A Single Retroviral Vector Design for the Simultaneous Expression of a Mir30 Based Shrna with An Oncogene – Identification of Raf-1 but Not BRAF as a Crucial Mediator for BCR-ABL Mediated Leukemogenesis.

Abstract

AbstractAbstract 3392 Introduction:Chronic myelogenous leukemia (CML) is characterized by the t(9;22)(q34;q11) chromosomal translocation and the expression of BCR-ABL, a fusion protein with tyrosine kinase activity. BCR-ABL activates various signaling cascades mediating signals for proliferation, transformation and anti-apoptosis. The BCR-ABL inhibitor imatinib is the standard therapy for CML. However, this treatment is assumed to be not curative since leukemia initiating cells cannot be completely eradicated by solely BCR-ABL inhibition. Identification of key mediators within the BCR-ABL signaling cascade thus remains crucial. The MEK/ERK cascade is one of the major promitogenic pathways activated in CML. Whether Raf-1, BRAF or both Raf isoforms are required for BCR-ABL mediated activation of this pathway is not known. As both Raf-1 and BRAF knockout mice are embryonic lethal, the role of Raf-1 and BRAF in BCR-ABL mediated leukemogenesis has not been investigated in appropriate in vivo models so far. Here we studied the impact of Raf-1 and BRAF for BCR-ABL dependent transformation by using a retroviral vector system, which allows to directly couple shRNA based target suppression to oncogene expression in a CML mouse model. Methods:We exerted an shRNA-based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the BCR-ABL oncogene and miR-30 based shRNAs (miR) for BRAF and Raf-1 respectively on a single construct resulting in one shared RNA transcript. This approach ensured knockdowns of more than 80–90% for the respective Raf protein in every BCR-ABL transformed cell. Result:Methylcellulose assays showed that primary bone marrow cells coexpressing Raf-1 miR and BCR-ABL had a 2 fold decreased colony forming ability, whereas BRAF knockdown had no impact on colony forming ability compared to control cells. We then transplanted murine bone marrow (BM), transduced with retrovirus coexpressing Raf-1 or BRAF miR and p185 BCR-ABL, to lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf-1 miR but not BRAF miR and BCR-ABL compared with the BCR-ABL transduced control miR group. Raf-1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival in comparison to control mice. However, BRAF knockdown had no significant effect on overall survival or disease progression in the bone marrow transduction transplantation model. We hypothesized that this impact of Raf-1 knockdown might be due to incomplete activation of the MEK/ERK cascade in the absence of Raf-1. We could demonstrate that Raf-1 is necessary for BCR-ABL dependent ERK activation in primary murine bone marrow as well as in cell lines. In contrast in BRAF knockdown BCR-ABL positive cells levels of phosphorylated and thereby activated ERK remained unchanged compared to control cells, indicating that BRAF is dispensable for BCR-ABL dependent ERK phosphorylation. Conclusion:Taken together our data demonstrate that primarily Raf-1 is responsible for BCR-ABL mediated activation of the promitogenic MEK/ERK signaling cascade. Raf-1 but not BRAF is also crucial for the development of a myeloproliferative disease by BCR-ABL in mice. Therefore, Raf-1 but not BRAF inhibition may be a potential interesting additional therapeutic approach in CML.The coexpression of an oncogene and a target specific miR-30 based shRNA from a single retroviral construct displays a powerful tool that can be used to systematically screen drugable signaling targets involved in CML and other leukemic malignancies. Disclosures:No relevant conflicts of interest to declare.