Targeting NFAT as a Novel Leukemia Treatment Method

• Lina Sora, Pharmaceutical Sciences, University of Pittsburgh

• Christian Fernandez, University of Pittsburgh School of PharmacologyAcute Lymphoblastic Leukemia is a bone marrow cancer characterized by increased immature B or T cell lymphocytes in the bloodstream. This type of cancer is most common in children and progresses quickly. Currently, the treatment methods for ALL are drugs such as Cyclosporin A and FK506. Cyclosporin A is a common drug used to prevent organ rejection. Another method of treatment is 11R-VIVIT, an NFAT inhibitor. These, however, inhibit NFAT translocation into the nucleus via blocking calcium binding to calcineurin and blocking calcineurin binding to NFAT, respectively. However, this inhibition prevents NFAT translocation into the nucleus and stops activation of all NFAT and calcineurin-activated genes pathways inducing adverse side effects and various sites of toxicity. This study has looked at a novel treatment method that targets the transcriptional ability of NFAT after translocation into the nucleus. The method of experimentation was to conduct electrophoretic mobility shift assays to observe the binding of NFAT to various DNA oligonucleotides. Each experiment contained lanes that demonstrated the ability of NFAT to bind to DNA and the confirmation of this interaction via the addition of an antibody which produced a super shift. The EMSAs consisted of extracted nuclear protein from Jurkat cells and mouse splenocytes incubated with oligonucleotides h-ARRE, h-ARRE no AP1 binding site, TNFa, and m-ARRE. Initially, the results made clear the ability of the extracted protein to bind to these sites and to determine the optimal concentration of NFAT for subsequent experiments. OR-1011 was added to the assay once this concentration was determined. This treatment allowed for the visualization of the mechanism of the drug OR-1011 regulates NFAT., University of Delaware

Acute Lymphoblastic Leukemia is a bone marrow cancer characterized by increased immature B or T cell lymphocytes in the bloodstream. This type of cancer is most common in children and progresses quickly. Currently, the treatment methods for ALL are drugs such as Cyclosporin A and FK506. Cyclosporin A is a common drug used to prevent organ rejection. Another method of treatment is 11R-VIVIT, an NFAT inhibitor. These, however, inhibit NFAT translocation into the nucleus via blocking calcium binding to calcineurin and blocking calcineurin binding to NFAT, respectively. However, this inhibition prevents NFAT translocation into the nucleus and stops activation of all NFAT and calcineurin-activated genes pathways inducing adverse side effects and various sites of toxicity. This study has looked at a novel treatment method that targets the transcriptional ability of NFAT after translocation into the nucleus. The method of experimentation was to conduct electrophoretic mobility shift assays to observe the binding of NFAT to various DNA oligonucleotides. Each experiment contained lanes that demonstrated the ability of NFAT to bind to DNA and the confirmation of this interaction via the addition of an antibody which produced a super shift. The EMSAs consisted of extracted nuclear protein from Jurkat cells and mouse splenocytes incubated with oligonucleotides h-ARRE, h-ARRE no AP1 binding site, TNFa, and m-ARRE. Initially, the results made clear the ability of the extracted protein to bind to these sites and to determine the optimal concentration of NFAT for subsequent experiments. OR-1011 was added to the assay once this concentration was determined. This treatment allowed for the visualization of the mechanism of the drug OR-1011 regulates NFAT.