Despite major advances in human to mouse xenografts, most of the human acute myeloid leukemia (AML) samples fail to adequately engraft available strains of mice. The reasons for the disparate results with AML samples remain unclear, but likely include elements of homing, survival in a foreign niche, expansion in the absence of specific human growth factors and supporting stromal cells, escape from immune surveillance and residual innate immunity in the mice, as well as intrinsic differences among AML samples. One general strategy for improving engraftment has been the development and use of increasingly immunodeficient mouse strains (reviewed in1). For example, targeting of the IL2RG gene in the NOD/SCID (NS) strain by either complete knockout or truncation of the intracellular portion (NOD/LtSz-scid IL2RG: NSG and NOD/shi-scid IL2RG, respectively) was shown to diminish innate immunity, in particular macrophage function and NK activity, thereby improving engraftment by normal human CD34+ cells and AML cells. 2, 3 Alternatively, transgenic expression of hSCF, hGM-CSF and hIL-3 (three non-or poorly cross-reacting cytokines) in the NOD/SCID background (NOD/LtSz-scid-SGM3, NSS) led to improvements in expansion of normal human myeloid cells, as well as slight gains in engraftment of AML samples. 4, 5

In this study, we have generated a new mouse strain by crossing the NSG mouse with the NSS mouse to generate the NOD/LtSz-scid IL2RG–SGM3 mouse (NSGS)(Figure 1a). Tail clip gDNA PCR and Enzyme-linked immunosorbent assay were used to identify mice with IL2RG knockout and human cytokine expression. Enzyme-linked immunosorbent assay assays for IL-3 and granulocyte macrophage-colony stimulating factor were well correlated and clearly differentiated cytokine homozygotes from heterozygotes and, therefore, were used to identify suitable breeder pairs for further colony expansion (Figure 1b). Although our Enzyme-linked immunosorbent assay measurements differed somewhat from those reported for the NSS mouse2, we observed very similar levels of expression between the newly generated NSGS mice and our line of NSS mice (Figure 1c). Peripheral blood (PB) complete blood counts yielded no consistent, meaningful differences among strains, indicating that no major changes in myeloid, lymphoid, erythroid or platelet levels occurred in the PB of these mice in response to the human cytokines (Figure 1d). In this study, we focused on the possible improvements that could be realized in human AML and pre-leukemia models, given the dependency of many of these cells on cytokine stimulation for growth and the already proven superiority of mice lacking IL2RG for AML xenografts. For these experiments, we first used a cord blood-derived AML driven by retroviral expression of the MLL-AF9 oncogene6 into which we subsequently transferred an activated NRas cDNA containing a codon 12 (G to D) point mutation (MA9-NRas). We have found that the NRas ‘second hit’significantly and reproducibly decreases latency of disease and allows quick development of AML in non-irradiated NS and NSG mice (MW and JCM, unpublished