Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer therapy and is showing promise for other non-cancer disease indications. CAR T cells are directed to a target antigen that is overexpressed by tumor cells. On-target toxicity can arise if the targeted protein is also expressed in normal cell populations. Additionally, off-target toxicity may occur if the CAR T cell recognizes or cross-reacts with a non-target protein thereby activating the T cells leading to subsequent adverse sequela. Given the nature of this biological class as a human cellular product, standard safety assessments in animals are largely not appropriate as the drug product is derived from human cells and the associated CAR often lacks reactivity with the animal homolog of the target protein. Undesired targeting of healthy/normal tissue that express the intended target antigen (on-target/off-tumor), as well as unintended targeting of other antigens expressed on healthy/normal tissue is a safety concern that may be explored using human induced pluripotent stem cell (hiPSC)-derived test systems as surrogates for various normal cell types. The panel of hiPSC-derived cells is intended to broadly represent different human cell types from vital organs that can be targets for severe adverse events. Herein the development of an orthogonal approach to an in vitro co-culture assay used to assess unintended CAR T cell cytotoxicity in normal cells is described. Experimental considerations including assay and cell model qualification are presented, and an orthogonal workflow described. Finally, an illustrative case of experimental CD33 CAR T cells co-cultured with a select panel of hiPSC-derived normal cells serves as a springboard for other CAR T cell developers to consider in their nonclinical safety programs.

Technological advances and the desire to reduce dependence on animal models have brought human-relevant models to the forefront of drug development. This paradigm shift is leveraging the advances in in vitro systems and new approach methodologies (NAMs), which was the focus of a workshop convened by the Health and Environmental Sciences Institute (HESI) in May 2024. Highlights included discussions on predicting cardiac failure modes and the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), microfluidic systems like BioFlux™, and engineered heart tissues in enhancing early-stage drug safety assessments. Regulatory perspectives underscored the challenges and potential for integrating NAMs into submissions, advocating for standardized reporting and validation protocols. Case studies where NAMs offered superior predictivity compared to traditional methods are emerging and offer insights into a roadmap forward. However, there remains a need for collaboration among academia, industry, and regulatory bodies to ensure robust validation and adoption. These efforts aim to refine cardiovascular drug discovery, reduce attrition rates, and accelerate the transition toward more ethical and efficient preclinical testing paradigms.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization focused on resolving global health challenges through collaborative science. HESI achieves this by engaging scientists from around the world in the public, including health regulatory authorities, and private sectors and coalescing around a specific topic. The HESI Cardiac Safety Committee (CSC) officially formed in 2008 to improve public health by reducing unanticipated cardiovascular-related adverse effects from pharmaceuticals or chemicals. Since the publication by Pierson et al. (2013) outlining achievements, the HESI CSC has continued to champion cardiac safety, having a major impact on the field of cardiovascular safety assessments. This has been attained through numerous prospective studies, retrospective analyses, workshops, symposia and more than 24 peer review publications. These publications detail results of the work over the past decade and while reporting results is important, the lasting impacts on the field of CV safety show the real value of collaborative science. Examples of this work include details of the Comprehensive In vitro Proarrhythmia Assay (CiPA) initiative and subsequent updated ICH E14/S7B Q&As, as well as the related stem cell validation studies and best practices. Similarly, challenges and opportunities in both manual and automated patch clamp studies for proarrhythmic prediction and use in an in silico model. A better understanding of assay sensitivity and specificity can lead to better interpretation and implementation. HESI studies on in vivo and in vitro structure and functional assessments have contributed in these areas. Earlier detection of potential failure modes allow for better drug development and design. HESI team are investigating promising new biomarkers to detect hemostatic changes earlier in the process. Answering challenging questions as a collaborative team, sharing data for the benefit of the larger scientific community, and helping young scientists advance their careers are just a few of the outcomes of the last 10 years of the CSC. Future challenges ahead and the direction of the CSC will be offered for input.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization dedicated to resolving global health challenges through collaborative scientific efforts across academia, regulatory authorities and the private sector. Collaborative science across non-clinical disciplines offers an important keystone to accelerate the development of safer and more effective medicines. HESI works to address complex challenges by leveraging diverse subject-matter expertise across sectors offering access to resources, data and shared knowledge. In 2008, the HESI Cardiac Safety Committee (CSC) was established to improve public health by reducing unanticipated cardiovascular (CV)-related adverse effects from pharmaceuticals or chemicals. The committee continues to significantly impact the field of CV safety by bringing together experts from across sectors to address challenges of detecting and predicting adverse cardiac outcomes. Committee members have collaborated on the organization, management and publication of prospective studies, retrospective analyses, workshops, and symposia resulting in 38 peer reviewed manuscripts. Without this collaboration these manuscripts would not have been published. Through their work, the CSC is actively addressing challenges and opportunities in detecting potential cardiac failure modes using in vivo, in vitro and in silico models, with the aim of facilitating drug development and improving study design. By examining past successes and future prospects of the CSC, this manuscript sheds light on how the consortium's multifaceted approach not only addresses current challenges in detecting potential cardiac failure modes but also paves the way for enhanced drug development and study design methodologies. Further, exploring future opportunities and challenges will focus on improving the translational predictability of nonclinical evaluations and reducing reliance on animal research in CV safety assessments.

Table S1. List of all 94 Oncolead cell lines tested with corresponding IC50 values for NCI-737 and NCI-006.

Combined Supplementary Data file containing: Figure S1. Proliferation assays using NCI-737 and NCI-006 in an expanded panel of EWS cell lines. Figure S2. Effects of additional siRNA sequences against LDHA and LDHB in an expanded panel of EWS cell lines. Figure S3. Effects of additional siRNA sequences against EWS-FLI1 on LDHA and LDHB and additional ChIP-seq data. Figure S4. LDH expression, basal activity and in vitro early time course inhibition in EWS cell lines. Figure S5. Steady state levels of pyruvate and lactate and 13C-tracing experiments from 13C-U-glucose. Figure S6. Effects of NCI-737 and NCI-006 on ECAR measurements. Figure S7. Effects of NCI-737 on OCR measurements. Figure S8. Effects of NCI-737 on ECAR measurements of expanded panel of EWS cell lines. Figure S9. Results of oral dosing of LDH inhibitors in EWS xenograft models. Figure S10. Results of IV pilot studies of LDH inhibitors in EWS xenograft models. Figure S11. Additional growth rate and pharmacokinetic data for EWS xenograft models. Figure S12. Additional growth rate and necrosis data for EWS xenograft models. Figure S13. Depiction of of changes in lactate and pyruvate peaks and lactate/pyruvate ratio by hyperpolarized 13C-MR spectroscopy in EWS xenograft models. Figure S14. Measurements of toxicity in animals treated with NCI-737.

A description of additional supplemental methods

Chemotherapy-induced peripheral neuropathy (CIPN) is widely recognized as a potentially severe toxicity that often leads to dose reduction or discontinuation of cancer treatment. Symptoms may persist despite discontinuation of chemotherapy and quality of life can be severely compromised. The clinical symptoms of CIPN, and the cellular and molecular targets involved in CIPN, are just as diverse as the wide variety of anticancer agents that cause peripheral neurotoxicity. There is an urgent need for extensive molecular and functional investigations aimed at understanding the mechanisms of CIPN. Furthermore, a reliable human cell culture system that recapitulates the diversity of neuronal modalities found in vivo and the pathophysiological changes that underlie CIPN would serve to advance the understanding of the pathogenesis of CIPN. The demonstration of experimental reproducibility in a human peripheral neuronal cell system will increase confidence that such an in vitro model is clinically useful, ultimately resulting in deeper exploration for the prevention and treatment of CIPN. Herein, we review current in vitro models with a focus on key characteristics and attributes desirable for an ideal human cell culture model relevant for CIPN investigations.

Abstract Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. To uncover cell type-specific dependencies to LDH, we screened a diverse panel of 94 cancer cell lines for responsiveness to two novel LDH A/B inhibitors developed through the NCI Experimental Therapeutics Program (NExT). We found that Ewing sarcoma (EWS) cell lines were exquisitely sensitive, with IC50 values approximately ten-fold below the median IC50 of the panel. To understand the mechanism behind this sensitivity, we genetically knocked down LDHA and LDHB using siRNA, and discovered that EWS cell lines were sensitive to loss of LDHA only, which inhibited proliferation and induced apoptosis. Notably, treatment of EWS cells with the LDH inhibitors phenocopied these effects. Additionally, genetic knockdown of EWS-FLI1, the oncogenic driver of EWS, resulted in loss of LDHA, but not LDHB. Analysis of publicly available ChIP-seq data generated using shFLI1-transfected EWS cells revealed that LDHA, but not LDHB, is directly regulated by EWS-FLI1. Functional mechanistic studies of glycolytic intermediates and cellular bioenergetics in EWS cells treated with the LDH inhibitors demonstrated that loss of viability was due to impairment of glycolysis, which occurred both in vitro and in vivo, and perturbation of the NAD+/NADH ratio. The translational potential of these compounds was next evaluated using in vivo analyses of pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Intravenous administration of the LDH inhibitors resulted in diminished LDH activity, reduction of the lactate-to-pyruvate ratio, tumor cell necrosis, and a decrease in tumor growth rate in aggressive xenograft models of EWS. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, our data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as EWS that exhibit oncogene-dependent expression of LDH and increased glycolytic activity. This abstract is also being presented as Poster B33. Citation Format: Choh Yeung, Anna E. Gibson, Sameer H. Issaq, Nobu Oshima, Marielle E. Yohe, Haiyan Lei, Ganesha Rai, Daniel J. Urban, Michelle S. Johnson, Gloria A. Benevides, Giuseppe L. Squadrito, Sandy Eldridge, John Hamre III, Arnulfo Mendoza, Jack F. Shern, Lee J. Helman, Murali C. Krishna, Matthew D. Hall, Victor M. Darley-Usmar, Leonard M. Neckers, Christine M. Heske. Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR08.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human- and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell-derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell-derived cardiomyocytes.

To achieve therapeutic goals, many cancer chemotherapeutics are used at doses close to their maximally tolerated doses. Thus, it may be expected that when therapies are combined at therapeutic doses, toxicity profiles may change. In many ways, prediction of synergistic toxicities for drug combinations is similar to predicting synergistic efficacy, and is dependent upon building hypotheses from molecular mechanisms of drug toxicity. The key objective of this initiative was to generate and make publicly available key high-content data sets for mechanistic hypothesis generation as it pertains to a unique toxicity profile of a drug pair for several anticancer drug combinations. The expectation is that tissue-based genomic information that are derived from target tissues will also facilitate the generation and testing of mechanistic hypotheses. The view is that availability of these data sets for bioinformaticians and other scientists will contribute to analysis of these data and evaluation of the approach.

Drug-induced effects on cardiac contractility can be assessed through the measurement of the maximal rate of pressure increase in the left ventricle (LVdP/dtmax) in conscious animals, and such studies are often conducted at the late stage of preclinical drug development. Detection of such effects earlier in drug research using simpler, in vitro test systems would be a valuable addition to our strategies for identifying the best possible drug development candidates. Thus, testing platforms with reasonably high throughput, and affordable costs would be helpful for early screening purposes. There may also be utility for testing platforms that provide mechanistic information about how a given drug affects cardiac contractility. Finally, there could be in vitro testing platforms that could ultimately contribute to the regulatory safety package of a new drug. The characteristics needed for a successful cell or tissue-based testing platform for cardiac contractility will be dictated by its intended use. In this article, general considerations are presented with the intent of guiding the development of new testing platforms that will find utility in drug research and development. In the following article (part 2), specific aspects of using human-induced stem cell-derived cardiomyocytes for this purpose are addressed.

Chemotherapy-induced peripheral neuropathy (CIPN) is an adverse effect caused by several classes of widely used anticancer therapeutics. Chemotherapy-induced peripheral neuropathy frequently leads to dose reduction or discontinuation of chemotherapy regimens, and CIPN symptoms can persist long after completion of chemotherapy and severely diminish the quality of life of patients. Differences in the clinical presentation of CIPN by widely diverse classifications of anticancer agents have spawned multiple mechanistic hypotheses that seek to explain the pathogenesis of CIPN. Despite its clinical relevance, common occurrence, and extensive investigation, the pathophysiology of CIPN remains unclear. Furthermore, there is no unequivocal gold standard for the prevention and treatment of CIPN. Herein, we review in vivo and in vitro models of CIPN with a focus on histopathological changes and morphological features aimed at understanding the pathophysiology of CIPN and identify gaps requiring deeper exploration. An elucidation of the underlying mechanisms of CIPN is imperative to identify potential targets and approaches for prevention and treatment.

The search for new chemical entities which are clinically effective and do not adversely affect the cardiovascular system is an ongoing objective. In vivo studies designed to detect potential drug-induced cardiovascular toxicity typically utilize both rodent and non-rodent species. An important component of such studies includes the microscopic evaluation of tissues for histopathologic changes. A factor which could potentially complicate this type of evaluation relates to the potential for laboratory animals to develop natural or spontaneous pathological cardiovascular lesions. Some types of these naturally occurring alterations are similar to those induced by chemical compounds and thus could confound accurate interpretation. Accurate morphologic analysis becomes contingent upon the ability to distinguish spontaneous cardiovascular changes from actual drug-induced lesions. A summary of some of the more frequently reported spontaneous cardiovascular alterations in commonly-used laboratory animals is presented below. Special emphasis is given to the spectrum of spontaneous background myocardial pathology that might be encountered during preclinical studies conducted to identify potential cardiotoxic actions of anticancer agents.

Abstract Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. Significance: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human-and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell-derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell-derived cardiomyocytes.

A comprehensive profiling of cardiotoxicity early in drug discovery and development can aid in reducing late-stage attrition and establishing risk-mitigation strategies during clinical development. In most cases, multiple assay platforms and instrument-specified plate formats are required for this type of approach. In this study, we evaluated both functional and structural endpoints associated with cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a single 384-well plate. We measured intracellular Ca2+ transit, caspase 3/7 activation and plasma membrane permeabilization sequentially in the same plate via a series of assay readouts. A set of cardiac ion channel modulators (dofetilide, sotalol, nifedipine and mexiletine) and chemotherapeutics (tamoxifen, nilotinib, sunitinib and doxorubicin) was tested at clinically relevant concentrations for effects on intracellular Ca 2+ transits after a short-term (30 minutes) exposure, and plasma membrane permeabilization and caspase 3/7 activation after a long-term (72 hours) exposure. Intracellular Ca2+ transits were monitored by fluorescent images taken with a high-speed camera in beating cardiomyocytes loaded with Cal520® Ca2+ dye, permeabilized plasma membrane (for dead-cell detection) was identified with live-stain DRAQ7TM nuclear dye and activation of caspase 3/7 was determined biochemically with the Caspase-Glo® 3/7 Assay kit. Multiple endpoints derived from Ca 2+ transits, including beat rate, calcium transit duration (CTD) measured at 30% or 90% from peak and corrected by inter-peak interval (IPI), along with CTD triangulation, beat rhythm, short- or long-term variability of CTD90 and IPI Poincare plots, were used to assess drug effects on intracellular Ca2+ cycling and arrhythmogenicity. Increases in positive nuclear staining for DRAQ7TM and caspase 3/7 activity represented structural cardiotoxicity. We found that increased CTD triangulation, development of arrhythmic events and both the short- and long-term variability of CTD90 or IPI were robust indicators of functional effects. Positive nuclear staining for DRAQ7TM was a robust indicator of structural effects. Accordingly, dofetilide and sotalol were identified as primarily arrhythmogenic, doxorubicin was primarily structurally toxic, while nilotinib and sunitinib were both arrhythmogenic and structurally toxic. The use of these endpoints in a single plate format simplifies the cardiotoxicity assessment and does not require multiple cell plates for measurements.

Targeting the anti-apoptotic protein Mcl-1 holds a promise to improve therapy of multiple types of Mcl-1 dependent cancers but raises concerns of on-target cardiotoxicity due to the presence and reported role of Mcl-1 in heart. Herein, we investigated the importance of Mcl-1 in the survival and contractile function of human pluripotent stem cell-derived cardiomyocytes in culture. Effective knockdown of Mcl-1 with siRNAs reproducibly resulted in early (measured at Day 3) marginal alterations in caspase 3/7 activity, LDH leakage, ATP content and cellular impedance. After 14 days of Mcl-1 knockdown, loss of mitochondrial membrane potential, deteriorating effects on mitochondrial ultrastructure, and alterations in beat rate and amplitude were revealed. Inhibition of Bcl-xL by siRNA-mediated knockdown or selective inhibitors did not cause any overt cellular responses except for a minimal increase in caspase 3/7 activity; however, loss of Mcl-1 concomitant with down-regulated Bcl-xL activated apoptosis and caused extensive cell death as reflected by an 80% loss in cell index, activation of caspase-3 with associated PARP cleavage, and a decrease in beat amplitude and mitochondrial membrane potential after 3 days of Mcl-1/Bcl-xL knockdown., Together, these findings suggest that Mcl-1 and Bcl-xL provide duplicate safeguard measures in maintaining structural and functional integrity of cardiomyocytes. Hence, BH3-mimetic drugs targeting Mcl-1 may be well tolerated in the presence of intact Bcl-xL.