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C. Di Genua et al.
In line with previous reports,18,19 expression of K- RasG12D (KM genotype) caused a myeloproliferative phenotype, consisting of leukocytosis, anemia, increased BM cellularity, increase in the Mac1+Gr1lo myeloid cells in the PB and spleen, splenomegaly, and thrombocytopenia (Figure 1B-I and Online Supplementary Table S2).
Strikingly, co-expression of Aml1-ETO and K-RasG12D did not result in a more aggressive disease, but ameliorat- ed key features of the phenotype associated with K- RasG12D expression, including restoration of white blood cell (WBC) count and hemoglobin levels (Figure 1B and C), reduction in the Mac1+Gr1lo myeloid cells in the PB and spleen, and reduced spleen weight (Figures 1E-H). Platelet count was, however, further reduced compared to CON (Figure 1I) and mice still showed an increase in BM cellu- larity (Figure 1D).
Myelo-erythroid progenitors were analyzed by flow cytometry (Figure 2 and Online Supplementary Figure S2). We found both Aml1-ETO (2-fold, P=0.0093) and K- RasG12D (8-fold, P<0.0001) expression decreased the number of megakaryocyte progenitors (MkP), and when
AB
co-expressed resulted in a further reduction (15-fold, P<0.0001) in the BM compared to CON (Figure 2A). This paralleled the decrease in platelet counts found in the PB (Figure 1I). Aml1-ETO also increased in the number of MkP in the spleen compared to CON (2-fold, P=0.005) (Figure 2B). BM colony forming unit – erythrocytes (CFU- E) were not affected in any genotypes compared to con- trols (Figure 2C). However, K-RasG12D caused an increase in CFU-E in the spleen compared to CON (36- fold increase, P=0.0004) (Figure 2D), indicating stress ery- thropoiesis, which was reversed in AKM mice. BM pre- granulocyte/macrophage progenitors (pre-GM) were unperturbed in all three genotypes compared to CON (Figure 2E). However, K-RasG12D expression caused an increase in pre-GM in the spleen compared to CON (9- fold increase, P<0.0001) (Figure 2F). The increase in pre- GM was reversed when K-RasG12D was co-expressed with Aml1-ETO (3-fold decrease, P<0.0001) (Figure 2F). There was no difference in BM lymphoid-primed multi- potent progenitor (LMPP) across all genotypes; however, there was an increase in the number of spleen LMPP in
Figure 2. Aml1-ETO reverses some of the myelo-erythroid progenitor cell phenotypes caused by K-RasG12D. (A, C, E) Absolute number of CD45.2 megakaryocyte progeni- tor (MkP) (A), CFU-E (C) and Pre-GM (E) in the bone marrow (BM) from recipients of CON (n=12), AM (n=14), KM (n=12) and AKM FL (n=14). Results were generated in three independent experiments. (B, D, F) Absolute numbers of CD45.2 MkP (B), colony forming unit-erythrocyte (CFU-E) (D) and pre-granulocyte-monocyte (Pre-GM). (F) in the spleen from recipients of CON (n=8), AM (n=10), KM (n=8) and AKM FL (n=10). Results were generated in two independent experiments. The results were analyzed using multiple comparison ANOVA and are presented as the mean±Standard
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Deviation. *P<0.05; ***P<0.001; ****P<0.0001.
**P<0.01;
EF
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haematologica | 2019; 104(11)