Enhanced and Accelerated Repopulation of Mutant C-Kit Immunodeficient Mice By Transplants of Primary Chronic and Acute Myeloid Leukemic Human Cells

Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγc^null (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a Sirpα^NOD-Rag1^-/--IL2Rγc^-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W⁴¹/W⁴¹genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols.

Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34⁺ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x10⁶ CD34⁺ cells from 1 CP-CML patient or ~10⁶ CD34⁺ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x10⁶ CD34⁺ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant.

Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible.