MISTRG6kitW41: Enhanced Engraftment in a Cytokine Humanized Patient-Derived Xenotransplantation Mouse Model

Myelodysplastic syndrome (MDS) is a heterogenous disorder of the hematopoietic stem cell (HSC) often resulting in bone marrow (BM) failure and progression to acute myeloid leukemia (AML). A lack of adequate mouse models that replicate the molecular and phenotypic diversity of MDS in patients has significantly limited preclinical studies. To fill this gap, we developed a novel MDS-patient-derived xenotransplantation (PDX) model in humanized mice, engineered via knock-in technology. Representation of the myeloid lineages is significantly improved in MISTRG compared to NSG mice with highly efficient and clonally faithful engraftment of MDS (Song et al. Nat Commun 2019) and that introduction of the humanized IL6 gene provides not only improved B-cell development but also overall enhanced engraftment (Yu et al. Blood, ASH 2017). We hypothesized that knocking in the murine c-kit^W41/W41 mutation into MISTRG6 (M6) mice, resulting in MISTRG6kit^W41 (M6k) mice, would further enhance engraftment of human HSCs and obviate the need for irradiation as was previously described when c-kit^W41/W41 was introduced into NSG mice (McIntosh et al. Stem Cell Reports, 2015).

To assess how the c-kit^W41/W41 mutation would function in the M6 mouse model, we engrafted both irradiated (IR) (80cGy) and non-irradiated (non-IR) M6/M6k mice with adult healthy donor peripheral blood mobilized stem cells (PBSCs), BM and fetal liver derived HSPCs. Engraftment levels were analyzed 12 weeks post engraftment via flow cytometry and validated by histology. Upon engraftment with fetal liver derived HSPCs, non-IR M6K mice demonstrated similar humanization levels as IR M6 mice with mean engraftment of 60% and 70% in peripheral blood (PB) and BM, respectively. Furthermore, non-IR M6k mice yielded better humanization than IR M6 mice when engrafted with adult cells. Mean PB and BM humanization levels in BM derived HSPC engrafted mice were 60% and 80% in non-IR M6K mice, respectively, compared to 20% and 40% in PB and BM of IR M6 mice, respectively. PBSCs engraftment in non-IR mice was significantly higher in M6K mice with increases in average engraftment levels from 1% to 15% and 5% to 30% in PB and BM, respectively, when compared to their M6 mice counterparts. Interestingly, even in the ckit^W41/W41 mutant background, low dose irradiation led to further increases in BM engraftment, from 30% to 57%.

For the study of MDS it is imperative to assess erythroid and megakaryocytic potential. Megakaryopoiesis was significantly increased in BM of non-IR M6K compared to M6 mice and was further enhanced by irradiation. Similarly, erythropoiesis in M6K mice was higher than that in M6 mice with a shift from CFU- erythroblast to Pro- and intermediate-erythroblasts.

Next we challenged M6k mice by engrafting MDS samples ranging from low to high grade MDS. We tested a variety of samples harboring various mutations including, but not limited to: SRSF2, U2AF1, TET2, RUNX1, IDH2, and ASXL1. In addition, samples represented different time points of the disease, including diagnosis, remission and progression to secondary AML. At 11 weeks post engraftment, xenografts of MDS samples at time of diagnosis and during remission had average BM engraftment levels of 13% and 5% huCD45⁺, with CD34⁺ cells representing an average of 11% and 17% of human CD45⁺ cells, respectively. At 13 weeks post engraftment, xenografts of secondary AML samples were assessed in BM and spleen. HuCD45⁺ engraftment levels in BM and spleen averaged to 89% and 73% with double positive CD33⁺CD34⁺cells representing on average 37% and 71% of human CD45⁺, respectively. Whereas healthy CD34⁺ grafts in MISTRG mice give rise to high percentage of lymphoid cells, MDS engrafted mice did not have human T-cells.

To determine whether CD34⁺ engrafted M6k mice would provide faithful replication of the primary human MDS, we engrafted sequential samples from a single patient, at time of MDS diagnosis and at time of progression to secondary AML. scRNA-seq analysis of CD34⁺ cells was performed to identify differentially expressed genes (DEGs) between patient sample/ xenografts and an age matched healthy control. Thereafter, DEGs from both comparisons were intersected. M6k engrafted MDS CD34⁺ gave rise to grafts highly faithful to the original patient sample confirming that MISTRG6 and in particular MISTRG6 enhanced by introduction of the ckit^W41/W41 are highly useful tools for the study of MDS.