Relapsed NRAS^G12V-independent MPD-like AML has become less myelosuppressive and more differentiated. (A) Modified Wright-Giemsa staining of peripheral blood shows more differentiated myeloid lineage cells in the relapsed disease. In the blood smear from a mouse with established AML, more than 80% of WBCs have undifferentiated blast morphology. In the blood smear from the same mouse with relapsed MPD-like AML after Dox treatment, most cells are differentiated granulocytes (neutrophils, eosinophils) and less than 20% are undifferentiated blasts. Images taken at ×1000 magnification. Bars represent 25 μm. (B) For 0 through 6 days during which the WBC concentration reaches 200 K/μL or more, the RBC concentration in the relapsed MPD-like AML mice is significantly higher than in the mice harboring AML expressing NRAS^G12V (P < .001). Narrow bars indicate standard deviations. (C) Western blot analysis shows that the Ras/Erk pathway is inactivated with Dox treatment during AML regression, but is reactivated in the NRAS^G12V-independent relapsed MPD-like AML. The NRAS^G12V protein is not detected in Dox-resistant relapsed MPD-like AML. The Erk1/2 proteins acted as loading controls. Vertical lines have been inserted to indicate a repositioned gel line.