Improving Generation of Cardiac Organoids from Human Pluripotent Stem Cells Using the Aurora Kinase Inhibitor ZM447439

Drug-induced cardiotoxicity reduces the success rates of drug development. Thus, the limitations of current evaluation methods must be addressed. Human cardiac organoids (hCOs) derived from induced pluripotent stem cells (hiPSCs) are useful as an advanced drug-testing model; they demonstrate similar electrophysiological functionality and drug reactivity as the heart. How-ever, similar to other organoid models, they have immature characteristics compared to adult hearts, and exhibit batch-to-batch variation. As the cell cycle is important for the mesodermal differentiation of stem cells, we examined the effect of ZM447439, an aurora kinase inhibitor that regulates the cell cycle, on cardiogenic differentiation. We determined the optimal concentration and timing of ZM447439 for the differentiation of hCOs from hiPSCs and developed a novel protocol for efficiently and reproducibly generating beating hCOs with improved electrophysiological functionality, contractility, and yield. We validated their maturity through electro-physiological- and image-based functional assays and gene profiling with next-generation sequencing, and then applied these cells to multi-electrode array platforms to monitor the cardio-toxicity of drugs related to cardiac arrhythmia; the results confirmed the drug reactivity of hCOs. These findings may enable determination of the regulatory mechanism of cell cycles underlying the generation of iPSC-derived hCOs, providing a valuable drug testing platform.

Elevated ABCB1 Expression Confers Acquired Resistance to Aurora Kinase Inhibitor GSK-1070916 in Cancer Cells

The emergence of multidrug resistance (MDR) has been a major issue for effective cancer chemotherapy as well as targeted therapy. One prominent factor that causes MDR is the overexpression of ABCB1 transporter. In the present study, we revealed that the Aurora kinase inhibitor GSK-1070916 is a substrate of ABCB1. GSK-1070916 is a newly developed inhibitor that is currently under clinical investigation. The cytotoxicity assay showed that overexpression of ABCB1 significantly hindered the anticancer effect of GSK-1070916 and the drug resistance can be abolished by the addition of an ABCB1 inhibitor. GSK-1070916 concentration-dependently stimulated ABCB1 ATPase activity. The HPLC drug accumulation assay suggested that the ABCB1-overexpressing cells had lower levels of intracellular GSK-1070916 compared with the parental cells. GSK-1070916 also showed high binding affinity to ABCB1 substrate-binding site in the computational docking analysis. In conclusion, our study provides strong evidence that ABCB1 can confer resistance to GSK-1070916, which should be taken into consideration in clinical setting.

The Evaluation of Effect of Aurora Kinase Inhibitor CCT137690 in Melanoma and Melanoma Cancer Stem Cell

Background: Dysregulation of the cell cycle is one of the main causes of melanomagenesis. Genome-wide studies showed that expression of Aurora -A and -B significantly has been upregulated in melanoma. However, there is no FDA approved drug targeting aurora kinases in the treatment of melanoma. In addition, the development of resistance to chemotherapeutic agents in the treatment of melanoma and, as a result, the relapse due to heterogeneous cell groups in patients is a second phenomenon that causes treatment failure. Therefore, there is an urgent need for therapeutic alternatives targeting both melanoma and melanoma cancer stem cells (MCSCs) in treatments. At this stage, cell cycle regulators become promising targets. Objective: In this study, we aimed to identify the effects of Aurora kinase inhibitor CCT137690 on the cytotoxicity, apoptosis, cell cycle, migration, and colony formation and expression changes of genes related to proliferation, cell death and cell cycle in melanoma and melanoma cancer stem cell. In addition, we investigated the apoptotic and cytostatic effects of CCT137690 in normal fibroblast cells. Methods: We evaluated the cytotoxic effect of CCT137690 in MCSCs, NM2C5 referring as melanoma model cells and WI38 cells by using the WST-1 test. The effect of CCT137690 on apoptosis was detected via Annexin V and JC-1 method; on cell cycle progression by cell cycle test; on gene expression by using RT-PCR, on migration activity by wound healing assay and clonal growth by clonogenic assay in NM2C5 cells and MCSCs. The effects of CCT137690 in WI-38, referring as healthy fibroblast cell, were assessed through Annexin V and cell cycle method. Results: CCT137690 was determined to have a cytotoxic and apoptotic effect in MCSCs and melanoma. It caused polyploidy and cell cycle arrest at the G2/M phase in MCSCs and melanoma cells. The significant decrease in the expression of MMP2, MMP7, MMP10, CCNB1, IRAK1, PLK2 genes, and the increase in the expression of PTEN, CASP7, p53 genes were detected. Conclusion: Aurora kinases inhibitor CCT137690 displays promising anticancer activity in melanoma and especially melanoma cancer stem cells. The effect of CCT137690 on melanoma and MCSC may provide a new approach to treatment protocols.

High NRF2 Levels Correlate with Poor Prognosis in Colorectal Cancer Patients and with Sensitivity to the Kinase Inhibitor AT9283 In Vitro

Aberrant hyperactivation of nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis; however, its relevance in colorectal tumours is not well-established. Measuring the expression of surrogate genes for NRF2 activity in silico, in combination with validation in patients’ samples, we show that the NRF2 pathway is upregulated in colorectal tumours and that high levels of nuclear NRF2 correlate with a poor patient prognosis. These results highlight the need to overcome the protection provided by NRF2 and present an opportunity to selectively kill cancer cells with hyperactive NRF2. Exploiting the CRISPR/Cas9 technology, we generated colorectal cancer cell lines with hyperactive NRF2 and used them to perform a drug screen. We identified AT9283, an Aurora kinase inhibitor, for its selectivity towards killing cancer cells with hyperactive NRF2 as a consequence to either genetic or pharmacological activation. Our results show that hyperactivation of NRF2 in colorectal cancer cells might present a vulnerability that could potentially be therapeutically exploited by using the Aurora kinase inhibitor AT9283.