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Investigation of the role of RCAN1 Gene in apoptosis

Our lab is interested in understanding the molecular basis of chemotherapeutic evoked apoptosis and resistance in leukemic lymphoblasts. The long‐term goal of our research is to improve the therapeutic effectiveness of chemotherapeutic agents. Antileukemic agents and signaling pathway inhibitors induce apoptosis by regulating gene expression; a drawback of this treatment is the development of resistance to these agents. The RCAN1 (Regulator of Calcineurin 1) gene is located on chromosome 21 and is one of many genes implicated in apoptosis. Upregulation of RCAN1 correlates with sensitivity to antileukemic agents and signaling pathway inhibitors. The protein product of this gene, RCAN1, binds to the protein phosphatase calcineurin, inhibiting its activity, and altering cellular responses to survival and death triggers. We are testing the hypothesis that if the protein function of the RCAN1 is knocked out, cells will be protected from apoptosis. We are screening a series of antileukemic agents and signaling pathway inhibitors, utilizing paired wild‐type and RCAN1 knockout HAP1 cell lines, to determine which agents might mediate an apoptotic response via RCAN1 involvement. HAP1 is a human near‐haploid cell line derived from the male chronic myelogenous leukemia (CML) cell line KBM‐7, allowing for efficient CRISPR‐mediated genome editing. Viability of cells exposed to various agents was evaluated by MTT assays and transcript levels were monitored via reverse‐transcription‐PCR analysis of RNA extracted from treated cells. MTT assays show that RCAN1 knockout offered the greatest protection from apoptosis caused by the AKT inhibitor MK‐2206, when compared to wildtype cells. Upregulation of BIM, a proapoptotic protein, by MK‐2206 was reduced in RCAN1 knockout HAP1 cells compared to the wild‐type HAP1 cells. Our results suggest that RCAN1 may influence the AKT pathway to mediate apoptosis.