Dominique R. Talbert

Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development.

Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types.

Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks. Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability.

A multi-parameter in vitro screen in human stem cell-derived cardiomyocytes identifies ponatinib-induced structural and functional cardiac toxicity.

Ponatinib, a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph + ALL and CML, was temporarily withdrawn from the U.S. market due to severe vascular adverse events. Cardiac-specific toxicities including myocardial infarction, severe congestive heart failure, and cardiac arrhythmias have also been shown with ponatinib. Targeted oncology agents such as ponatinib have transformed cancer treatment but often induce toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells. These toxicities are often missed by the standard preclinical toxicity assessment methods, which include human Ether-à-go-go-related gene (hERG) and animal toxicity testing. In this study, we show that a multiparameter in vitro toxicity screening approach using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) accurately predicted the cardiac toxicity potential of ponatinib. This in vitro model evaluated ponatinibs effect on the overall cell health, mitochondrial stress, and function of hiPSC-CM and also provided mechanistic insight into the signaling pathways and cellular structures altered with treatment. We show here that ponatinib rapidly inhibits prosurvival signaling pathways, induces structural cardiac toxicity (as shown by actin cytoskeleton damage, mitochondrial stress, cell death, and troponin secretion), and disrupts cardiac cell beating. Most of these effects occurred at doses between 10× and 50× ponatinibs Cmax, a dose range shown to be relevant for accurate prediction of in vivo toxicity. Together these studies show that a comprehensive in vitro screening tool in a more relevant human cardiac cell model can improve the detection of cardiac toxicity with targeted oncology agents such as ponatinib.

Abstract 2611: A multi-parameter in vitro screen demonstrates increased cardiac toxicity with pan-HER inhibitors afatinib and neratinib when used in combination with chemotherapy

The human epidermal growth factor receptor 2 (HER2) oncogene is amplified and overexpressed in 25 to 30% of breast cancers and is associated with poor prognosis. Currently, two HER2-targeting agents have been FDA-approved for the treatment of these breast cancers: trastuzumab and lapatinib. Despite their proven clinical benefit, acquired resistance and reports of cardiotoxicity have limited the utility of both drugs which has led to the development of pan-HER inhibitors such as afatinib and neratinib. However, little is known about the cardiac safety profile of pan-HER inhibitors when used alone or in combination with chemotherapeutic agents as they are often utilized in the clinic. To better understand the effect of these compounds on cardiac cells, we utilized a multi-parameter in vitro toxicity screen in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This in vitro model evaluated each drug9s effect, both alone and in combination with chemotherapy, on the overall health, metabolic stress, and function of hiPSC-CM. Our results showed that although afatinib and neratinib were relatively cardiac safe at doses near their Cmax (∼150nM), higher doses of both drugs (>30X Cmax) induced a range of damaging effects on cardiac health and function. Interestingly however, combination treatment with lower doses of afatinib and neratinib followed by chemotherapeutic stress led to significant cardiotoxicity including alterations in cardiac cell beating, increased lipid accumulation (indicative of mitochondrial damage), and decreased cell viability. These results suggest that although afatinib and neratinib are relatively cardiac safe when used alone at lower doses, combination regimens with these drugs and chemotherapeutic agents may increase cardiac toxicity risk. These effects are likely mediated by the inhibition of survival pathways in cardiomyocytes by pan-HER inhibitors which exacerbates chemotherapy-mediated damage. Similar effects have been shown previously for the HER2-targeted antibody trastuzumab when used in combination with anthracyclines. Together this study shows the utility of a multi-parameter in vitro screen using a more relevant hiPSC-CM model to detect potential cardiac risk for both single drug and novel combination treatments. Since many targeted therapies are given with other oncology treatments, comprehensive safety screening for combination therapies is warranted to illuminate any potential synergies on cardiotoxicity. Citation Format: Dominique R. Talbert, Kimberly Doherty, Patricia Trusk, Diarmuid Moran, Sarah Bacus. A multi-parameter in vitro screen demonstrates increased cardiac toxicity with pan-HER inhibitors afatinib and neratinib when used in combination with chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2611. doi:10.1158/1538-7445.AM2015-2611

Abstract 3674: Targeted oncology therapeutics show unique cardiotoxic profiles: Ponatinib as a case study

Although tyrosine kinase inhibitors (TKI) have greatly improved the treatment and prognosis of multiple cancer types, unexpected clinical cardiotoxicity has occurred with some of these agents that was not predicted by standard preclinical toxicity assessment methods. Since cardiotoxicity with TKI are often caused by the inhibition of signaling pathways that are critical to cardiac cell function, multi-targeted TKI that block multiple pathways increase the likelihood for cardiac cell damage. We recently demonstrated that an in vitro multi-parameter test panel assessing the effect of drug treatment on overall cardiac health and function could accurately predict the cardiotoxicity of four FDA-approved TKI. The multi-targeted TKI that are clinically associated with cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while a more targeted and relatively cardiac-safe drug, erlotinib, showed only minor changes in cardiac cell health. The present studies expand upon this initial observation using a broad panel of drugs such as TKI (including the recently withdrawn TKI ponatinib), chemotherapeutic agents, and non-oncology compounds, which were grouped based on their known clinical toxicity profiles ranging from cardiac safe to withdrawn. We assessed each compound9s cardiotoxic potential by examining their effect on cell viability, reactive oxygen species (ROS) generation, lipid formation, troponin secretion and beating activity in a human induced pluripotent-derived cardiac stem (iPS) cell model. The results showed a unique cardiotoxic signature of oncology therapeutics that was significantly different from other drug classes. Oncology compounds, including ponatinib, induced metabolic stress (ROS generation and lipid formation) resulting in potent cardiac cell death and troponin secretion while other classes of drugs disrupted cardiac cell beating with minimal impact on cardiac cell health. In comparison, cardiac-safe drugs, including non-oncology compounds and more targeted TKI, did not impact any of the endpoints that assessed cardiac cell health and function. Our results demonstrate unique toxicity profiles for different drug classes which may reflect multiple mechanisms of cardiotoxicity. For oncology compounds specifically, multi-targeted TKI and chemotherapeutic agents show potent cardiac toxicity while more targeted agents are less cardiac toxic. These studies show that a multi-parameter approach provides a comprehensive and robust assessment of cardiotoxic potential by evaluating several parameters that are important to cardiac cell health and function in a more predictive adult cardiac cell model. A panel such as this aids in early risk assessment of cardiotoxic compounds and may reduce drug failure due to toxicity discovered in later phases of development or after drug approval such as that shown with ponatinib. Citation Format: Dominique Talbert, Kimberly Doherty, Patricia Trusk, Diarmuid Moran, Scott Shell, Sarah Bacus. Targeted oncology therapeutics show unique cardiotoxic profiles: Ponatinib as a case study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3674. doi:10.1158/1538-7445.AM2014-3674

Abstract C280: Oncology therapeutics show unique cardiotoxic profiles in human cardiac cells.

Tyrosine Kinase Inhibitors (TKI) have greatly improved the treatment and prognosis of multiple cancer types. However unexpected clinical cardiotoxicity has been shown with some of these agents that was not wholly predicted by current preclinical toxicity assessment methods. Since cardiotoxicity with TKI are often caused by the inhibition of signaling pathways critical to cardiac cell function, multi-targeted TKI that block multiple pathways increases the likelihood for cardiac cell damage. We recently demonstrated that an in vitro multi-parameter test panel assessing the effect of drug treatment on overall cardiac health and function could accurately predict the cardiotoxicity of 4 FDA-approved TKI. The 3 multi-targeted TKI clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the more targeted and relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. The present studies expand upon this initial observation to include other TKI, chemotherapeutic agents, and non-oncology compounds which were grouped based on their known clinical toxicity profiles ranging from cardiac safe to withdrawn. We then assessed the cardiotoxic potential of these compounds by examining their effect on cell viability, reactive oxygen species (ROS) generation, lipid formation, troponin secretion and beating activity in a human induced pluripotent-derived cardiac stem (iPS) cell model. The results showed a unique cardiotoxic signature of oncology therapeutics that was significantly different from other classes of drugs such as anti-arrhythmics. Oncology compounds induced potent cell death and lipid formation (metabolic stress) in iPS cells while anti-arrhythmics more potently disrupted cardiac cell beating with minimal impact on cell viability. In addition, troponin secretion was directly correlated with cardiac cell death for all of the oncology agents. In comparison, the cardiac-safe drugs, including non-oncology compounds and more targeted TKI, did not negatively affect any of the endpoints that assessed cardiac cell health and function. Thus different classes of drugs appear to show unique toxicity profiles that may reflect multiple mechanisms which lead to cardiotoxicity. Specifically our data suggest that more promiscuous TKI which inhibit the function of multiple oncogenic targets may be affecting pathways which are critical for cardiac cell health while more targeted agents are less cardiac toxic. These studies show that a multi-parameter approach can provide a robust characterization of drug-induced cardiac cell toxicity that can be leveraged to improve drug safety during early phase development. Furthermore, these studies emphasize the importance of developing more targeted oncology agents which are effective at killing cancer cells but lack toxic effects on cardiac cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C280. Citation Format: Dominique Talbert, Kimberly Doherty, Diarmuid Moran, Robert Wappell, Patricia Trusk, Scott Shell, Sarah Bacus. Oncology therapeutics show unique cardiotoxic profiles in human cardiac cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C280.

Abstract 4423: Implications for cardiotoxicity with targeted therapies.

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen that was not wholly predicted by current FDA mandated pre-clinical tests. Thus a more robust tool to predict the cardiotoxic potential of these compounds is warranted. For this purpose, we developed a panel of in vitro tests in a clinically relevant human cardiomyocyte model that assesses drug impact on overall cardiac health (cell viability, apoptosis, and morphology), mitochondria and metabolic intactness (reactive oxygen species (ROS) generation, AMPK activity, and lipid accumulation), and electrophysiological function (impedance (beat rate) and ion channel blockage). To demonstrate the utility of our panel, we examined the effect of 4 FDA-approved TKi with known clinical outcome on cardiac health. The 3 drugs with known cardiac adverse events (crizotinib, sunitinib, and nilotinib) all proved to be cardiotoxic by our panel although each showed distinct mechanisms of toxicity. However erlotinib, a cardiac-safe drug that targets EGFR and is used primarily in non-small cell lung cancer (NSCLC), did not show any indications of toxicity. Surprisingly, the most cardiotoxic drug by our panel was crizotinib, an ALK/ MET inhibitor that has revolutionized treatment for ALK+ NSCLC patients. Crizotinib potently impaired overall cardiac health by increasing ROS and apoptosis but also impeded electrophysiological function by inducing ion channel blockage and reducing cardiac beat rate. Interestingly crizotinib also induced lipid and cholesterol accumulation which was shown to be correlated with increased expression of the sterol-regulatory binding protein (SREBP) pathway by transcriptome analysis. The multi-targeted TKi sunitinib (used in renal cell carcinoma and gastrointestinal stromal tumors) and nilotinib (used in chronic myelogenous leukemia) also showed unique cardiotoxicity profiles. In conclusion, our studies showed a distinct cardiotoxicity profile for each TKi that correlates with clinical outcome. These results suggest that caution should be taken with these TKi especially when used long-term or in the adjuvant setting. Furthermore, these effects may be exacerbated in patients with adverse cardiac genetic predispositions and studies are ongoing in our lab on patient-derived cardiac cell lines to investigate this possibility. This multi-parameter screening approach allows for a more complete assessment of the potential for drug-induced cardiotoxicity and may allow for earlier detection in the drug development process. In addition, understanding the mechanisms of cardiac toxicities may lead to the discovery of novel mitigation strategies and combination therapies to improve cardiac cell health during treatment with oncology therapies. Citation Format: Kimberly R. Doherty, Robert L. Wappel, Dominique R. Talbert, Patricia B. Trusk, Scott A. Shell, Diarmuid M. Moran, James W. Kramer, Arthur Brown, Sarah Bacus. Implications for cardiotoxicity with targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4423. doi:10.1158/1538-7445.AM2013-4423

Abstract 3101: The Role of MYC and the miR-17∼92 cluster in histone deacetylase inhibitor-induced apoptosis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In recent years histone deacetylase inhibitors (HDACis) have emerged as promising therapeutics for cancer. While favorable responses to HDACis as single agents have been shown in several hematological malignancies, very little efficacy has been demonstrated in solid tumors. c-Myc (Myc), an oncoprotein commonly over-expressed in cancer, has been shown by several studies to play a critical role in HDACi-mediated cellular death. To expand upon these findings and determine the role that Myc plays in this process in solid tumors, we compared the effect of two HDAC inhibitors, SAHA and LAQ824, on the proliferation of solid tumor cell lines expressing high versus low levels of Myc. We found that cells expressing high levels of Myc were more sensitive to HDACi. In addition, there were significant differences in the type of response to HDACi treatment between the two cell types with prominent apoptosis in cells expressing higher levels of Myc while cell cycle arrest was more commonly observed in cells expressing lower levels of Myc. Interestingly, HDACi reduced the expression of Myc and one of its well-known oncogenic miRNA targets, miR-17∼92 cluster, resulting in an increase in the expression of the master pro-apoptotic protein Bim. We propose that this novel mechanism may play a role in the potent anti-proliferative effects mediated by HDACi. Furthermore, these studies suggest that Myc expression could be used as a predictive biomarker to select patients with solid tumors who may be more responsive to HDACi treatment. Citation Format: Dominique R. Talbert, Robert L. Wappel, Diarmuid M. Moran, Scott A. Shell, Sarah S. Bacus. The Role of MYC and the miR-17∼92 cluster in histone deacetylase inhibitor-induced apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2013-3101 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1146: Multiple HDAC inhibitors (HDACi) reduce the expression of the oncogenic miR-17∼92 cluster; a pan HDACi class effect

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL miR-17∼92 is an oncogenic polycistronic cluster encoding six mature miRNAs (MIR-17, MIR-20A, MIR-18A, MIR-19A, MIR-19B, and MIR-92A). This oncogenic cluster is driven by MYC activity and is highly-expressed in many tumor types where it has been shown to promote tumorigenesis through the suppression of oncogene-induced senescence. We recently observed reduced expression of the miR-17∼92 miRNA cluster in a miRNA microarray analysis of MCF-7 and MDA-MB-231 breast cancer cells treated with the pan-HDAC inhibitors (HDACis) Vorinostat, MS-275, and LAQ824. Treatment with all three HDACis, at concentrations shown to induce the expression of p21 and acetylated histone-H3, demonstrated this effect which was further validated by qRT-PCR. Furthermore, significant cell cycle arrest and induction of apoptosis and/or senescence were shown with treatment of both MCF-7 and MDA-MB-231 cells with the HDACis. There was also significant reduction of MYC expression upon treatment with HDACis which is consistent with studies showing that the miR-17∼92 cluster is driven by MYC. The miR-17∼92 cluster has also been shown to suppress the expression of several cellular targets important in tumor suppression including p21, PTEN, E2F1, and BIM. The expression of these proteins will also be assessed in MCF-7 and MDA-MB-231 cells after treatment with HDACi. The modulation of MYC, the miR-17∼92 cluster and its oncogenic targets will also be confirmed in MDA-MB-231 and MCF-7 tumor xenograft mice treated with the HDACis. Lastly, antagomirs will be used to inhibit the expression of the miR-17∼92 cluster in MCF-7 and MDA-MB-231 cells in order to compare the functional (apoptosis, senescence, and cell cycle arrest) and cellular (repression of target proteins) effects elicited by HDACi treatment. Together, we have shown that pan-HDAC inhibitors inhibit the expression of MYC and the oncogenic miR-17-92 cluster. This finding could have important utility in the clinical development of pan-HDACis by identifying MYC and/or the miR-17-92 cluster as potential biomarkers to select patients more likely to respond to pan-HDACi treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1146. doi:10.1158/1538-7445.AM2011-1146