Negative Feedback By Dusp6 Modulates Myeloproliferation Induced By Oncogenic Nras

Hematological malignancies frequently contain oncogenic mutations in NRAS. Expression of Nras^G12D/+ from the endogenous locus in mice causes a spectrum of neoplastic phenotypes, with some mice developing a myeloproliferative neoplasm (MPN) that recapitulates human chronic and juvenile myelomonocytic leukemia and others developing lymphoid neoplasia. Recent findings show that hematological disease (MPN and T-ALL) is accelerated in Mx1-Cre Nras^G12D/G12D mice, clearly demonstrating that the expression level of the Ras oncoprotein is critical to Ras-induced transformation and cellular responses. Ras signal output is tightly regulated by negative feedback mechanisms. Dusp6 (also known as MKP-3 or Pyst-1) is both a transcriptional target of Ras and an Erk1/2 phosphatase, effectively forming a negative feedback loop that dampens Ras signal output. However, the physiological function of Dusp6 in Ras transformation and hematological disease remains poorly characterized. We previously showed that Dusp6 loss exacerbates anemia in mice expressing oncogenic Kras, but there was not effect observed on peripheral myeloid cell counts. Here we examined the functional consequence of Dusp6 loss in Nras-driven disease. We generated Dusp6 knockout mice that conditionally express Nras^G12D/+ from the endogenous locus (Dusp6^-/-; Mx1-Cre Nras^G12D/+) and found that, although survival is minimally impacted, Dusp6 loss in the hematopoietic compartment accelerates the development of MPN. Peripheral blood from 32-week-old Dusp6^-/-; Mx1-Cre Nras^G12D/+ mice showed an elevated leukocyte count (42.6 ± 14.2 K/ul, n=7) compared to age-matched Dusp6^+/+; Mx1-Cre Nras^G12D/+ mice (8.6 ± 1.2 K/ul, n=7) and wild-type mice (10.6 ± 1.0 K/ul, n=9). Ineffective erythropoiesis is also evident at 32 weeks of age as the level of hemoglobin was decreased in Dusp6^-/-; Mx1-Cre Nras^G12D/+ mice (9.8 ± 1.4 g/dl, n=7), whereas Dusp6^+/+; Mx1-Cre Nras^G12D/+ mice contained hemoglobin amounts similar to wild-type animals (13.3 ± 1.0g/dl, n=7 and 13.7± 0.3g/dl, n=9, respectively). A concomitant rise in reticulocytes was observed in Dusp6^-/-; Mx1-Cre Nras^G12D/+ mice (8.0 ± 1.7%, n=7) versus Dusp6^+/+; Mx1-Cre Nras^G12D/+ mice (4.5 ± 1.1%, n=4) and wild-type mice (2.7 ± 0.15%, n=9). Although Dusp6^+/+; Mx1-Cre Nras^G12D/+ mice showed splenomegaly at 32 weeks of age (321.1 ± 91.8 mg, n=3), the spleens from Dusp6^-/-; Mx1-Cre Nras^G12D/+ mice were 3.5-fold larger (1154.0 ± 119.7 mg, n=5). No difference in thymus weight was observed at 32 weeks, suggesting that Dusp6 loss does not accelerate lymphoproliferation, a characteristic often observed in Nras mutant mice at 24 weeks of age. Together, these data show that Dusp6 plays a critical role in Ras transformation. While the role of Dusp6 in anemia is consistent in both Kras^G12D/+ and Nras^G12D/+ models, the leukocytosis in  Nras^G12D/+ mice newly demonstrates a functional role for Dusp6 in modulating myeloid phenotypes in MPN.