Supplementary Materials 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@stanford.edu

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

The 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.

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

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 (32–50)

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/9/377/eaaf2584/DC1)

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

Supplementary Materials

Methods

Derivation of human induced pluripotent stem cells (hiPSCs). All the protocols for this study were approved

by the Stanford University Institutional Review Board. Briefly, peripheral blood was obtained via standard

blood draw from 8 healthy control patients and 2 patients receiving TKI as part of their cancer treatment

regimen. Approximately 10 mL of blood was collected in Vacutainer tubes (BD Biosciences). Using a Ficoll-

Paque PLUS gradient (GE Healthcare), we isolated peripheral blood mononuclear cells (PBMCs) and

subsequently cultured them at 1 million cells per mL in a humanized blood cell culture medium (Life

Technologies). PBMCs were reprogrammed using a Sendai virus vector expressing OCT4, KLF4, SOX2, and

MYC (OKSM) (Life Technologies) following manufacturer’s instructions. Three additional patient-specific,

healthy control lines were derived from human skin fibroblasts using a lentivirus reprogramming vector

expressing OKSM. To obtain skin fibroblasts, skin punch biopsies were digested with Collagenase IV and were

retained in GlutaMAX-containing DMEM cell culture medium (Life Technologies) supplemented with 10%

fetal bovine serum (FBS, Gibco) in sterile conditions at standard 37 °C and 5% CO2 cell culture incubator

conditions. Subsequently, hiPSC clones were picked and grown on growth factor-reduced Matrigel (Corning)-

coated 6-well tissue culture dishes (Greiner) in E8 pluripotent stem cell culture medium (Life Technologies).

Immunofluorescence and laser confocal microscopy. Beating hiPSC-CM sheets were incubated in TrypLE

for 2 min at 37 °C followed by mechanical dissociation for the next 6 min using a P1000 pipettor, centrifuged at

200 x g for 4 min, and plated on 0.1-0.2% gelatin-coated glass coverslips following TrypLE deactivation.

Immunostaining was performed per standard protocols using 4% paraformaldehyde for fixation, 0.2% Triton X-

100 for membrane permeabilization, and 4% bovine serum albumin for blocking. Cardiac troponin T (Abcam

AB45932 1:200) and alpha-actinin (Sigma A7811 1:200) antibodies were used for qualitative imaging. Imaging

was performed using a DMIL-LED inverted tissue culture microscope (Leica Microsystems), BioTek Cytation

5 (BioTek), or a Zeiss LSM 510Meta confocal microscope (Carl Zeiss AG) using Zen imaging software.

hiPSC-CM culture for contractility analysis. Day 30-40 post-differentiation patient-specific, healthy control

hiPSC-CMs or, where indicated, commercially available, healthy control iCell hiPSC-CMs were used for

contractility analysis. Initially, iCell Cardiomyocytes (Cellular Dynamics International, CMC-100-010-000.5,

lot 1031999) or patient-specific healthy control hiPSC-CMs were transferred to liquid nitrogen storage. The

hiPSC-CMs were thawed and plated at a density of 5,000 cell/well into 0.1% gelatin (Stem Cell Technologies,

07903)-coated 384 well plates (Greiner Bio-One, 781091). The cardiomyocytes were maintained for 48 hours in

iCell Cardiomyocyte plating media at 37 °C and 5% CO2. After 48 hours, the media was changed to iCell

Cardiomyocyte maintenance media (Cellular Dynamics International, CMM-100-120-001) supplemented with 5

mM D-(+)-glucose (Sigma-Aldrich, G7021). The cardiomyocytes were maintained at 37 °C and 5% CO2 for 10

days with media changes every other day prior to addition of the library of tyrosine kinase inhibitors (TKIs) and

subsequent imaging.

Quantification of cell motion for contractility analysis. Deformation vector maps were obtained from

analysis of image time series using a particle image velocimetry (PIV) routine (32). To quantify the

deformation, a two-step approach was implemented. The aim of the first step was to select a reference frame

that represented the diastolic relaxation of the cells. This was achieved by running PIV using the previous frame

as reference, resulting in a measurement of the instantaneous contraction/relaxation velocity vector map. The

chosen interrogation window size was 128x128 pixels with 64-pixel overlap. The spatially averaged value of

the magnitude of the velocity fields measured the overall motion of the cell culture at each instant of time. The

resulting signal presented two periodic peaks that corresponded to a cardiac cycle (contraction and relaxation).

In the time elapsed between consecutive contraction cycles, the cells are mostly at rest. These periods of

inactivity were automatically identified to choose several reference frames. Ensemble-PIV was run again using

the selected reference frame to obtain the deformation fields and a smaller interrogation window (64x64 pixel

with 32-pixel overlap) to obtain finer spatial resolution and reduced noise levels.

Extraction of contractility signals. Using the cell deformation vector maps obtained from PIV, we applied

Gauss’ divergence theorem to automatically quantify cell contractility as the relative change in area of beating

cells. Contractility signals were obtained as the spatial average of the magnitude of relative change in area,

resulting in periodic signals with one peak per cycle (Fig. S5) whose magnitude is proportional to the overall

axial tension of the cell culture.

Retrieval of contractility parameters. The contractility signals presented periodic peaks related to each

contraction/relaxation cycle. Several parameters were extracted from these signals to perform quantitative and

statistical comparisons. To improve the robustness of the parameter retrieval, all the peaks present in one

contractility signal were collapsed into a most-representative peak by performing time-dependent conditional

averaging (Fig. S6A). As a byproduct of conditional averaging, the mean time between contractility peaks

(Tpeak) was obtained. The rest of the parameters were measured from the single-cycle contractility profile

obtained after conditional averaging (Fig. S6B).

Differentiation and characterization of hiPSC-ECs. To derive hiPSC-ECs, hiPSCs were maintained in E8

medium until the start of differentiation, at which time they were treated with 6 µM GSK3β inhibitor

CHIR99021 in CDM3 medium on day 0 and 2 µM CHIR99021 on day 2. Medium was changed on day 4 and

day 6. On day 7 post-differentiation, cells were treated with 20 µg/mL FGF2 and with 25 µg/mL VEGFA

(Peprotech). Medium was changed every other day. At day 12 post-differentiation, cells were sorted using a

magnetic-activated cell sorter (Miltenyi Biotech) for the endothelial cell surface markers CD31 and CD144.

Cells were then plated on 384-well plates (Greiner) in EGM-2 endothelial medium (Lonza) supplemented with

50 ng/mL VEGFA (Peprotech). To further functionally characterize hiPSC-ECs, cells were subjected to a tube

formation assay. Briefly, 100,000 hiPSC-ECs were plated into a 24-well culture plate coated with Matrigel

(Corning). After 24 hours, spontaneous tube formation was examined.

Differentiation and characterization of hiPSC-CFs. To derive hiPSC-CFs, hiPSCs were maintained in E8

medium until the start of differentiation, at which time they were treated first with GSK3β inhibitor CHIR99021

on day 0 and then with Wnt signaling inhibitor Wnt-C59 on day 2, both in CDM3 medium. At day 4 post-

differentiation, cells were switched to FibroLife medium (Lifeline Cell Technology) for human fibroblast

expansion. This medium was changed every other day. At day 8, cells were re-passaged at low confluency to

selectively facilitate hiPSC-CF adhesion over hiPSC-CM. Cells were also negatively-sorted for CD31 and

CD144 using a magnetic-activated cell sorter to eliminate endothelial cells. Cells were cultured for the next 8

days, and FibroLife was changed every 2 days to maintain and expand the hiPSC-CF population. Primary

human CFs for comparison to hiPSC-CFs were obtained from ATCC.

RNA-seq data analysis. RNA was isolated using miRNeasy kit (Qiagen) following the manufacturer’s protocol

and DNase treatment was performed using RNase-free DNase kit (Qiagen). 100 ng of total RNA was converted

to cDNA and amplified using NuGen V2 RNA-seq kit (NuGen, San Carlos, CA). cDNA was fragmented to an

average of 300 bps using the Covaris S2, and Illumina sequencing adapters were ligated to 500 ng of cDNA

using NEBNext® mRNA Library Prep Reagent Set (New England Biolabs, Ipswich, MA). PCR was performed

on the adapter-ligated cDNA using the following conditions (denaturation at 98 °C for 30 seconds, following 12

cycles of denaturation at 98 °C for 10 seconds, annealing at 65 °C for 30 seconds, and extension at 72 °C for 30

seconds, ending with an extension at 72 °C for 5 min). Libraries were submitted to the Stanford Stem Cell

Institute Genome Center for sequencing using Illumina’s HiSeq2000 platform using paired-in reads at an

average length of 100 bps (2x100). Reads were mapped using Tophat 2.0.8b with the hg19 reference annotation.

Cuffcompare and Cuffdiff were then used to determine which gene levels were significantly different (p<0.05).

RNA-seq expression data was annotated by the Affymetrix Expression Console software (Affymetrix). The

Pearson Correlation Coefficient was calculated for each pair of samples using the expression level of transcripts

that showed standard deviation greater than 0.2 among all samples. Heat maps were generated with TM4 suite

in MeV software (Multiple Experiment Viewer, MeV team).

SUPPLEMENTARY FIGURES

Fig. S1. hiPSCs exhibit characteristic morphologies and markers of pluripotent stem cells. (A) In this

study, eight patient-specific healthy control hiPSC lines were derived from human peripheral blood

mononuclear cells (PBMCs) using Sendai virus reprogramming vectors expressing OCT4, KLF4, SOX2, and

MYC (OKSM). An additional three patient-specific healthy control lines were derived from human skin

fibroblasts using a lentivirus vector expressing OKSM. Two hiPSC lines from patients treated with TKIs

sunitinib and axitinib were also derived using PBMCs/Sendai virus reprogramming. (B) hiPSCs from all

individuals exhibited standard human induced pluripotent stem cell colony morphology and expressed typical

markers for pluripotency such as NANOG and TRA-1-81.

Fig. S2. Quantitative and qualitative cell viability assays illustrate sorafenib, regorafenib, and ponatinib

cytotoxicity in hiPSC-CMs. (A) Dose response curves quantifying TKI toxicity in hiPSC-CMs from healthy

control patients using the CCK-8 cell viability assay, which measures intracellular dehydrogenase activity in

viable cells. Purified day 30 hiPSC-CMs were treated with TKIs for 72 hours. n = 5 biological replicates

conducted per line. (B) Dose response curves representing quantification of TKI toxicity in hiPSC-CMs from

multiple patients using CellTiter-Glo luminescence-based cell viability assay measuring ATP output. Day 30

hiPSC-CMs were treated with TKI for 72 hours. n = 5 biological replicate experiments were conducted for each

line. (C) High-throughput fluorescence imaging of purified day 30 hiPSC-CMs, stained with cardiomyocyte specific markers TNNT2 and α-actinin (ACTN2), treated for 72 hours from 0 to 100 µM, and doxorubicin as a

positive control for toxicity. All data are expressed as means ± SEM.

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

inhibiting TKI toxicity. Six additional patient-specific, healthy control hiPSC-CM lines derived from

PBMC/Sendai virus hiPSC reprogramming were treated with the three most cytotoxic TKIs in our panel

(sorafenib, regorafenib, and ponatinib) as well as with anthracycline doxorubicin as positive control for 72

hours. CellTiter-Glo luminescence-based viability assay was used to confirm cytotoxicity on these three drugs

on these six additional hiPSC-CM lines. All three TKIs demonstrated severe cytotoxicity at less than 10 µM. n

= 5 biological replicate experiments were conducted for each hiPSC-CM line. Data expressed as means ± SEM.

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

TKI treatment. CellTiterGlo cytotoxicity analysis in hiPSC-CMs and hiPSC-ECs from two patients receiving

VEGFR2/PDGFR-inhibiting TKIs (sunitinib and axitinib) as part of their kidney cancer treatment regimen.

Sunitinib was first line treatment and axitinib was the second line treatment. The hiPSC-CMs and hiPSC-ECs

from TKI patients 1 and 2, as well as control patient 1, were treated with sunitinib and axitinib for 72 hours. n =

5 biological replicate experiments were conducted for each line. All data are expressed as means ± SEM.

Fig. S5. Commercially-available, healthy control hiPSC-CMs exhibit alterations in cellular contractility

after 72 hour TKI treatment. Raw contractility tracings from commercially available hiPSC-CMs treated with

TKIs for 72 hours from 0 to 100 µM. Representative tracings from triplicate wells at each concentration shown.

Contractility was measured using a Kinetic Image Cytometer IC 200 at 6.5 second time series (Vala Sciences).

For detailed methods in regards to contractility measurements, see Materials and Methods.

Fig. S6. Heatmaps of high-throughput contractility analysis on commercially-available, healthy control

hiPSC-CMs treated with TKIs. (A) Illustration of representative hiPSC-CM contraction tracing with

associated contractility parameters. (B) Evaluation of hiPSC-CM contractility parameters following 72 hour

TKI treatment using Vala Sciences IC200 Kinetic Imaging Cytometer. Average results from triplicate wells are

shown at each concentration. Readings were also normalized to untreated controls. Red shift indicates decreased

hiPSC-CM contraction rate, whereas green shift indicates increased hiPSC-CM contraction rate.

Fig. S7. Extended calculations for TKI safety index after 72 hour TKI treatment on commercially-

available, healthy control hiPSC-CMs. (A) For the panel of TKIs and doxorubicin, Cessation of Beating,

Effective Concentration, and Amplitude of Effect contractility parameters were obtained from the hiPSC-CM

contractility evaluation using an IC200 Kinetic Imaging Cytometer. LD50 values were obtained from dose

response curves quantifying cytotoxicity following 72 hour TKI treatment in hiPSC-CMs from multiple

individuals using PrestoBlue viability assay, which measures the reducing environment in metabolically active,

viable cells. Cmax obtained from FDA literature for each compound (see Table S1). (B) For calculating the

safety index: cessation of beating, effective concentration, and LD50 values were first divided by Cmax to give

updated values (A), (B), and (D), respectively. The effective concentration (B) was divided by amplitude of

effect to give updated effective concentration (C). Updated cessation of beating (A) was normalized by ibrutinib

value, updated effective concentration (C) was normalized by ponatinib value, and updated LD50 viability (D)

was normalized by dasatinib value. These values were chosen for normalization because they were the highest

values for (A), (C), and (D), respectively, excluding outliers (e.g., trametinib in (A), trametinib/axitinib in (C),

trametinib in (D)). Outliers and N/A were set to 1.000 following normalization. Final safety index was obtained

by averaging normalized cessation of beating ((A) normalized)), effective concentration ((C) normalized)), and

LD50 viability values ((D) normalized)). Red shift indicates higher cardiotoxicity, and green shift indicates

lower cardiotoxicity. N/A indicates that we were unable to measure that parameter.

Fig. S8. hiPSC-CMs exhibit alterations in cellular contractility after 72 hour treatment with known QT

interval-prolonging TKIs. Patient-specific, healthy control hiPSC-CMs were derived from 4 individuals.

Similar to the commercially-available hiPSC-CM data, these patient-specific hiPSC-CMs were subjected to 72

hour treatment with two “clean” compounds, DMSO and axitinib, not associated with alteration in QT interval

at patient-relevant doses, and two known QT interval-prolonging TKIs, nilotinib and vandetanib. Both nilotinib

and vandetanib have associated black box warnings for causing QT interval prolongation in patients. Cells were

treated with TKIs from 0 to 100 µM. Heatmaps generated examining alteration in total contraction time per

single hiPSC-CM contraction, averaged over triplicate wells per dose point. Red box in heatmap indicates an

increase in total contraction time, green indicates a decrease, and white indicates no signal detected.

Fig. S9. hiPSC-derived endothelial cells exhibit endothelial cell characteristics and demonstrate

cytotoxicity in response to TKI treatment. (A) Workflow for hiPSC-EC production. F indicates FGF2 and V

indicates VEGF. (B) hiPSC-ECs exhibited characteristic “cobblestone” morphology. They also absorbed low

density lipoprotein and formed characteristic tube-like structures when replated onto Matrigel basement membrane. (C) hiPSC-ECs stained positive for the endothelial-specific cell surface markers CD31 (PECAM1)

and CD144 (VE-Cadherin). (D) hiPSC-ECs stained positive for von Willebrand Factor (vWF), another marker

for endothelial cells. (E) Dose response curves representing quantification of TKI toxicity in hiPSC-ECs from

multiple patients using the PrestoBlue viability assay. n = 5 biological replicate experiments conducted for each

line. All data expressed as means ± SEM.

Fig. S10. hiPSC-derived cardiac fibroblasts exhibit properties of adult cardiac fibroblasts and

demonstrate cytotoxicity in response to TKI treatment. (A) Workflow for hiPSC-CF production. (B) hiPSC-

CFs expressed the mesodermal and mesenchymal marker vimentin. (C) A subset of hiPSC-CFs expressed

vimentin (VIM) and alpha smooth muscle actin (ACTA2), markers of myofibroblast differentiation. (D) hiPSC-

CFs exhibited a similar proliferative rate to primary CFs. n = 3 biological replicate experiments. (E) hiPSC-CFs

exhibited whorl-like morphologies, like primary CFs. (F) Dose response curves representing quantification of

TKI toxicity in hiPSC-CFs from multiple patients using the PrestoBlue viability assay. n = 5 biological replicate

experiments conducted per line. Data expressed as means ± SEM.

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

ECs, and hiPSC-CFs. Dose response curves representing quantification of TKI toxicity in undifferentiated

hiPSCs from multiple patients using CellTiter-Glo luminescence-based cell viability assay which measures ATP

output. Initially, hiPSCs were seeded at 1,000 cells per well in a 384-well plate and allowed to grow for 3 days.

Then, hiPSCs were treated with TKI for 72 hours. n = 5 biological replicate experiments were conducted for

each line. All data are expressed as means ± SEM.

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

compensatory insulin/IGF1 signaling. Representative kinase assays on day 30 post-differentiation hiPSC-

CMs can elucidate changes in receptor tyrosine kinase phosphorylation in response to treatment with

VEGFR2/PDGFR-inhibiting TKIs sorafenib, axitinib, regorafenib, cabozantinib, ponatinib, and sunitinib.

Specifically, this kinase assay examines phosphorylation changes in EGFR, ErbB2, PDGFRα, ErbB4,

VEGFR2, INSR, IGF1R, and Axl. (A) Sorafenib treatment resulted in dose dependent inhibition of

VEGFR2/PDGFR phosphorylation, but no significant compensatory increase in INSR/IGF1R signaling. (B)

Axitinib treatment resulted in strong dose dependent inhibition of VEGFR2/PDGFRA phosphorylation and a

strong compensatory increase in INSR/IGF1R signaling. (C) Regorafenib treatment resulted in dose dependent

inhibition of VEGFR2/PDGFR phosphorylation and a minor compensatory increase in INSR/IGF1R signaling.

(D) Cabozantinib treatment resulted in dose dependent inhibition of VEGFR2/PDGFR phosphorylation, but no

significant compensatory increase in INSR/IGF1R signaling. (E) Ponatinib treatment resulted in dose dependent

inhibition of VEGFR2/PDGFRA phosphorylation and a compensatory increase in INSR/IGF1R signaling. (F)

Sunitinib treatment resulted in dose dependent inhibition of VEGFR2/PDGFR phosphorylation and a minor

compensatory increase in INSR/IGF1R signaling.

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

Representative phosphorylated kinase array assessing for phosphorylation of a broad range of human kinases

(listed) after 12 hour treatment with IGF1 at 200 ng/mL. Treatment with IGF1 led to phosphorylation of Akt

signaling network members such as Akt, WNK1, and PRAS40. (B) Representative phosphorylated kinase

arrays assessing for phosphorylation of a broad range of human kinases (listed) after 12 hour treatment with

insulin at 20 µg/mL. Treatment with insulin led to increased phosphorylation of Akt signaling network members

such as Akt, WNK1, and PRAS40. Significant differences (*) defined by P < 0.05 per Student’s t-test. n = 3

biological replicate experiments, as shown in Figure 6.

Fig. S14. IGF1 and insulin treatment rescue doxorubicin toxicity in hiPSC-CMs. (A) Dose response curves

quantifying cytotoxicity in hiPSC-CMs following 72 hour treatment with 3 µM doxorubicin at increasing doses

of IGF1 (12 hour pretreatment). PrestoBlue viability assay was used, and n = 5 biological replicates were

conducted for each line. (B) Dose response curves quantifying cytotoxicity in hiPSC-CMs following 72 hour

treatment with 0-100 µM doxorubicin and 12 hour pretreatment with either 20 µg/mL insulin or 200 ng/mL

IGF1. PrestoBlue viability assay was used, and n = 5 biological replicates were conducted. All data are

expressed as means ± SEM.

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

Dose response curves representing quantification of ponatinib toxicity in hiPSC-CMs with and without insulin

or IGF1 co-treatment using the CellTiter-Glo viability assay. A significant increase in cell viability was

observed in response to insulin and IGF1 co-treatment as early as 12 hours post-ponatinib treatment. n = 3

biological replicate experiments were conducted. All data are expressed as means ± SEM.

Fig. S16. RNA-sequencing of hiPSC-CMs treated with VEGFR2/PDGFR-inhibiting TKI sorafenib

illustrates compensatory hyperactivation of VEGF signaling. (A) Heatmap illustrating alterations in gene

expression for five patient-specific, healthy control hiPSC-CM lines treated with 1 µM VEGFR2/PDGFR-

inhibiting TKI sorafenib for 72 hours. Red indicates increase in gene expression, blue indicates decrease. (B)

Heatmap illustrating alterations in VEGF pathway genes following sorafenib treatment. (C) Diagram

illustrating alterations in expression of VEGF pathway network genes following 1 µM sorafenib treatment. Red

indicates increase in gene expression, blue indicates decrease.

SUPPLEMENTARY TABLES

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

TKI Trade name Release

Date

Inhibitor

Target

Primary

FDA

Indication

Reported Cardiotoxicity

(reference #)

Link or

Pubmed ID for

Cmax Value

Afatinib Gilotrif 2013 EGFR/

HER2

EGFR+

NSCLC N/A

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2013/201292

Orig1s000ClinP

harmR.pdf

Erlotinib Tarceva 2004 EGFR NSCLC Myocardial infarction (rare)

(33)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2004/21-

743_Tarceva_bi

opharmr.PDF

Gefitinib Iressa 2003 EGFR NSCLC N/A 16231967

Lapatinib Tykerb 2007 EGFR, ERBB2 Breast

HER2+

QT prolongation, LVEF

decrease (34) (BOXED

WARNING:

HEPATOTOXICITY)

15955900

Axitinib Inlyta 2012 VEGFR1/2/3

PDGFR, c-Kit RCC

Hypertension, Heart Failure

(35)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2012/202324

Orig1s000ClinP

harmR.pdf

Cabozantinib Cometriq 2012

VEGFR2,

PDGFR

Axl

Thyroid

N/A

(BOXED WARNING:

GASTROINTESTINAL)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2012/203756

Orig1s000ClinP

harmR.pdf

Pazopanib Votrient 2009 VEGFR1/2/3,

PDGFR, c-Kit

RCC, STS,

thyroid,

QT prolongation, LVEF

decrease (36) (BOXED

WARNING:

HEPATOTOXICITY)

19509175

Ponatinib Iclusig 2012

VEGFR2,

PDGFR,

FGFR1, Abl, Src

CML, Ph+

ALL

Severe narrowing of vessels,

heart failure, left ventricular

dysfunction, hypertension (37)

(BOXED WARNING:

CARDIOVASCULAR

TOXICITY)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2012/203469

Orig1s000ClinP

harmR.pdf

Regorafenib Stivarga 2012

VEGFR1/2/3,

PDGFR, Kit,

Ret, Raf

GIST

Hypertension, myocardial

ischemia, myocardial infarction

(38) (BOXED WARNING:

HEPATOTOXICITY)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2012/203085

Orig1s000ClinP

harmR.pdf

Sorafenib Nexavar 2005

VEGFR2,

PDGFR,

Raf

HCC, RCC,

thyroid,

GIST

Hypertension, QT prolongation,

LVEF decrease, congestive

heart failure, myocardial

infarction (39)

16006586

Sunitinib Sutent 2006 VEGFR2,

PDGFR, c-Kit

GIST,

PNET, RCC

Hypertension, congestive heart

failure, LVEF decrease,

myocardial infarction, QT

prolongation (39) (BOXED

WARNING:

HEPATOTOXICITY)

16314617

Vandetanib Calprelsa 2011

VEGFR2,

PDGFR EGFR,

Ret

Thyroid

Hypertension, QT prolongation,

heart failure, sudden death,

torsades de pointes (40)

(BOXED WARNING:

CARDIOVASCULAR

TOXICITY)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2011/022405

Orig1s000ClinP

harmR.pdf

Bosutinib Bosulif 2012 Abl/Src Ph+ CML Pericardial effusion (41)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2012/203341

Orig1s000ClinP

harmR.pdf

Dasatinib Sprycel 2006 Abl, Src, c-Kit Ph+ CML,

Ph+ ALL

QT prolongation, pericardial

effusion, hypertension (42) 18420784

Imatinib Gleevec 2001 v-Abl, c-Kit,

PDGFR

Ph+ CML,

ALL, GIST LV dysfunction (rare) (43) 14990650

Nilotinib Tasigna 2007 Bcr-Abl Ph+ CML,

GIST

QT prolongation, Vascular

occlusive events (44) (BOXED

WARNING:

CARDIOVASCULAR

TOXICITY)

19924121

Dabrafenib Tafinlar 2013 BRAFV600

Melanoma

BRAFV600

E+

LVEF decrease (45)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2013/202806

Orig1s000ClinP

harmR.pdf

Vemurafenib Zelboraf 2011 B-Raf Melanoma QT prolongation (46)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2011/202429

Orig1s000ClinP

harmR.pdf

Trametinib Mekinist 2013 MEK1/2

Melanoma

BRAFV600

E+

Left ventricular dysfunction

(47) 23583440

Ibrutinib Imbruvica 2013 BTK CLL, MCL Atrial fibrillation (48)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2013/205552

Orig1s000ClinP

harmR.pdf

Crizotinib Xalkori 2011 c-Met, ALK NSCLC

ALK+

Sinus bradycardia, QT

prolongation (49)

http://www.acce

ssdata.fda.gov/dr

ugsatfda_docs/n

da/2011/202570

Orig1s000ClinP

harmR.pdf

Note: Additional cardiotoxicity information obtained from NIH DailyMed, the official provider of FDA drug

label information for drugs marketed in the United States.

https://dailymed.nlm.nih.gov/

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

cardiotoxicity screen

Cancer therapy Associated with Heart failure/Left Ventricular Dysfunction

Chemotherapy agents Incidence (%) Frequency of Use Doxorubicin (Adriamycin®) 3-26 b ++++

Dabrafenib (Tafinlar®) 8-9# ++++

Dasatinib (Sprycel®) 2-4# ++++

Lapatinib (Tykerb®) 0.9-4.9# ++++

Pazopanib (Votrient®) 0.6-11# ++++

Ponatinib (Iclusig®) 3-15 b +

Sorafenib (Nexavar®) 1.9-11 ++++

Sunitinib (Sutent®) 1-27# ++++

Trametinib (Mekinist®) 7-11# ++++

Cancer therapy Associated with Myocardial Infarction/Ischemia Chemotherapy agents Incidence (%) Frequency of Use Nilotinib (Tasigna®) 5-9.4# ++++

Ponatinib (Iclusig®) 12 b +

Cancer therapy Associated with Bradycardia Chemotherapy agents Incidence (%) Frequency of Use Crizotinib (Xalkori®) 11# ++

Pazopanib (Votrient®) 2-19 ++++

Trametinib (Mekinist®) Up to 10 ++++

Cancer Therapy Associated with QT Prolongation Chemotherapy agents Incidence (%) Frequency of Use Dabrafenib (Tafinlar®) 2-13 ++++

Dasatinib (Sprycel) < 1-3# ++++

Lapatinib (Tykerb®) 16# ++++

Nilotinib (Tasigna®) < 1-4.1 b ++++

Trametinib (Mekinist®) 4-13 ++++

Vandetanib (Caprelsa®) 8-14 b ++++

Vemurafenib (Zelboraf®) NR# ++++

# - Listed as a warning/precaution in package insert

b - Black box warning in package insert

Frequency of Use: This was quantified using inpatient and outpatient doses dispensed at MD Anderson Cancer Center during the

period of January 1, 2014 through December 21, 2014.

+ = < 1,000 doses dispensed

++ =1,000-5,000 doses dispensed

+++ = 5,000-10,000 doses dispensed

++++ = > 10,000 doses dispensed

Adapted from:

(50) E. T. Yeh. “MD Anderson Practices in Onco-Cardiology” (Department of Cardiology, The University of Texas MD Anderson

Cancer Center, 2016).

SUPPLEMENTARY MOVIES

Movie S1. hiPSC-CMs prior to purification via glucose deprivation. hiPSC-CMs begin to spontaneously

contract at approximately day 8-10 after cardiac differentiation is initiated. Cell sheets consistently contain 85-

95% hiPSC-CMs. Movie at 10x magnification.