P883: TARGETING CALCINEURIN/NFATC1 SIGNALING SENSITIZES PROTEASOME INHIBITION TREATMENT IN MULTIPLE MYELOMA
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Background: Proteasome inhibitors (PIs) (e.g., Bortezomib) are key treatments to improve the clinical outcome of multiple myeloma (MM). However, disease relapse from the emergence of PI-resistant clones is the main problem. Insights into the mechanisms underlying PI-resistance will provide novel druggable targets to prevent relapse. It was well-recognized that PIs induce endoplasmic reticulum (ER) stress in MM cells. ER stress is associated with the Calcineurin/nuclear-factor-of-activated-T-cells (NFAT) signaling. Accumulating studies indicated the importance of Calcineurin/NFAT signaling in tumorigenesis and drug resistance. However, the involvement of Calcineurin/NFAT signaling in PI-resistance in MM has not yet been investigated. Aims: To dissect the role of the Calcineurin/NFAT axis in Bortezomib (BTZ) treatment response and explore the therapeutic potential by combining Bortezomib and Calcineurin/NFAT inhibitors in MM. Methods: We studied the expression pattern of PPP3CA and NFATC1 from MM datasets and MM cell lines and their association with BTZ responses in MM. The expression of the four principal NFAT members was detected by expression microarray and their activation status by western blotting in MM cell lines. We identified the most prominent NFAT member and assessed its activation under BTZ treatment. A lentivirus-transduction system was used for overexpressing genes, and siRNA transfection was applied to induce NFATC1 knockdown in a BTZ-resistant cell line ARH77. Flow cytometry and western blotting assessed the combinational effect of Calcineurin/NFAT inhibitors and BTZ on MM cells. Results: The expression level of PPP3CA, which encodes Calcineurin Catalytic subunit A (CnA), was correlated to the poor clinical outcome of recurrent MM patients (MMRF cohort). Calcineurin was highly expressed in ARH77, which is less sensitive to BTZ treatment than other MM cell lines. Among the four NFAT members, we found that NFATC1 and NFATC3 were the most highly expressed members in MM cell lines. However, NFATc1 was the only member suppressed by CsA but activated by the calcium channel agonist Ionomycin in ARH77. Consistent with Calcineurin, NFATc1 was highly expressed in ARH77, with reference to a confirmed NFATc1-positive cell line Raji and the nuclear-NFATc1 overexpressing HEK-293T. Additionally, BTZ activated CnA and NFATc1 time-dependently in ARH77 but failed to activate CnA and NFATc1 in the BTZ-sensitive cell line NCI-H929. Immunofluorescence showed that BTZ triggered the nuclear translocation of NFATc1. From GEO datasets, NFATC1 was overexpressed in patients less responsive to BTZ, but was unchanged in patients subjected to conventional therapies. The Calcineurin/NFATc1 was thus a potential mechanism of BTZ-resistance in MM. Depleting NFATc1 enhanced BTZ-induced DNA damage-related apoptosis in ARH77. The combinational treatment of Calcineurin/NFAT inhibitors (CsA and FK506) and BTZ decreased the nuclear/cytosolic ratio of NFATc1 and promoted apoptosis in two MM cell lines compared to the treatment of BTZ alone. Image:Summary/Conclusion: The activation of the Calcineurin/NFATc1 axis is an important mechanism that contributes to BTZ-resistance in MM. Targeting this axis confers translational potential for improving BTZ-based therapies.Keywords:
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Biomechanical stress on the heart results in activation of numerous signaling cascades, leading to cardiomyocyte hypertrophy, apoptosis, and ultimately, heart failure. The Ca2+-dependent phosphatase calcineurin is an essential mediator of cardiac hypertrophy, and in most but not all studies, calcineurin inhibition attenuated cardiac hypertrophy in vivo. However, calcineurin inhibition has been reported to have adverse effects on cardiac remodeling and cardiomyocyte apoptosis. Calcineurin regulates the activity of a number of downstream targets, including the transcription factors NFAT, MEF2, and NF-kappaB, and the apoptotic factor Bad. To evaluate the contribution of NFAT activation by calcineurin to cardiomyocyte responses to hypertrophic stimulation, we used adenovirus to express VIVIT, a selective peptide inhibitor of calcineurin-mediated NFAT activation. We found that selective NFAT inhibition during phenylephrine stimulation inhibited hypertrophy but resulted in increased cardiomyocyte apoptosis. In contrast, nonselective inhibition of calcineurin by cyclosporin A did not cause cardiomyocyte apoptosis after phenylephrine stimulation. Cyclosporin A suppressed the effect of VIVIT on cardiomyocyte apoptosis. These results demonstrate that during phenylephrine stimulation calcineurin activates both pro- and antiapoptotic pathways in cardiomyocytes, and that NFAT activity is a critical component of the antiapoptotic pathway that regulates whether the outcome of calcineurin activation is cardiomyocyte apoptosis or survival.
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Deregulated calcium signaling is observed at different stages of tumorigenic processes. An important signaling pathway activated in response to calcium involves the protein phosphatase calcineurin and NFAT transcriptional factors. We review here recent data that indicate an important role of the calcineurin/NFAT pathway in lymphoma/leukemogenesis and discuss the potential therapeutic implications of these findings.
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Abstract Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently characterized. The peptide could selectively interfere with the calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase, similar to 11R-VIVIT. RCAN-11R did not affect cell viability when it was used at a higher concentration than the toxic concentration of 11R-VIVIT. RCAN-11R could therefore be useful as a therapeutic agent that is less toxic than current drugs or 11R-VIVIT.
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In noncontractile cells, a sustained increase in total cytoplasmic Ca(2+) concentration is typically needed to activate the intracellular protein phosphatase calcineurin, leading to dephosphorylation of the transcription factor nuclear factor of activated T cells (NFAT), its nuclear translocation, and induction of gene expression. It remains a mystery exactly how Ca(2+)-dependent signaling pathways, such as that mediated by calcineurin-NFAT, are regulated in contracting cardiac myocytes given the highly specialized manner in which Ca(2+) concentration rhythmically cycles in excitation-contraction coupling. Here, we critically review evidence that supports the hypothesis that calcineurin-NFAT signaling is regulated by contractile Ca(2+) transients in cardiac myocytes.
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ABSTRACT Cardiovascular disease is the major cause of death in industrialized nations. Targeted intervention in calcineurin, a calmodulin‐dependent, calcium‐activated phosphatase and its substrate, nuclear factor of activated T cells (NFAT), was demonstrated to be effective in the treatment of cardiovascular diseases. Although effective in the disruption of calcineurin phosphatase activity, cyclosporin A (CsA) and FK506 also resulted in undesired side effects and toxicity, prompting the discovery of VIVIT, a novel peptide inhibitor. VIVIT selectively and potently inhibits calcineurin/NFAT interaction, but does not compromise calcineurin phosphatase activity and non‐NFAT‐mediated signaling. VIVIT displays a favorable therapeutic profile as a potential drug candidate and constitutes a useful tool in exploring calcineurin‐NFAT functionality. This review describes the development of VIVIT peptide as a selective NFAT inhibitor and its application as a therapeutic agent in cardiovascular disorders including cardiac hypertrophy, restenosis, atherosclerosis, and angiogenesis.
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Thin spongy myocardium is critical at the early embryonic stage [before embryonic day (E) 13.5 in mice] to allow diffusion of oxygen and nutrient to the developing cardiomyocytes. However, establishment of compact myocardium at later stage (~E16.5) during development is necessary to prepare for the increase in demand for blood circulation. But molecular targets of the spongy-compact myocardium transition at E13.5–E16.5 in heart development remain elusive. Here, we report transcription targets, independently and dependently, regulated by the calcineurin/NFAT signaling during E13.5–E16.5 myocardium transition. We have uncovered that expression of one-third of the induced genes during myocardium transition is calcineurin/NFAT dependent. Among these calcineurin/NFAT dependent transcription targets, there is a dose-dependent regulation. Thus, in addition to temporal and spatial regulation, dose-dependent threshold requirement provides another mechanism to modulate transcription response mediated by the calcineurin/NFAT signaling. This research is supported, in part, by a grant from the American Heart Association.
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Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity.
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