Early and Late Leukemic Stem Cells in Murine Leukemia Models

Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood.

In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 10⁵ BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia.

In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML.

In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations.

Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation.

In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse.