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a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph + ALL and CML, was temporarily withdrawn from the U.S. market due to severe vascular adverse events.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 ponatinib's 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× ponatinib's 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.

KRAS mutation status is associated with enhanced dependency on folate metabolism pathways in non-small cell lung cancer cells.

KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non-small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism- and purine synthesis-related pathways in KRAS-mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS-mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other antifolates in KRAS-mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS-mutant cells by antifolates, which may also contribute to higher efficacy of antifolates in KRAS-mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS-mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher antifolate activity in this subtype of NSCLC.

Endoglin-mediated suppression of prostate cancer invasion is regulated by activin and bone morphogenetic protein type II receptors.

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor ß (TGFß) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFß receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFß receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

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.

Nutlin-3a induces cytoskeletal rearrangement and inhibits the migration and invasion capacity of p53 wild-type cancer cells.

MDM2 is an E3 ubiquitin ligase that binds and ubiquitinates the tumor suppressor protein p53, leading to its proteasomal degradation. Nutlin-3a (Nutlin) is a preclinical drug that binds MDM2 and prevents the interaction between MDM2 and p53, leading to p53 stabilization and activation of p53 signaling events. Previous studies have reported that Nutlin promotes growth arrest and/or apoptosis in cancer cells that express wild-type p53. In the current study, Nutlin treatment caused a cytoskeletal rearrangement in p53 wild-type human cancer cells from multiple etiologies. Specifically, Nutlin decreased actin stress fibers and reduced the size and number of focal adhesions in treated cells. This process was dependent on p53 expression but was independent of p21 expression and growth arrest. Consistent with this, Nutlin-treated cells failed to form filamentous actin-based motility structures (lamellipodia) and displayed significantly decreased directional persistence in response to migratory cues. Finally, chemotactic assays showed a p53-dependent/p21-independent decrease in migratory and invasive capacity of Nutlin-treated cells. Taken together, these findings reveal that Nutlin treatment can inhibit the migration and invasion capacity of p53 wild-type cells, adding to the potential therapeutic benefit of Nutlin and other small molecule MDM2 inhibitors. Mol Cancer Ther; 9(4); 895-905. (c)2010 AACR.

Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes.

BACKGROUND: Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation. RESULTS: In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells. CONCLUSION: Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.

Transient nutlin-3a treatment promotes endoreduplication and the generation of therapy-resistant tetraploid cells.

p53 Activity is controlled in large part by MDM2, an E3 ubiquitin ligase that binds p53 and promotes its degradation. The MDM2 antagonist Nutlin-3a stabilizes p53 by blocking its interaction with MDM2. Several studies have supported the potential use of Nutlin-3a in cancer therapy. Two different p53 wild-type cancer cell lines (U2OS and HCT116) treated with Nutlin-3a for 24 hours accumulated 2N and 4N DNA content, suggestive of G(1) and G(2) phase cell cycle arrest. This coincided with increased p53 and p21 expression, hypophosphorylation of pRb, and depletion of Cyclin B1, Cyclin A, and CDC2. Upon removal of Nutlin-3a, 4N cells entered S phase and re-replicated their DNA without an intervening mitotic division, a process known as endoreduplication. p53-p21 pathway activation was required for the depletion of Cyclin B1, Cyclin A, and CDC2 in Nutlin-3a-treated cells and for endoreduplication after Nutlin-3a removal. Stable tetraploid clones could be isolated from Nutlin-3a treated cells, and these tetraploid clones were more resistant to ionizing radiation and cisplatin-induced apoptosis than diploid counterparts. These data indicate that transient Nutlin-3a treatment of p53 wild-type cancer cells can promote endoreduplication and the generation of therapy-resistant tetraploid cells. These findings have important implications regarding the use of Nutlin-3a in cancer therapy

Molecular pathogenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common life-threatening malignancies in the world. This cancer generally arises within the boundaries of well-defined causal factors, of which viral hepatitis infection, aflatoxin exposure, chronic alcohol abuse, and nonalcoholic steatohepatitis are the major risk factors. Despite the identification of these etiological agents, hepatocarcinogenesis remains poorly understood. The molecular mechanisms leading to the development of HCC appear extremely complex and only recently have begun to be elucidated. Currently, surgical resection or liver transplantation offer the best chance of cure for the patient with HCC; however, these therapies are hindered by inability of many of these patients to undergo liver resection, by tumor recurrence and by donor shortages. A lack of suitable therapeutic strategies has led to a greater focus on prevention of HCC using antiviral agents and vaccination. Overall, the current outlook for patients with HCC is bleak; however, a better understanding of the molecular and genetic basis of this cancer should lead to the development of more efficacious therapies.

Microencapsulation of engineered cells to deliver sustained high circulating levels of interleukin-6 to study hepatocellular carcinoma progression.

Interlukin-6 (IL-6) is a pleitropic cytokine that plays a central role in normal and abnormal hepatic function and response. The aims of the current study were to determine the viability of using cell encapsulation technology to introduce a genetically modified xenogeneic (CHO) cell population to elevate circulating levels of rhIL-6 in a rat model and determine the effects of sustained high rhIL-6 levels on hepatocellular carcinoma (HCC) progression in vivo. An alginate matrix was combined with transfected CHO cells, selected for their ability to synthesize rhIL-6, and used to generate uniform alginate-cell beads. Once encapsulated transfected cells continued to undergo replication, formed colonies within the bead, and synthesized/released large quantities of rhIL-6 into culture medium in vitro. Intraperitoneal implantation of beads into rats resulted in significantly increased circulating and intrahepatic levels of rhIL-6 up to 4 days postimplantation. Prolonged implantation led to the escape of CHO cells from the bead, resulting in a host response and CHO cell death within the bead. Subsequently CHO-IL-6 encapsulated cells were implanted into rats previously inoculated intrahepatically with the H4IIE HCC cell line. These studies demonstrated the maintenance of high circulating/intrahepatic rhIL-6 levels in this model. Despite significantly increased rhIL-6, this technique did not significantly alter the rate of net tumor progression. However, Stat3 activity was significantly increased in both normal liver and HCC tissue resected from animals implanted with CHO-IL-6 cells. Collectively these data demonstrate the short-term viability of using cell encapsulation technology to generate high levels of active circulating and intrahepatic cytokines and raise the possibility of modifying specific signal transduction cascades identified to be important during tumor progression.

Interleukin-6 inhibits cell proliferation in a rat model of hepatocellular carcinoma.

BACKGROUND: Interleukin-6 (IL-6) plays a critical role in normal hepatic growth and liver regeneration. The aims of the present study are to determine the expression of components of IL-6 signaling in an in vivo model of hepatocellular carcinoma (HCC) and address the role of IL-6 signaling in the progression of HCC. METHODS: An in vivo rat HCC model was established and IL-6 receptor (IL-6R) and downstream signaling pathway expression and activity were determined in HCC and normal liver specimens. Tumorigenic HCC cells from resected HCC samples and normal hepatocytes were then isolated and cultured in the presence and absence of recombinant human IL-6 (rhIL-6). RESULTS: HCC specimens demonstrated decreased IL-6Ralpha/gp130 expression as compared with the normal liver. In contrast, HCC samples had significantly increased IL-6 messenger RNA expression and signal transducers and activators of transcription (STAT)3 activity. Using in vitro cell cultures, we demonstrated that IL-6 stimulated STAT3 and extracellular regulated kinase (ERK) activity in both HCC cells and isolated hepatocytes. However, while STAT3 activation profiles were similar, IL-6 stimulated ERK activity in a biphasic manner in HCC cells and a monophasic, sustained ERK activation in hepatocytes. In HCC cells, a significant induction of cyclin-dependent kinase (CDK) inhibitors, p21(waf1/cip1) and p27(Kip1) occurred, an effect that was not observed in normal hepatocytes. Finally, we established that IL-6 acted to inhibit serum-stimulated DNA synthesis and cell mitogenesis in HCC cells in vitro. CONCLUSIONS: These data demonstrate altered expression of components of IL-6 signaling in HCC in vivo. IL-6 treatment of HCC cells inhibits serum-stimulated mitogenesis, possibly via differences in activation profiles of intracellular signaling pathways and their effect on CDK inhibitor expression/activity.