Continued Expression of the NRAS(G12V) Oncogene Is Required for the Maintenance of Acute Myeloid Leukemia Induced in Cooperation with MLL-AF9.
Authors: Kim Won-Il , Matise Ilze , Diers Miechaleen , Largaespada David 
Published: April 2020

Abstract
To study the role of the NRAS(G12V) oncogene in the context of acute myeloid leukemia (AML) cells developing with in cooperation with MLL fusion oncogene (MLL-AF9), we used a Vav promoter-Tet transactivator (Vav-tTA)-driven repressible system of NRAS(G12V) expression in Mll-AF9 mice. Vav-tTA; TRE-NRAS(G12V); Mll-AF9 triply-transgenic mice were generated by crossing the Vav-tTA; TRE-NRAS(G12V) doubly-transgenic FVB/n and Mll-AF9 knock-in BL6 mice. The triply-transgenic FVB/n × BL6 F1 mice expressing both the NRAS(G12V) and Mll-AF9 transgenes developed AML, which showed a trend of decreased latency compared with those carrying only the Mll-AF9 knock-in transgene. Mast cell disease also occurred accordingly in the Vav-tTA; TRE-NRAS(G12V) co-transgenic mice. Since the mastocytosis disease is not transplantable, we transplanted bone marrow cells from four independent AML mice into recipient SCID mice to determine whether NRAS(G12V) expression is necessary to maintain AML in the recipient mice without mastocytosis. Continuously treating the transplanted SCID mice with doxycycline (Dox) in drinking water, we found the expression of NRAS(G12V) oncogene was required for AML persistence in three out of the four independent primary AML cells. Furthermore, we transplanted the AML bone marrow cells previously xenografted in the recipient SCID mice into other SCID mice to conditionally repress NRAS(G12V) expression only after the transplanted AML was fully established. We found the number of WBC cells was greatly decreased 4–6 days after the Dox treatment and this was correlated with the significant increase of apoptotic cells in bone marrow and peripheral bloods. The transplanted AML blast cells underwent apoptosis and were mostly removed from the circulating blood, bone marrow, and spleen after 8 days post Dox treatment. In 2–3 weeks after beginning Dox treatment and observing AML remission, Dox-resistant leukemia relapse was observed in recipient SCID mice. The relapsed leukemia failed to express NRAS(G12V) and showed significantly reduced aggressiveness along with less myelosuppression and more differentiated myeloid lineage cells than AML prior to repression of NRAS(G12V) expression. The NRAS(G12V)-independent relapsed disease histopathologically resembles an aggressive myeloproliferative disease (MPD) rather than AML, because the proportion of AML blast cells was less than 20% of myeloid lineage cells. The NRAS(G12V)-independent MPD could be transplanted into recipient SCID mice, but the subsequent anemia was greatly attenuated compared to transplant of the same AML clone expressing NRAS(G12V). We conclude that NRAS(G12V) can be a good molecular target to treat AML, because NRAS(G12V) expression is required for persistence and specific malignant features in AML induced in cooperation with MLL-AF9. Targeting NRAS(G12V) can strongly disturb the maintenance of AML blast cells and myelosuppression, although leukemia cells can relapse without NRAS(G12V) expression.