Developing a Humanized Murine Model for Co-Transplantation of Acute Myeloid Leukemia

Humanized mouse models and primary disease xenografts have produced significant translational insight into the biology of the bone marrow (BM) niche and aided drug discovery in acute myeloid leukemia (AML). To enable the study of cell-cell interactions between hematopoietic stem and progenitor cells (HSPCs) and AML as an emerging source of drug resistance, we describe two complementary approaches to humanized AML xenotransplantation models. One strategy utilizes the sequential transplantation of humanized hCD34+ cells followed by human AML cells. Recipient NBSGW mice (NOD.Cg-Kit W-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ), known for high rates of medullary engraftment, were conditioned with 15mg/kg busulfan followed by injection with either granulocyte colony-stimulating (G-CSF)-mobilized or BM-derived human CD34+ HSPCs. Flow cytometric analysis of the BM following injection of hCD34+ cells revealed efficient BM engraftment by both G-CSF- and BM- derived hCD34+ cells, demonstrated by BM human chimerism (hCD45%) of 63.7±10.0% by 8-weeks post-injection. Further BM hCD45 subtype analyses revealed B-cell (hCD45+ hCD19+) dominant repopulation (78.3±4.48%), followed by myeloid cell (hCD45+ hCD33+) repopulation (18.12±4.19%). T-cell repopulation was predictably minimal (0.008±0.01%). At 9- and 15- weeks post hCD34+ cells injection, GFP-expressing MOLM-14 AML cells (MOLM-14-GFP; 1E5, 1E6, or 2E6 cells) were injected. Flow cytometric analyses of MOLM-14-GFP BM chimerism up to 21 days post-injection showed complete absence of MOLM-14-GFP cells in the BM with no significant changes in overall hCD45 chimerism and hCD45 subtype composition compared to a PBS-injected control. These observations are consistent with a potential role for residual healthy hematopoietic cells in suppressing AML engraftment in the BM, previously shown in congenic studies. To test this hypothesis, we developed a second humanized AML model designed to investigate immunotherapeutic approaches for AML, where AML blasts are engrafted first, then followed by a human immune cell challenge. FA-AML1 or Kasumi-1 AML cells were injected into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) at 8 weeks of age. Injected mice were monitored for AML engraftment by HLA-DR expression in peripheral blood (PB). Engraftment occurred 6-12 weeks post-injection. After AML engraftment, we reconstituted a human immune system in the mice with a one-time injection of peripheral blood mononuclear cells (PBMCs), confirmed by flow cytometry for CD3+ T-cells (14.7±9.2%), CD33+ myeloid cells, and CD19+ B-cells. AML burden in the PB decreased 5-10% upon PBMC engraftment. In this model, a PB burden of <10% AML engraftment confers durable leukemic remission, whereas at >60% AML engraftment, leukemic cells persist in the periphery for over 100 days. Flow cytometric analysis demonstrated immune cell numbers peak in week 1 post-injection (30%), then decrease by week 4 to less than 1% in both the BM and PB, but persist at this low level for over 100 days. Altogether, these models confirm the ability of human hematopoietic cells to actively suppress leukemic engraftment in the BM microenvironment. More broadly, we demonstrate the development of two complementary humanized AML co-transplantation models that can attain dynamic ranges of human immune cell and AML cell engraftment to facilitate preclinical therapeutic development studies.