Cbfb-MYH11 + Leukemia Stem Cells Are Heterogeneous and Show Dynamic Marker Expression

Leukemia stem cells (LSCs) are the population of cells that are resistant to chemotherapy and the likely cause of relapse. Therefore, eradication of LSCs is a principle aim of many novel therapeutics currently under development. LSCs are thought to share some properties with normal hematopoietic stem cells (HSCs), including the ability to self-renew and the expression of similar cell surface markers. However, recent work in xenograft models has raised the possibility that the LSC compartment may be more heterogeneous than previously thought. In previous work using knockin mice expressing the Cbfb-MYH11 oncogene, we found that cells lacking the cell surface marker CD131 are enriched for LSC activity in vivo . Interestingly, this population is heterogeneous with respect to other cell surface markers. Two of the markers differentially expressed within the LSC enriched population are CKIT, a marker of HSCs, and IL1RL1, which is expressed on both progenitor and mature myeloid cells. To test whether these markers can be used to further enrich for LSCs, we sorted CD131^- cells into HSC-like (CD131^- IL1RL1^- CKIT⁺), early progenitor-like (CD131^- IL1RL1⁺ CKIT⁺, and CD131^- IL1RL1^- CKIT^-), and late progenitor-like (CD131^- IL1RL1⁺ CKIT^-) populations. Using in vitro colony forming assays, we found that cells with an HSC-like immunophenotype gave rise to more colonies than the cells with a more differentiated immunophenotype, consistent with previous work by others. To test whether colony forming ability correlates with LSCs activity in vivo, we transplanted each sub-population into congenic mice. Surprisingly, we found that each of these sub-populations caused leukemia with the CD131^-IL1RL1⁺ CKIT⁺ population appearing to have the highest LSC frequency and the CD131^- IL1RL1^- CKIT^- population appearing to have lowest LSC frequency. This finding implies that both HSC-like and more differentiated populations have LSCs activity, and that colony forming assays may underestimate LSC activity and frequency. To address which of these populations may be the most likely responsible for relapse, we measured the proliferation rate of each sub-population in vivo . We found that the CD131^- IL1RL1⁺ CKIT⁺ population had the highest proliferation rate, and the CD131^- IL1RL1^-CKIT^- populationshowed lowest proliferation rate. To evaluate the chemosensitivity of each sub-population, we examined the viability of Cbfb-MYH11⁺ leukemia cells in vitro with the standard chemotherapy drugs, cytarabine and doxorubicin. As expected, we found that the less proliferative populations had higher viability in the presence of either drugs, as compared to the more highly proliferative populations. Recent work in ALL indicates that there is a degree of plasticity in LSCs with respect to cell surface marker expression. To test whether cell surface marker expression is dynamic in AML, we transplanted mice with equal numbers of each sorted LSC sub-population and examined marker expression 24 hrs later. We found that each sub-population altered their cell surface marker expression, re-establishing the marker distribution found in the unsorted leukemia cells. To investigate whether marker plasticity is affected by drug treatment, we examined cell surface marker expression in sorted cells 24 hrs after incubation with doxorubicin. Surprisingly, we saw that the relatively highly proliferating subpopulations acquired the marker profile of the more slowly proliferating subpopulations. These data indicate that changes of cell surface marker in the presence of drug may lead to chemoresistance.

Collectively, this study demonstrates that multiple leukemia populations have LSC activity. This implies that, unlike normal HSCs, LSCs are likely not at the apex of a hierarchical differentiation scheme. We also found that different LSC sub-populations display differences in proliferation rates and LSC frequency. Finally, we show that LSCs cell surface marker expression is dynamic, especially in response to chemotherapy. These findings may in part explain the difficulties in developing LSC targeted therapies based on cell surface receptors.