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issues by conditionally introducing both mutant genes from the respective genomic loci in mice in order to reproduce physiological oncogene expression levels. They then subsequently performed competitive trans- plantation assays to be able to directly compare stem cell activity in wild-type and transgenic cells (Figure 1B).
AML1-ETO expression on its own affected platelet, B- and T-cell development, and led to an increased number of functional HSC together with enhanced myeloid reconstitution in secondary recipients. Expression of mutant K-RAS on its own resulted in a myeloproliferative phenotype, led to a lack of HSC expansion, decreased engraftment and diminished in vitro re-plating potential, which, importantly, was seen regardless of the presence of AML1-ETO. Interestingly, co-expression of both onco- proteins led to a milder myeloproliferative phenotype but not overt AML, as seen in patients, and only aggravated the defect in platelet development. The authors suggest that the competitive advantage observed in AML1-ETO- expressing HSC was due to an enhanced self-renewal capacity. To further elucidate the mechanisms behind the functional impairment of K-Ras(G12D)-expressing HSC, they compared gene expression in HSC from genetically modified and control cells using transplantation assays. The presence of mutant K-RAS conferred increased cell cycle activity as well as upregulation of the expression of checkpoint associated genes, such as E2F, Myc and G2M- associated genes, as compared to HSC harboring the AML1-ETO transgene only. Moreover, the presence of mutated K-RAS protein resulted in the downregulation of gene expression signature associated with self-renewal activity and acquisition of a GMP-associated transcrip- tional signature. Overall, the transcriptional profiles of K- RAS(G12D)-expressing HSC resembled those of myeloid progenitors. Gene expression changes in double mutant cells were distinct from those of cells carrying the individ- ual mutations. For example, two genes, Gja1 and Gzmb, were up-regulated in the double mutant HSC and the authors suggest that they regulate the p53 pathway and oxidative phosphorylation, respectively, both increasing cell death and apoptosis.
Taken together, Di Genua et al.23 show that expression of a mutant K-RAS is not compatible with a pre-leukemic state of murine AML1-ETO-expressing HSC. Although AML1-ETO alone confers a competitive advantage to HSC, the additional presence of K-RAS(G12D) results in loss of HSC function, most likely by exhaustion. The study shows that this phenomenon is explained by an increase in cell cycle activity leading to a loss of quies- cence in HSC co-expressing both mutations. Therefore, they hypothesize that acquisition of mutant K-RAS in t(8;21) AML occurs at the myeloid progenitor stage rather than within pre-leukemic HSC compartment harboring the t(8;21) translocation. Overall, this study demonstrates that the order of appearance of each class of mutations is also relevant for leukemic transformation in AML.
Additional questions arise from this and previous work (Figure 1C). Firstly, Di Genua et al. show that, even in the presence of K-RAS(G12D), AML1-ETO is unable to cause an overt leukemic phenotype in vivo. The same was found in xenotransplantation experiments with retrovirally transduced human CD34+ cord blood cells expressing
AML1-ETO together with N-RAS(G12D).26 This notion ties in with the observation of Cabezas-Wallscheid et al.16 that the development of AML in an AML1-ETO mouse model is a slow process, probably requiring multiple mutations or epigenetic reprogramming events, which were not examined in their study. Moreover, it has been previously shown that expression of AML1-ETO in an inducible transgenic mouse model leads to a block in dif- ferentiation, but not to an enhanced proliferation.17 Di Genua et al.23 add to this result that AML1-ETO-express- ing pre-leukemic stem cells have a quiescent phenotype, leading to an accumulation of cells with high re-plating activity that are capable of going into cycle in a transplan- tation setting. Given that the double oncogenic event studied here is not enough to cause AML, the question that is now apparent is which additional events cause AML in patients. This work demonstrates that, even with a type of AML that has been studied in fine molecular detail for decades, the players of selection and clonal evo- lution in patients still have additional cards up their sleeve.
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