Abstract A55: Induced hyperactivation of Ras-MAPK in chronic myeloid leukemia: role of negative feedback signaling

Background: Negative regulator molecules are well known essential keys for cell proliferation, survival and homeostasis balance. DUSP6 and SPRY2 are both tumor suppressors' genes which play an important role in RAS feedback signaling pathway by targeting different effector molecules. In this study, we evaluated the mechanisms under RAS hyperactivation scenario driven by BCR-ABL1 and the outcomes of an imbalance feedback signal in a chronic myeloid leukemia (CML) model. Experimental design: to study the function of DUSP6 and SPRY2 molecules we designed a genetic mouse model of CML. Briefly, bone marrow cells from DUSP6-/- and SPRY2fl/fl mice were cultured with recombinant mouse cytokines (IL-3, IL-6, SCF) and retrovirally transduced with BCR-ABL1 fusion oncogene. For the SPRY2 conditional knockout, cells were transduced with Cre-ERT2 retroviral recombinant vector or empty vector (ERT2) and deletion was induced by a 3-day treatment with 4-Hydroxytamoxifen (4–OHT). Lineage identity of CML-like cells was confirmed by flow cytometry and gene deletion was confirmed by western blot. Results: In previous gene expression analysis by microarray we showed that DUSP6 and SPRY2 negative regulator molecules are highly expressed in kinase -driven malignancies, including chronic myeloid leukemia. Subsequently, evaluating Dusp6 deficiency we observed a profound signaling imbalance in CML-like cells, resulting in cell cycle arrest in G2/M phase and increased susceptibility to cellular senescence as compared to DUSP6 wild type cells. Absence of this negative regulator molecule also decreased the self-renewal capability of DUSP6-/- CML-like cells, which failed to form colonies in methylcellulose culture. However, when assessing the impact on Erk activation, Dusp6 deletion unexpectedly led to Erk phosphorylation inhibition and Stat5 phosphorylation, suggesting a crosstalk between both pathways. Inducible deletion of SPRY2 surprisingly had opposite effects and once deleted resulted in a significant increase of self-renewal capability observed in colony formation assay. Lack of Spry2 had no major effects in Erk activation and cell growth. Conclusion: the negative feedback mediators analyzed in our work, showed that feedback signaling is a complex network and distinct molecules acting as negative regulators in the same pathway can actually lead to different outcomes in the same type of leukemia. Overall, we suggest that Dusp6 can play an important role in oncogenic signaling by preventing cellular senescence and maintaining cellular self-renewal. The negative regulator Spry2, however, seems to have an essential function as tumor suppressor gene in CML cells and its deletion can lead to a favorable homeostasis imbalance for leukemic cells. Citation Format: Vanessa Silveira, Seyedmehdi Shojaee, Markus Muschen. Induced hyperactivation of Ras-MAPK in chronic myeloid leukemia: role of negative feedback signaling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A55.