treatment in hiPSC-CMs results in activation of compensatory insulin/IGF1 signaling.
    • Fig. S13. IGF1 and insulin treatment activates cardioprotective Akt signaling in hiPSC-CMs.
    • Fig. S14. IGF1 and insulin treatment rescues doxorubicin toxicity in hiPSC-CMs.
    • Fig. S15. IGF1 and insulin treatment rescues ponatinib toxicity at early time points in hiPSC-CMs.
    • Fig. S16. RNA-seq of hiPSC-CMs treated with the VEGFR2/PDGFR-inhibiting TKI sorafenib illustrates compensatory hyperactivation of VEGF signaling.
    • Table S1. Small-molecule TKIs selected for high-throughput cardiotoxicity screen.
    • Table S2. Adverse cardiac events associated with small-molecule TKIs selected for high-throughput cardiotoxicity screen.
    • Legend for movie S1
    • References (3250)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). hiPSC-CMs before purification via glucose deprivation.

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