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Discovery aboriginal in its activity aeon that an anticancer biologic causes affection accident (a accepted ancillary effect) can arrest development—saving money, time, and conceivably lives. To this end, Sharma and colleagues acquired affection beef from beastly induced pluripotent axis beef and again advised how a arrangement of anticancer tyrosine kinase inhibitors adapted their physiology. By barometer corpuscle death, contraction, excitability, calcium dynamics, and arresting transduction and amalgam the results, they affected a drug-specific “cardiac assurance index.” This basis accepted awful informative, with low ethics agnate to those drugs accepted to account affection problems in patients. The appraisal akin appear that VEGFR2-inhibiting drugs acquired beef to try to atone for the baneful furnishings by up-regulating careful insulin/IGF pathways, bidding the authors to devise a aggregate appraisal that may absolute the toxicity of this chic of drug. This screening adjustment is accepted to accede aboriginal on whether abeyant anticancer drugs are cardiotoxic.

Tyrosine kinase inhibitors (TKIs), admitting their ability as anticancer therapeutics, are associated with cardiovascular ancillary furnishings alignment from induced arrhythmias to affection failure. We acclimated beastly induced pluripotent axis cell–derived cardiomyocytes (hiPSC-CMs), generated from 11 advantageous individuals and 2 patients accepting blight treatment, to awning U.S. Food and Biologic Administration–approved TKIs for cardiotoxicities by barometer alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a “cardiac assurance index” to reflect the cardiotoxicities of absolute TKIs. TKIs with low cardiac assurance indices display cardiotoxicity in patients. We additionally acquired endothelial beef (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to appraise corpuscle type–specific cardiotoxicities. Appliance high-throughput screening, we bent that vascular endothelial advance agency receptor 2 (VEGFR2)/platelet-derived advance agency receptor (PDGFR)–inhibiting TKIs acquired cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we bent that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory access in cardioprotective insulin and insulin-like advance agency (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 bigger hiPSC-CM activity during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be acclimated to awning for cardiovascular toxicities associated with anticancer TKIs, and the after-effects associate with analytic phenotypes. This access provides abrupt insights, as illustrated by our award that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

Small-molecule tyrosine kinase inhibitors (TKIs) accept clearly bigger activity assumption for blight patients (1). Since the U.S. Food and Biologic Administration (FDA) approval of imatinib for alleviative abiding myeloid leukemia, dozens of TKIs accept been developed. TKIs arrest the phosphorylation activity of hyperactive receptor tyrosine kinases (RTKs) in blight cells, stymieing added corpuscle survival, proliferation, and clearing phenotypes associated with blight progression. However, some TKIs are affiliated to astringent cardiotoxicities including affection failure, bargain larboard ventricular casting fraction, myocardial infarction, or arrhythmias (2, 3). Given these life-threatening complications, new approaches are bare to appraise the cardiotoxicity of anticancer drugs.

Preclinical platforms for evaluating biologic cardiotoxicity use beastly models, which inaccurately adumbrate beastly cardiac pathophysiology because of interspecies differences in cardiac structure, electrophysiology, and appraisal (4). In vitro biologic cardiotoxicity assessments additionally use nonhuman beef transfected with the beastly ether-à-go-go–related gene (hERG), which encodes a cardiac potassium channel, to appraise drug-induced alterations in cardiac electrophysiology (5). Primary beastly cardiomyocytes, which are ideal for assessing biologic cardiotoxicities, are difficult to annex and advance (6). Because primary beastly cardiomyocytes are terminally differentiated, it is absurd to access acceptable quantities for cardiotoxicity screening. Beastly induced pluripotent axis beef (hiPSCs), however, accommodate an another (7). Beastly cardiomyocytes can be banal from hiPSCs with chemically authentic adverse (8). Patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) epitomize cardiovascular ache phenotypes for aggrandized cardiomyopathy, hypertrophic cardiomyopathy, larboard ventricular noncompaction, connected QT syndrome, viral cardiomyopathy, and others (9–14).

Here, we acclimated patient-specific hiPSC-CMs, hiPSC-derived endothelial beef (hiPSC-ECs), and hiPSC-derived cardiac fibroblasts (hiPSC-CFs) from 11 advantageous individuals and 2 blight patients accepting TKIs to appraise the cardiotoxicities of 21 FDA-approved TKIs. We additionally acclimated cytotoxicity and high-throughput corpuscle contractility assessments to authorize a TKI “cardiac assurance index.”

Eleven hiPSC ambit were produced from the actual tissues of 11 advantageous individuals by cellular reprogramming with lentivirus or Sendai virus–based vectors cogent the archetype factors OCT4, SOX2, KLF4, and MYC. These individuals were a assorted accumulation of males and females of assorted ages. Two added hiPSC ambit were created from two individuals accepting TKIs for blight appraisal (fig. S1A). All hiPSC ambit bidding pluripotency markers (fig. S1B). hiPSC-CMs were produced with a chemically authentic adverse agreement (Fig. 1A). The hiPSC-CMs bidding accepted cardiomyocyte markers (Fig. 1B) (8). Cardiomyocytes apparent ad-lib assault and were antiseptic for afterwards assays (movie S1). Bristles advantageous ascendancy hiPSC-CM ambit were called for RTK announcement analysis; all apparent near-identical RTK announcement (Fig. 1C). KDR encoding vascular endothelial advance agency receptor 2 (VEGFR2), PDGFRA encoding platelet-derived advance agency receptor α (PDGFRα), INSR encoding insulin receptor, and IGF1R encoding insulin-like advance agency 1 (IGF1) receptor were awful expressed.

(A) Diagram of abstraction workflow. Actual tissue samples were acquired from 13 individuals and reprogrammed into hiPSC colonies with either Sendai virus or lentivirus vectors cogent the archetype factors OCT4, SOX2, KLF4, and MYC (OKSM). hiPSCs were differentiated into hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. Antiseptic cardiomyocytes were advised with TKIs and advised for alterations in corpuscle viability, contractility, cellular signaling, and gene expression. (B) Confocal diminutive immunofluorescence images of differentiated hiPSC-CMs cogent the sarcomeric markers cardiac troponin T (TNNT2) and α-actinin (ACTN2). DAPI, 4′,6-diamidino-2-phenylindole. (C) Day 30 hiPSC-CMs from bristles advantageous ascendancy ambit cogent above RTKs including INSR, IGF1R, PDGFRA, and KDR. Aerial TNNT2 and low PECAM1 announcement indicates a authentic hiPSC-CM citizenry bare of ECs. n = 3 biological replicates conducted for gene announcement appraisal in anniversary hiPSC-CM line. Abstracts are agency ± SEM. RPKM, reads per kilobase per million.

Twenty-one small-molecule TKIs were acclimated for a high-throughput cardiotoxicity awning in hiPSC-CMs (table S1). Abounding TKIs arrest assorted RTK families and abet cardiotoxicities including larboard ventricular dysfunction, myocardial infarction, and arrhythmias. However, the net account with account to blight appraisal outweighs these risks, and these drugs are frequently assigned at above blight appraisal centers (table S2). We included the awful cardiotoxic anthracycline doxorubicin as a absolute ascendancy for toxicity. Appliance the PrestoBlue corpuscle activity assay, we begin that the VEGFR2/PDGFR-inhibiting TKIs sorafenib, regorafenib, and ponatinib induced the best corpuscle afterlife in hiPSC-CMs, with boilerplate baleful dosage (LD50) ethics of 3.4, 7.1, and 4.3 μM, appropriately (Fig. 2A). Doxorubicin was acutely cytotoxic to hiPSC-CMs at an LD50 of 0.78 μM. TKIs not acerb associated with cytotoxicity, such as imatinib or erlotinib, had LD50 ethics of 78.20 and 87.60 μM, respectively. Sorafenib, regorafenib, and ponatinib were awful cytotoxic in all 11 advantageous ascendancy hiPSC-CM lines, as abstinent with quantitative and qualitative activity assays (figs. S2, A to C, and S3). We additionally performed cytotoxicity assays in hiPSC-CMs and hiPSC-ECs acquired from two individuals with branch blight (fig. S4). These individuals accustomed two TKIs each: sunitinib as first-line appraisal and axitinib as second-line treatment. These patients accomplished no cogent analytic cardiotoxicity from either agent. As expected, we did not beam a cogent aberration in cytotoxicity amid TKI-receiving accommodating hiPSC-CMs and advantageous ascendancy hiPSC-CM ambit afterwards subjecting them to sunitinib or axitinib.

(A) Dose-response curves quantifying cytotoxicity afterwards a 72-hour TKI appraisal of bristles advantageous ascendancy hiPSC-CM ambit appliance a PrestoBlue activity assay. n = 5 biological replicates conducted per line. Abstracts are agency ± SEM. (B) Evaluation of hiPSC-CM contractility afterwards a 72-hour TKI appraisal with the IC200 Kinetic Imaging Cytometer. Boilerplate after-effects from leash wells apparent at anniversary concentration. Red indicates decreased abbreviating rate, admitting blooming indicates added abbreviating rate. (C) Ethics aggregate from cytotoxicity and contractility analyses in hiPSC-CMs. Blooming concealment indicates ethics associated with beneath cardiotoxicity. Red concealment indicates ethics associated with college cardiotoxicity. Abeyance of assault is the absorption at which >50% of leash wells accomplished beating. Able absorption is the absorption at which a cogent about-face in all listed contractility ambit was detected (see fig. S7 and Abstracts and Methods for details). Amplitude of aftereffect is the amount to which all listed contractility ambit were adapted at the able absorption (see Abstracts and Methods for details). LD50 is the TKI absorption at which a 50% accident in activity is empiric from activity assays, averaged beyond accommodating hiPSC-CM lines. Accommodating Cmax represents the best TKI claret claret absorption accomplished by patients appear in FDA literature. The cardiac assurance basis is a amount from 0 to 1 that normalizes contractility and activity ambit to accommodating Cmax and combines these ambit to accommodate a about metric for TKI cardiotoxicity. Highlighted drugs (surrounded by a red rectangle) accept a assurance basis at or beneath 0.10, our beginning for awful cardiotoxic compounds. Clinically appear cardiotoxicities are alterations in accommodating cardiac activity (see table S1). QT, QT breach prolongation; Hy, hypertension; LV, larboard ventricular casting atom decrease; HF, affection failure; MI, myocardial infarction; TdP, Torsades de pointes; SCD, abrupt cardiac death; Brady, bradycardia; PE, pericardial effusion; Vas, vascular abnormalities; Afib, atrial fibrillation; **cardiovascular toxicity–associated boxed warning; #noncardiovascular toxicity–associated boxed warning.

To abstain lab-to-lab biases and variations in hiPSC-CM quality, we performed contractility appraisal in CMs acquired from commercially available, advantageous ascendancy hiPSCs. We empiric alterations in hiPSC-CM assault amount and added ambit at doses lower than the LD50 cytotoxicity ethics afterwards appraisal with assorted TKIs such as nilotinib and vandetanib, suggesting that aberrant assault arises afore cardiomyocyte afterlife (Fig. 2B and fig. S5). We additionally bent hiPSC-CM contractility ambit in acknowledgment to accretion TKI concentrations, able biologic concentrations at which contractility alterations initially appeared, and TKI concentrations at which hiPSC-CM abbreviating accomplished (Fig. 2C and figs. S6 and S7). To accurately appraise TKI toxicity, we advised whether baneful TKI concentrations empiric in cytotoxicity and contractility assays akin doses accomplished by patients. We acquired accommodating Cmax ethics from FDA literature, accouterment an appraisal of best TKI claret claret concentrations in patients (Fig. 2C). By normalizing our in vitro abstracts on abeyance of beating, able concentration, and LD50 cytotoxicity ethics to literature-reported Cmax values, we developed a cardiac assurance index, a metric that identifies clinically cardiotoxic TKIs (see Abstracts and Methods, Fig. 2C, and fig. S7 for details).

Three of seven compounds with cardiac assurance indices at or beneath 0.10 (doxorubicin, nilotinib, and vandetanib) were ahead labeled with FDA atramentous box cardiotoxicity warnings. A assurance basis amount of 0.10 was called as our beginning for awful cardiotoxic drugs because it apparent a break in the assurance basis amid clinically cardiotoxic, atramentous boxed drugs (doxorubicin, nilotinib, and vandetanib) and added compounds not frequently associated with cardiotoxicity. Nilotinib and vandetanib, which account QT breach assiduity and arrhythmias, were called for added analysis. Three of the TKIs with assurance indices beneath 0.10 were VEGFR2/PDGFR-inhibiting TKIs (regorafenib, sorafenib, and vandetanib). Regorafenib and sorafenib had assurance indices commensurable to that of the cardiotoxic anthracycline doxorubicin. Thus, VEGFR2/PDGFR-inhibiting TKIs induced cardiotoxicities in hiPSC-CMs at clinically accordant concentrations commensurable to the doses that patients experience. In patients, VEGFR2/PDGFR-inhibiting TKIs account assorted toxicities including hypertension, affection failure, and QT breach assiduity (15).

QT breach assiduity charcoal a above affair during biologic development (16). Because nilotinib and vandetanib account alarming QT breach assiduity and arrhythmias clinically, we conducted added contractility, calcium imaging, and electrophysiological analyses in four advantageous ascendancy hiPSC-CM ambit advised with nilotinib or vandetanib (fig. S8). We called dimethyl sulfoxide (DMSO) and axitinib, which are not associated with contractility abnormalities at clinically accordant doses assigned to patients (per analytic abstract and our antecedent data), as abrogating controls for toxicity. We empiric a assiduity in cardiomyocyte abbreviating time afterwards nilotinib or vandetanib appraisal at clinically accordant concentrations as low as 3.7 μM (fig. S8). Neither DMSO nor axitinib elicited abbreviating irregularities at clinically accordant concentrations.

We additionally conducted calcium imaging of hiPSC-CMs afterwards a 2-hour nilotinib or vandetanib appraisal (Fig. 3A). At clinically accordant concentrations (Fig. 2C and table S1), nilotinib and vandetanib abiding calcium brief continuance and decreased exhausted amount in hiPSC-CMs (Fig. 3, B and C). The electrophysiologically “safe” drugs (DMSO control, imatinib, and axitinib) did not decidedly adapt calcium brief amplitude, brief duration, or assault amount (Fig. 3C); nilotinib and vandetanib adapted cardiomyocyte electrophysiology (Fig. 4). We subjected hiPSC-CMs to an astute TKI appraisal up to 10 min or to a best appraisal for 2 hours and recorded cellular electrophysiology with appliance anchor (Fig. 4A). With astute treatment, TKIs at clinically accordant concentrations did not adapt activity abeyant (AP) continuance (Fig. 4B). However, afterwards 2 hours, the QT interval–prolonging TKIs nilotinib and vandetanib decidedly abiding AP continuance and decreased cellular assault amount (Fig. 4, C and D). Drugs not accepted to adapt cardiac electrophysiology, such as DMSO, imatinib, and axitinib, did not adapt AP duration. These after-effects authenticate that adverse arrhythmogenic furnishings of accepted QT interval–prolonging TKIs such as nilotinib and vandetanib can be recapitulated in hiPSC-CMs, as adjourned by contractility assays, calcium imaging, and appliance catch electrophysiology.

(A) Schematic illustrating TKI appraisal dieting for hiPSC-CMs afore calcium imaging. (B) Raw band scans of alone hiPSC-CM calcium transients afterwards TKI appraisal at adumbrated clinically accordant concentrations and calcium dye appraisal over assorted beats. (C) Altitude of hiPSC-CM calcium imaging ambit afterwards a 2-hour TKI treatment. n = 10 beef recorded for anniversary condition. Abstracts are presented as box-and-whisker plots assuming the minimum, aboriginal quartile, median, mean, third quartile, and best of the abstracts set. Student’s t appraisal indicates acceptation compared to ascendancy (*P < 0.05 and **P < 0.01).

(A) Schematic illustrating bureaucracy for astute and 2-hour TKI appraisal afore AP recording. (B) Representative AP tracings afterwards astute TKI appraisal for up to 10 min at clinically accordant concentrations in hiPSC-CMs. (C) Representative AP tracings afterwards a 2-hour TKI appraisal at clinically accordant concentrations in hiPSC-CMs. (D) Altitude of hiPSC-CM electrophysiological ambit afterwards a 2-hour TKI treatment. Abstracts are agency ± SEM. *P < 0.05, compared to DMSO, Student’s t test. n = 10 beef recorded for anniversary condition.

We abutting acquired endothelial beef (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) from hiPSCs to actuate corpuscle type–specific differences in cardiotoxicity. hiPSC-ECs were produced with a chemically authentic adverse agreement appliance small-molecule Wnt signaling modulators, fibroblast advance agency (FGF) and VEGF stimulation, and magnetic-activated corpuscle allocation of CD31 /CD144 populations (fig. S9A). These hiPSC-ECs apparent accepted EC morphologies, markers, and functionality (fig. S9, B to D). As in hiPSC-CMs, sorafenib, regorafenib, and ponatinib were the best cytotoxic TKIs in hiPSC-ECs (fig. S9E). We additionally developed a custom hiPSC-CF adverse agreement appliance small-molecule Wnt signaling modulation, FGF2 and VEGFA stimulation, and abrogating allocation for ECs (fig. S10A). These hiPSC-CFs were abrogating for cardiomyocyte markers, bidding mesenchymal and myofibroblast markers, and were morphologically agnate to primary cardiac fibroblasts (fig. S10, B to E). These hiPSC-CFs apparent TKI cytotoxicity profiles agnate to those of hiPSC-CMs and hiPSC-ECs, with sorafenib, regorafenib, and ponatinib eliciting the accomplished cytotoxicities (fig. S10F). We abutting advised akin hiPSCs with our TKI console to actuate whether noncardiovascular corpuscle types display toxicities agnate to hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs (fig. S11). hiPSCs apparent a adapted TKI cytotoxicity profile, assuming college toxicity from VEGFR2/PDGFR bifold inhibitors than did cardiovascular derivatives (hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs). For example, axitinib, the atomic cytotoxic TKI in hiPSC-CMs, was acutely baneful to hiPSCs. Doxorubicin was additionally essentially added baneful in hiPSCs than in cardiovascular corpuscle types. These after-effects advance that the VEGFR2/PDGFR-inhibiting TKIs sorafenib, regorafenib, and ponatinib display corpuscle type–specific cytotoxicities that adapt amid cardiovascular and noncardiovascular corpuscle types.

To annotate TKI-induced signaling alterations, we acclimated an RTK proteome profiler to appraise RTK phosphorylation afterwards appraisal with VEGFR2/PDGFR-inhibiting TKIs (Fig. 5 and fig. S12). Drugs were added to hiPSC-CMs at subcytotoxic concentrations. We empiric a dose-dependent abatement in VEGFR2 and PDGFRα phosphorylation afterwards VEGFR2/PDGFR-inhibiting TKI treatment, with axitinib eliciting the arch bifold inhibition, suggesting that these TKIs can arrest functionally accordant signaling pathways in hiPSC-CMs. ErbB2, ErbB4, and epidermal advance agency receptor 2 (EGFR2) phosphorylation remained connected over accretion TKI concentrations. Cabozantinib, a accepted Axl inhibitor, decreased Axl phosphorylation. Notably, we empiric added INSR and IGF1R phosphorylation afterwards appraisal with ponatinib and axitinib, suggesting a compensatory accretion in insulin/IGF signaling during VEGFR2/PDGFR inhibition.

Normalized altitude of RTK phosphorylation in antiseptic hiPSC-CMs advised with 0 to 1 μM of the VEGFR2/PDGFR-inhibiting TKIs sorafenib, cabozantinib, ponatinib, axitinib, regorafenib, or sunitinib for 72 hours. Phosphorylation arrangement blots are apparent in fig. S12. n = 3 biological replicates conducted. Abstracts are agency ± SEM.

Treatment with insulin or IGF1 can enhance cardiac activity during adverse contest (17, 18). Given that insulin/IGF signaling was up-regulated afterwards appraisal with VEGFR2/PDGFR-inhibiting TKIs, we accepted that this compensatory up-regulation protects hiPSC-CMs from TKI toxicity. To actuate whether exogenous insulin or IGF1 could enhance cardioprotective signaling in hiPSC-CMs, we acclimated a high-throughput kinase phosphorylation arrangement (Fig. 6A and fig. S13). Both IGF1 and insulin added phosphorylation of the antiapoptotic Akt protein network. Corpuscle adaptation was added back hiPSC-CMs apparent to ponatinib were accordingly advised with IGF1 or insulin (Fig. 6B). This ascertainment was accepted quantitatively with CellTiter-Glo activity assays (Fig. 6C). Additionally, we empiric that IGF1 and insulin appraisal rescued hiPSC-CMs from doxorubicin cytotoxicity (fig. S14). To affirm that the aftereffect of insulin/IGF1 was a aftereffect of added cardiomyocyte adaptation rather than proliferation, we adjourned the corpuscle cardinal at aboriginal time credibility afterwards TKI treatment. We empiric an access in activity alone 12 hours afterwards TKI treatment, acknowledging that insulin and IGF augment cardiomyocyte adaptation (fig. S15). We abutting evaluated the gene announcement acknowledgment in hiPSC-CMs during appraisal with the VEGFR2/PDGFR-inhibiting TKIs sorafenib, regorafenib, and ponatinib and empiric an access in advance agency receptor gene announcement (Fig. 7). NRP2, encoding for the noncanonical VEGFR neuropilin 2, was up-regulated in our microarray. We afterwards conducted RNA sequencing (RNA-seq) appraisal of bristles advantageous ascendancy hiPSC-CM ambit advised with 1 μM VEGFR2/PDGFR-inhibiting TKI sorafenib for 72 hours and empiric added announcement of KDR, encoding for the VEGFR2 receptor, and VEGFC, encoding for the VEGFC ligand (fig. S16). These gene announcement analyses advance that noncanonical VEGF-binding receptors and VEGF signaling alleyway associates are up-regulated to atone for accident approved VEGFR signaling afterwards appraisal with VEGFR2/PDGFR-inhibiting TKIs. A arbitrary of the compensatory cardioprotective signaling archetypal is apparent in Fig. 8. Together, our abstracts advance that VEGFR2/PDGFR-inhibiting TKIs arm-twist a compensatory access in cardioprotective insulin/IGF1 signaling (phosphorylation) in hiPSC-CMs. This cardioprotective signaling can be acclimatized with exogenous insulin/IGF1 ligands to enhance cardiomyocyte survival. Simultaneously, VEGFR2/PDGFR-inhibiting TKIs access the afterwards gene announcement of VEGF alleyway associates to atone for an upstream accident in VEGF arresting transduction.

(A) Phosphorylation arrays demonstrating alterations in hiPSC-CM kinase activity afterwards a 12-hour IGF1 or insulin treatment. n = 3 biological carbon phosphorylation arrays conducted. Abstracts are agency ± SEM. *P < 0.05, Student’s t test. We empiric a cogent access in phosphorylation of the afterwards protein amino acerbic residues afterwards IGF1 appraisal (P ethics 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 empiric a cogent access in phosphorylation of the afterwards protein amino acerbic residues afterwards insulin appraisal (P ethics 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 advised with sorafenib, regorafenib, or ponatinib at accretion concentrations for 72 hours in the attendance of IGF1 or insulin. Calcein-AM stains applicable cells. (C) CellTiter-Glo altitude of hiPSC-CM activity with or afterwards insulin/IGF1 cotreatment during TKI treatment. n = 5 biological replicates conducted. IGF and insulin appraisal decidedly rescued ponatinib toxicity (P = 0.004). Abstracts are agency ± SEM.

(A) Microarray calefaction map illustrating differentially bidding genes afterwards a 72-hour sorafenib, regorafenib, and ponatinib appraisal in hiPSC-CMs. Beef were advised with 1 μM TKI to abstain cytotoxicity at college doses. Red indicates aerial gene expression, and dejected indicates low gene expression. (B) Graph represents bend announcement change (compared to control) of decidedly adapted genes afterwards biologic treatment. Decidedly adapted genes authentic by P < 0.05 compared to basic control. Assorted P-value comparisons bogus appliance one-way between-subject appraisal of about-face (ANOVA).

(A) In hiPSC-CMs, the RTKs VEGFR2, PDGFRα, INSR, and IGF1R are upstream of prosurvival signaling pathways. (B) Our after-effects advance that VEGFR2/PDGFR-inhibiting TKIs up-regulate INSR and IGF1R signaling (phosphorylation) to atone for the accident of VEGFR/PDGFR signaling (phosphorylation). This compensatory aftereffect can augment cardiomyocyte adaptation during TKI appraisal via addition of exogenous insulin and IGF1 ligands. We empiric an access in the afterwards gene announcement of noncanonical VEGF-binding receptors and VEGFR alleyway members, apparently to atone for VEGFR2/PDGFR-inhibiting TKI-induced accident in upstream VEGFR signaling (phosphorylation).

TKIs are a above chic of blight therapeutics, with revenues from these drugs annually extensive billions of dollars (19). However, abounding TKIs, like added chemotherapeutics, display abundant cardiotoxicities (3). Our after-effects authenticate that hiPSC-CMs can appraise TKI cardiotoxicity in a high-throughput fashion. We evaluated 21 FDA-approved TKIs appliance hiPSC-CMs acquired from 11 advantageous individuals and 2 patients accepting TKIs as blight therapy. From the abstracts obtained, we developed a cardiac assurance basis amalgam TKI-induced cytotoxicity measurements, contractility assessments, and literature-reported TKI claret claret concentrations in patients. We additionally accurate the abrogating furnishings of accepted cardiotoxic TKIs.

Previous studies evaluated TKI cardiotoxicity appliance animals and added in vitro models (20). Sorafenib, one of the three best cytotoxic TKIs in our study, induces cardiomyocyte afterlife and contractility defects in the zebrafish affection and causes ventricular dysfunction and affection abortion clinically (table S2) (20). Ponatinib, aloof briefly for adverse vascular events, was appear to abet mitochondrial accent and arm-twist abbreviating abnormalities in hiPSC-CMs, acceptance our after-effects (21, 22). Although regorafenib has a atramentous box admonishing for alarmist toxicity, our abstraction demonstrates that regorafenib can abet cardiotoxicity at clinically accordant doses agnate to added VEGFR2/PDGFR-inhibiting TKIs (23). This is reasonable because sorafenib and regorafenib (also accepted as fluoro-sorafenib) accept agnate atomic structures (24).

The seven drugs with cardiac assurance indices at or beneath 0.10, our beginning for awful cardiotoxic compounds, account analytic cardiotoxicities including affection failure, ventricular dysfunction, myocardial infarction, and arrhythmias (table S2). Six of these seven drugs are broadly assigned at above blight appraisal centers at greater than 10,000 doses annually (table S2). Three of the seven compounds with assurance indices at or beneath 0.10 (doxorubicin, vandetanib, and nilotinib) accept cardiotoxicity-associated FDA atramentous box warnings, lending authority to our cardiac assurance index. We additionally accepted the analytic arrhythmia-inducing abeyant of nilotinib and vandetanib by contractility assay, appliance catch electrophysiology, and calcium imaging in hiPSC-CMs.

Three of the four TKIs with the everyman assurance indices (sorafenib, regorafenib, and vandetanib) were VEGFR2/PDGFR-inhibiting TKIs, abrupt our absorption in added belief this chic of drugs. Clinically, these TKIs account cardiovascular toxicities including hypertension, affection failure, and QT assiduity (15). The VEGFR2/PDGFR-inhibiting TKIs sorafenib, regorafenib, and ponatinib induced the accomplished levels of cytotoxicity in hiPSC-ECs, hiPSC-CFs, and hiPSC-CMs alike. The aerial hiPSC-EC toxicity we empiric with VEGFR2/PDGFR-inhibiting TKIs such as ponatinib is constant with the vascular toxicity empiric clinically with these TKIs (1). The cytotoxicity contour for akin hiPSCs was essentially adapted from that of hiPSC-derived cardiovascular derivatives. For example, the VEGFR2/PDGFR inhibitor axitinib was the atomic cytotoxic TKI in hiPSC-CMs. However, it was acutely baneful to hiPSCs, conceivably because hiPSCs are awful acute to alterations to RTK signaling due to their pluripotent state. Doxorubicin was baleful to hiPSCs akin at 0.1 μM, acceptable because it is a awful able DNA intercalating abettor for killing hyperproliferative corpuscle types such as blight beef and hiPSCs. The hiPSC-CMs additionally bidding the above RTK kinase families targeted by TKIs in our console (INSR, IGF1R, PDGFRα, and VEGFR2), lending authority to our model. However, we did not beam a subject-specific aberration in TKI-induced cardiotoxicity amid the 11 hiPSC-CM ambit bogus from advantageous individuals and the 2 bogus from TKI-treated patients. This was accepted because neither of the individuals accepting TKI appraisal developed astringent analytic cardiotoxicity from TKI treatment, as would accept been apparent if any of the capacity apparent a abiogenetic predisposition to TKI-induced cardiotoxicity that could be recapitulated in vitro.

The VEGF and PDGFR signaling pathways are analytical regulators of cardiovascular development. VEGF regulates EC activity and promotes cardiomyocyte adaptation (25, 26). Although nanomolar concentrations of sorafenib, regorafenib, and ponatinib acquired complete VEGFR2/PDGFR inhibition, cytotoxicity was empiric at micromolar concentrations, suggesting that VEGFR2/PDGFR inhibition may not anon account hiPSC-CM cytotoxicity. However, VEGFR2/PDGFR inhibition may accommodate a accessory account to hiPSC-CMs. VEGFR2/PDGFR-inhibiting TKIs such as ponatinib and axitinib led to compensatory hyperactivation of cardioprotective insulin/IGF1 signaling. Axitinib, the arch inhibitor of VEGFR2 and PDGFRα phosphorylation per our kinase assays, induced the arch compensatory aftereffect forth with ponatinib, acceptable INSR/IGF1R phosphorylation twofold. This TKI-induced compensatory aftereffect is accidental because insulin and IGF1 are cardioprotective during adverse cardiac contest (17, 18). Although VEGFR2/PDGFR-inhibiting TKIs may account cardiomyocyte cytotoxicity, they may “prime” hiPSC-CMs for dispatch with prosurvival factors.

We acclimatized this compensatory aftereffect to enhance hiPSC-CM adaptation during VEGFR2/PDGFR-inhibiting TKI appraisal with either insulin or IGF1, assiduity antiapoptotic Akt signaling. We empiric a cardioprotective aftereffect with insulin and IGF1 during ponatinib appraisal but not with sorafenib or regorafenib. This may be because, per our RTK phosphorylation arrays, ponatinib elicited the arch compensatory access in INSR/IGF1R phosphorylation amid these three TKIs. We additionally empiric bargain doxorubicin cytotoxicity afterwards IGF1 and insulin pretreatment, suggesting that these advance factors ability allay anthracycline cardiotoxicity in cardiomyocytes, acceptance added studies (27). Phosphorylation of RTKs such as ErbB2, ErbB4, EGFR2, and Axl was not decidedly adapted by VEGFR2/PDGFR-inhibiting TKIs, except by the accepted Axl inhibitor cabozantinib. Because insulin and IGF1 additionally abet corpuscle aeon activity, we advised whether they enhance hiPSC-CM admeasurement instead of adaptation (28). To appraisal for a prosurvival effect, we essentially beneath the TKI appraisal but still empiric an access in hiPSC-CM activity afterwards insulin/IGF1 pretreatment. To added aegis adjoin abashing furnishings of insulin/IGF1-induced proliferation, we acclimated day 30 postdifferentiation hiPSC-CMs, which display lower mitotic activity than do adolescent beef (28). Finally, we conducted gene announcement appraisal to annotate gene networks adapted by TKI treatment. The VEGFR2/PDGFR-inhibiting TKIs sorafenib, regorafenib, and ponatinib induced a bifold access in the announcement of neuropilin 2, a noncanonical VEGFR (29). Thus, hiPSC-CMs may atone for VEGFR2 signaling accident by up-regulating abetting and approved VEGFR expression, appropriately facilitating VEGF signaling in the absence of VEGFR2 kinase activity. RNA-seq appraisal appear an up-regulation of KDR (VEGFR2 receptor) and an up-regulation in the announcement of VEGF ligands. This suggests that in hiPSC-CMs, the upstream barricade of the phosphorylation avalanche by VEGFR2/PDGFR-inhibiting TKIs causes a afterwards access in the gene announcement of VEGF alleyway members, potentially to atone for the accident of upstream VEGFR signaling (Fig. 8).

In summary, we acclimated hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs to awning for the cardiotoxicity of 21 FDA-approved TKIs and accustomed a cardiac assurance basis for TKI toxicity. We additionally accurate the accepted cardiotoxicities of FDA atramentous boxed TKIs in our hiPSC-CM platform. Although TKIs ambition assorted RTK families, we empiric that VEGFR2/PDGFR-inhibiting TKIs apparent aerial cardiotoxicity on assorted cardiovascular corpuscle types. These cardiotoxicities could be rescued by co-opting compensatory prosurvival insulin/IGF1 signaling pathways. There is abundant absorption in anticipation hiPSCs from a broader accomplice of individuals to authorize a population-level, prospective, preclinical toxicity appraisal to bigger acquaint biologic development. As presented here, a aggregate of electrophysiological, functional, and abiogenetic assays and connected advances in hiPSC assay will accredit the development of able high-throughput platforms for preemptively screening abeyant chemotherapeutic compounds for cardiotoxicities.

To investigate the cardiotoxicity of FDA-approved TKIs in hiPSC-CMs, 11 advantageous individuals and 2 individuals accepting TKIs for blight assay were recruited and consented. Either bark bite biopsies or claret draws were performed to access primary tissue samples for hiPSC production. Reprogramming of either bark fibroblasts or borderline claret mononuclear beef to hiPSCs was conducted according to ahead appear protocols (8, 30). A minimum of n = 3 biological replicates were conducted for anniversary experiment, with capacity in the amount legends.

The hiPSCs were differentiated into hiPSC-CMs appliance a chemically authentic agreement and maintained in boilerplate supplemented with beastly albumin and ascorbic acerbic (8). This cardiomyocyte aliment boilerplate was bare of advance factors such as insulin and IGF1. Differentiated beef were glucose-starved and supplemented with 5 mM sodium dl-lactate to metabolically baddest hiPSC-CMs (31). Back replating, hiPSC-CMs were dissociated with TrypLE Accurate (Life Technologies) and reseeded on Matrigel-coated plates.

TKI stocks (LC Laboratories) were resuspended in 10 mM DMSO and stored at −80°C. Insulin (Life Technologies) was stored at −20°C, and Long-R3 IGF1 (Sigma-Aldrich) was stored at 4°C. For corpuscle activity accomplishment experiments, day 30 to 35 postdifferentiation hiPSC-CMs were pretreated with insulin or IGF1 for 12 hours afore TKI treatment, back insulin or IGF1 was resupplemented.

Day 30 to 35 postdifferentiation hiPSC-CMs were argent on Matrigel at 25,000 beef per able-bodied of a 384-well bowl (Greiner Bio-One). Beef were advised with TKIs at 0 to 100 μM for 72 hours unless contrarily specified. Immunostaining qualitatively adjourned corpuscle activity per antecedent protocols (8). For quantitative activity measurements, beef were advised with CellTiter-Glo Activity Appraisal (Promega), CCK8 (Dojindo), or PrestoBlue reagent (Life Technologies) per manufacturer-recommended procedures. High-throughput imaging and activity assays were conducted appliance a Cytation 5 bowl reader/imager (BioTek Instruments). Prism (GraphPad) was acclimated for ambit fitting, LD50 calculations, and statistical analysis.

After a 72-hour TKI treatment, hiPSC-CMs were done with Tyrode’s solution. Imaging dye was able by diluting Hoechst 33258 (H3569, Activity Technologies) to 4 μg/ml and aureate antibiotic agglutinin–Alexa Fluor 488 conjugate (W11261, Activity Technologies) to 5 μg/ml in Tyrode’s solution. Band-aid was added to hiPSC-CMs and incubated at 37°C and 5% CO2 for 15 min. Afterwards rewashing with Tyrode’s solution, hiPSC-CMs were incubated for 15 min afore imaging. The IC200 Kinetic Imaging Cytometer (Vala Sciences) recorded a 6.5-s time alternation of appliance hiPSC-CMs at 100 Hz at 20× deepening per able-bodied of a 384-well plate.

Safety coefficients presented in this cardboard are computed as follows. Abeyance of assault is the absorption at which >50% of leash wells accomplished beating. Able absorption for the ith metric (ECi) is a absorption that presents a statistically cogent contractility aberration (P < 0.05) from baseline. Amplitude of the aftereffect (AEi) quantifies the consequence of such departures from baseline as the log2 of the arrangement of the metric amount EVi at absorption ECi, hereinafter referred to as able amount (EVi), to the amount of the metric BVi at baseline concentration. Additionally, i = 1…M, area M is the cardinal of metrics acquired from the contractility appraisal (M = 9 in this paper: Tpeak, Trise, Tfall, absolute abbreviating time, Dpeak, Dvalley, Dp2v, abbreviating rate, and alleviation rate).

Average amplitude of aftereffect (AE) is acquired by averaging the adapted AEi’s.

Average able absorption (EC) is a abounding boilerplate of the adapted able concentrations appliance anniversary agnate amplitude of aftereffect as weight.

The account for this EC announcement is that it considers all adapted ECs and introduces a bent against the ECs agnate to metrics that best acutely adapt accustomed corpuscle behavior.

Day 30 to 40 postdifferentiation hiPSC-CMs were reseeded in Matrigel-coated eight-well Lab-Tek II accommodation (Nalge Nunc International) and were advised with TKIs for 2 hours. Beef were advised with 5 μM Fluo-4 AM and 0.02% Pluronic F-127 (Molecular Probes) in Tyrode’s band-aid for 15 min at 37°C and done with Tyrode’s band-aid afterward. Ca2 imaging was conducted appliance a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss AG) and analyzed appliance Zen software. Ad-lib Ca2 transients were acquired at 37°C appliance a single-cell band browse mode.

Whole-cell APs were recorded with appliance catch technique, as ahead declared (8). Cultured hiPSC-CMs were dissociated appliance TrypLE and argent as distinct beef on bottle coverslips coated with Matrigel. Beef were placed in an RC-26C recording alcove (Warner) and army assimilate an astern microscope (Nikon). The alcove was continuously perfused with balmy (35° to 37°C) extracellular band-aid of the afterwards composition: 150 mM NaC1, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 1.0 mM Na pyruvate, 15 mM Hepes, and 15 mM glucose; pH was adapted to 7.4 with NaOH. Bottle micropipettes (2- to 3-megohm tip resistance) were bogus from accepted bank borosilicate bottle capillary tubes (Sutter BF 100-50-10) and abounding with the afterwards intracellular solution: 120 mM KCl, 1.0 mM MgCl2, 10 mM Hepes, 10 mM EGTA, and 3 mM Mg-ATP; pH was adapted to 7.2 with KOH. Single-beating cardiomyocytes were selected, and APs were recorded in whole-cell accepted catch approach appliance an EPC 10 appliance catch amplifier (HEKA). External band-aid absolute 0.1% DMSO (vehicle) was activated to authorize the baseline. Then, beef were advised with TKI band-aid absolute imatinib, axitinib, nilotinib, or vandetanib (LC Laboratories). Abstracts were acquired appliance PATCHMASTER software (HEKA), digitized at 1.0 kHz, and analyzed appliance FITMASTER (HEKA), Igor Pro (WaveMetrics), and Prism 5 (GraphPad). For recordings on differentiated ventricular-like hiPSC-CMs, the best diastolic abeyant of distinct cardiomyocytes assorted from −70 to −50 mV, activity abeyant amplitude (APA) was greater than 90 mV, and activity abeyant continuance (APD)90/APD50 was beneath than 1.20. Only cardiomyocytes acceptable the above belief were ventricular-like cardiomyocytes and called for assessing the furnishings of TKIs. Baseline APs were recorded for 3 min afore appliance of the biologic and at 3, 5, and 10 min while befitting a connected perfusion with the drug. In a abstracted alternation of experiments, the drugs were added for 2 hours at 37°C afore patching. Boilerplate responses of n = 10 APs were analyzed per treatment. Cogent APD90 assiduity is authentic as >10% change in APD90.

Phosphorylation of beastly RTKs and added phosphoproteins was bent appliance Beastly Phospho-RTK Arrangement or Beastly Phospho-Kinase Antibiotic Arrangement (R&D Systems). Day 30 to 35 postdifferentiation hiPSC-CMs were advised with TKIs for 72 hours and lysed. Lysate was incubated brief on an RTK or phosphokinase console and afterwards with an anti–phosphotyrosine–horseradish peroxidase antibiotic to appraise phosphorylation. Blots were developed appliance Gel Doc XR (Bio-Rad). Phosphorylation acuteness was bent appliance ImageJ software.

RTK announcement in hiPSC-CMs was bent appliance Ion AmpliSeq (Life Technologies). RNA was extracted appliance the RNeasy Micro Kit (Qiagen). Complementary DNA libraries were actinic appliance the Ion AmpliSeq Transcriptome Beastly Gene Announcement Kit. Libraries were added to Ion PI chips and loaded assimilate an Ion Chef apparatus for arrangement preparation. Transcriptome sequencing was conducted on an Ion Proton sequencing arrangement (Life Technologies). For announcement appraisal of hiPSC-CMs afterwards TKI treatment, a GeneChip Beastly Gene 1.0 ST DNA microarray was acclimated (Affymetrix).

Data are presented as agency ± SEM unless contrarily specified. Comparisons are conducted via Student’s t test, unless contrarily specified, with cogent differences authentic by *P < 0.05 or **P < 0.01. For microarray experiments, assorted P-value comparisons were bogus appliance a one-way between-subject ANOVA (P < 0.05) and Affymetrix Transcriptome Appraisal Console 2.0 software.

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

Materials and Methods

Fig. S1. hiPSCs display appropriate morphologies and markers of pluripotent axis cells.

Fig. S2. Quantitative and qualitative corpuscle activity assays allegorize sorafenib, regorafenib, and ponatinib cytotoxicity in hiPSC-CMs.

Fig. S3. Quantitative corpuscle activity assays on added hiPSC-CM ambit authenticate VEGFR2/PDGFR-inhibiting TKI toxicity.

Fig. S4. Quantitative corpuscle activity assays in hiPSC-CMs and hiPSC-ECs acquired from patients accepting TKI treatment.

Fig. S5. Commercially available, advantageous ascendancy hiPSC-CMs display alterations in cellular contractility afterwards a 72-hour TKI treatment.

Fig. S6. Calefaction maps of high-throughput contractility appraisal on commercially available, advantageous ascendancy hiPSC-CMs advised with TKIs.

Fig. S7. Extended calculations for TKI assurance basis afterwards a 72-hour TKI appraisal on commercially available, advantageous ascendancy hiPSC-CMs.

Fig. S8. hiPSC-CMs display alterations in cellular contractility afterwards a 72-hour appraisal with accepted QT interval–prolonging TKIs.

Fig. S9. hiPSC-ECs display EC characteristics and authenticate cytotoxicity in acknowledgment to TKI treatment.

Fig. S10. hiPSC-CFs display backdrop of developed cardiac fibroblasts and authenticate cytotoxicity in acknowledgment to TKI treatment.

Fig. S11. hiPSCs authenticate a TKI cytotoxicity contour that is adapted from those of hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs.

Fig. S12. VEGFR2/PDGFR-inhibiting TKI appraisal in hiPSC-CMs after-effects in activation of compensatory insulin/IGF1 signaling.

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

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

Fig. S15. IGF1 and insulin appraisal rescues ponatinib toxicity at aboriginal time credibility in hiPSC-CMs.

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

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

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

Movie S1. hiPSC-CMs afore ablution via glucose deprivation.

References (32–50)

Acknowledgments: We accede K. Red-Horse for abetment with confocal imaging. We additionally accede E. Yeh from the MD Anderson Blight Center for advice apropos TKI acceptance and cardiotoxicity prevalence. We accede the Stanford High-Throughput Bioscience Center for abetment with high-throughput imaging and bowl clairvoyant assays. We accede A. Olson from the Stanford Neuroscience Microscopy Service for advice with calcium imaging. Funding: We accede abutment from the American Affection Association Predoctoral Fellowship (13PRE15770000) and NSF Graduate Research Fellowship (DGE-114747) (A.S.); NIH (K99/R00 HL121177) and American Affection Association Beginning Grant-in-Aid (14BGIA20480329) (P.W.B.); NIH Director’s Pioneer Award, American Affection Association Grant-in-Aid, and Endowed Faculty Scholar Award of the Lucile Packard Foundation for Children and Child Health Research Institute at Stanford (S.M.W.); and Burroughs Wellcome Foundation Innovation in Regulatory Science, American Affection Association Accustomed Investigator Award, and NIH (R01 HL132875, R01 HL130020, R01 HL128170, R01 HL123968, and R24 HL117756) (J.C.W.). Author contributions: A.S., P.W.B., and J.C.W. advised the abstraction and alternate in abstracts appraisal and arrangement writing. A.S. and P.W.B. alternate in all beginning work. W.L.M. and R.S. conducted high-throughput contractility assessments. P.S. performed appliance catch electrophysiology. N.S. assisted with anticipation hiPSC-CMs and hiPSC-ECs from patients accepting TKI treatment. J.M.C. performed microarray and RNA-seq analysis. T.K. assisted with high-throughput toxicity analysis. H.W. conducted calcium imaging. A.H. conducted hiPSC-EC adverse and characterization. E.M. assisted with gene announcement analysis. Y.Z. performed immunocytochemistry and edited the manuscript. A.K. performed immunocytochemistry and edited the manuscript. A.C.F. assisted with accommodating recruitment. J.C.d.A., S.M.W., J.J.M., M.M., and J.C.W. assisted with abstraction architecture and arrangement editing. Aggressive interests: M.M. holds disinterestedness in and is on the accurate advising lath for Vala Sciences, a aggregation alms high-content screening services, decidedly chart acclimated for barometer the electrical and contractile appraisal of cardiomyocytes, and is on the accurate advising lath of Axis Corpuscle Theranostics, a aggregation that uses patient-specific hiPSC-CMs for biologic discovery. J.C.W. is a cofounder and is on the accurate advising lath of Axis Corpuscle Theranostics. Added authors acknowledge that they accept no aggressive interests. Abstracts and abstracts availability: The gene announcement abstracts are begin at Gene Announcement Omnibus with accretion no. GSE8894 and GSE89411.

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