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K-Ras mutation depletes pre-leukemic HSC
leukemic HSC, it is preferable that the model used retains a relatively unperturbed hematopoietic hierarchy without overt leukemia or other malignancy. In addition, the use of “knock-in” models of oncogenes that are expressed from their own promoter at their original loci is impor- tant to retain faithful expression patterns within the hematopoietic hierarchy. Both these conditions are ful- filled by the Aml1ETO/+;KrasG12D/+;Mx1-Cretg/+ model used here.
The results from this study differs to other models that demonstrate that Ras mutations collaborate with pre- leukemic mutations to develop an AML, such as Dnmt3a- /-;KrasG12D/+ and CbfbMYH11/+;NrasG12D/+, where both models develop a more aggressive disease when combined.43,44 Both models lead to transformation of myeloid progeni- tors, in contrast to the lack of transformation seen in AKM mice, even after serial transplantation. Both Dnmt3a-/-;KrasG12D/+ and CbfbMYH11/+;NrasG12D/+ models do, however, result in a loss of LT-HSC which is consistent with our results. The lack of transformation of myeloid progenitors in AKM mice gave us a unique opportunity to study pre-leukemic stem cells functionally in the absence of progenitor cell transformation as seen in the other models.
RNA sequencing revealed genes that may underlie the observed HSC phenotype of AM and AKM mice. Gzmb is a serine protease that has recently been reported to be important in HSC function.35 Knock-out of Gzmb confers enhanced self-renewal to HSC in a cell-intrinsic manner.
Figure 6. Schematic summarizing the effect of K-RasG12D on pre-leukemic hematopoietic stem cells (HSC). HSC that acquire Aml1-ETO gain a competitive advantage, leading to an expansion in HSC number. Acquisition of K-RasG12D and Aml1-ETO concurrently leads to HSC depletion. It remains to be determined whether sequential acquisition of Aml1-ETO followed-by K-RasG12D might sup- port development of leukemia. As this was not tested in the current study, this is depicted as a dotted arrow.
Gzmb deficient mice also had a better survival rate after administering 5-FU.35 Gja1 encodes gap junction channel protein connexin 43 found on HSC. Gja1 deficient HSC were shown to be more quiescent after 5-FU treatment. Gja1 deficient HSC also developed an accumulation of ROS.37 ROS levels in HSC have been shown to play an important role in hematopoietic reconstitution;45 however, oxidative phosphorylation gene expression was not enriched in AM HSC versus CON suggesting down-regu- lation of Gja1 in AM HSC did not lead to an increase in ROS. GJA1 has lower expression on CD34+ BM cells from AML patients with AML1-ETO compared to WT CD34+ BM cells.46 Down-regulation of GZMB and Gja1 have also both been identified in human and murine leukemic stem cells, respectively.47-49 Together, down-regulation of GZMB and GJA1 may contribute to the AML1-ETO-associated competitive advantage of HSC that we observed, and could potentially be important for pre-leukemic HSC per- sistence after chemotherapy. Importantly, expression of both Gzmb and Gja1 were increased in the presence of K- RasG12D, indicating that re-expression of these genes may have contributed to the loss of HSC function and self-renewal. However, as we identified disruption of mul- tiple genes and pathways in AKM HSC, it seems likely that the underlying mechanistic basis for loss of functional Aml1-ETO expressing HSC associated with K-RasG12D is complex, and is unlikely to be attributable to one single target gene and more likely to involve an interplay of many genes and pathways.
Mutations are acquired in a stepwise manner in AML, and the consequence of the type and order of the muta- tions acquired will make the HSC more or less likely to facilitate subsequent evolution to leukemia.50 Our find- ings help to provide a cellular and molecular basis for the observed patterns of clonal evolution during AML devel- opment. HSC that acquire Aml1-ETO gain a competitive advantage, leading to an expansion in HSC number, increasing the pool of cells available to acquire additional mutation that could eventually promote leukemia devel- opment. HSC that acquire a Kras mutation, either alone or in combination with Aml1-ETO, are depleted. A potential limitation of our study is that the mutations are introduced simultaneously rather than sequentially. New model systems that allow knock-in mutations to be intro- duced sequentially, and potentially also in specific cellular compartments, warrant further investigation (Figure 6).
In summary, our findings help to explain why signaling mutations such as KRAS are not observed within pre- leukemic HSC in AML patients and usually occur as a late event in leukemogenesis. The distinct molecular signa- tures associated with pre-leukemic mutations in HSC suggest that approaches to target leukemic versus pre- leukemic stem cell expansion are likely to be different.
Acknowledgments
A.J.M received funding from a Medical Research Council Senior Clinical Fellowship (MR/L006340/1). This work was supported by the Medical Research Council (MC_UU_12009, and G0701761, G0900892 and MC_UU_12009/7 to CN). The authors acknowledge the contributions of the WIMM Flow Cytometry Facility, supported by the MRC HIU; MRC MHU (MC_UU_12009); NIHR Oxford BRC and John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170) and by the WIMM Strategic Alliance awards G0902418 and MC_UU_12025.
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