Feedback Through Dusp6 Modulates Ineffective Erythropoiesis Caused By Mutant Kras

Somatic mutations that activate Ras signaling are common in hematologic malignancies. They are particularly associated with juvenile and chronic myelomonocytic leukemias, which are classified as ‘overlap’ myelodysplastic syndromes / myeloproliferative neoplasms (MDS/MPN). These diseases are characterized by myelomonocytic proliferation and dysplasia leading to anemia and thrombocytopenia. We have previously shown that conditional expression of Kras^G12D in mice models MDS/MPN, producing a fatal disease with increased granulocyte/macrophage (G/M) progenitors and a reduction in the megakaryocyte/erythroid (Meg/E) lineage. Despite intensive study of Ras signaling, the mechanisms by which oncogenic Ras regulates cell fate decisions in hematopoiesis remain unclear; better understanding of these processes may allow development of novel therapies for MDS/MPN. We found that mRNA and protein levels of dual specificity phosphatase-6 (Dusp6), a negative regulator of MAPK signaling, is elevated in the bone marrow of Kras^G12D mice. This suggested that a negative feedback loop generated by Dusp6 might regulate the proliferation and differentiation of hematopoietic progenitors in MDS/MPN. To test this hypothesis, we conditionally expressed Kras^G12D from its endogenous locus in the bone marrow of wild type and Dusp6 knockout mice. Interestingly, many characteristics of MDS/MPN were more severe in Dusp6^-/-; Kras^G12D mice. For example, we observed significantly lower hemoglobin levels in 3-4 week old Dusp6^-/-; Kras^G12D mice (8.35g/dL, n=4) compared to Dusp6^+/+; Kras^G12D mice (11.7g/dL, n=10). Moreover, the peripheral blood of Dusp6^+/+; Kras^G12D mice contained approximately 13% of reticulocytes (n=12), whereas Dusp6^-/-; Kras^G12D mice had nearly 36% reticulocytes (n=4) in the peripheral blood. Furthermore, we observed acanthocytes, poikilocytes and other structural abnormalities in the red blood cells from Dusp6^-/-; Kras^G12D mice and not in mice wild type for Dusp6. Thus, Dusp6 loss accelerates the onset of anemia caused by Kras^G12D, which suggests an exacerbation of the erythroid differentiation defect. We did not observe any significant differences in white blood count or spleen size attributable to Dusp6 genotype. Differentiation of hematopoietic progenitors was assessed in the bone marrow by flow cytometry. The frequency of pre-Meg/E progenitors (Lin^-Sca1^-Kit⁺CD34⁺FcgR^-CD150⁺CD105^-) from Dusp6^-/-; Kras^G12D mice was reduced by approximately 50% when compared to Dusp6^+/+; Kras^G12D mice. Consistent with this result, Dusp6^-/-; Kras^G12D mice possessed a significantly greater percentage of immature red blood cells (proerythroblasts; CD71^hi Ter119⁺) and fewer mature red blood cells (orthochromatic erythroblasts; CD71⁺ Ter119⁺) than bone marrow cells from Dusp6^+/+; Kras^G12D mice. By contrast, Dusp6 expression does not have an effect on the frequency of pre-G/M cells (Lin^-Sca1^-Kit⁺CD34⁺FcgR^-CD150^-CD105^-), which remains unchanged between Dusp6^+/+; Kras^G12D and Dusp6^-/-; Kras^G12D mice. Together, these findings demonstrate that negative feedback through Dusp6 functions to maintain erythropoiesis the context of oncogenic Ras signaling. Futher, these data strengthen the association between excessive MAPK activity and dyserythropoiesis, and further support development of inhibitors of MEK and/or ERK to treat MDS/MPN.