MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

Abstract

The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

Complexity of cancer stem cells

“…Consecutive repetition of such assays (replating) can result in the isolation of subpopulations of hematopoietic cancer cells with self-renewal ability and also allows determination of whether genes of interest have the potential to confer selfrenewal activity in non-transformed hematopoietic cells. 22,23 Spheroid formation assays were performed originally to isolate neural stem or progenitor cells and are now often used to evaluate stem-like characteristics of tumor cells. Suspension culture in medium not containing serum but supplemented with basic fibroblast growth factor and epidermal growth factor allows the isolation of tumor cells with self-renewal capacity.…”

“…8,38 On the other hand, some studies have suggested that the cells of origin for mouse AML stem cells are not only restricted to HSCs but also include progenitor cells committed to the myeloid lineage using bone marrow transplantation (BMT) model. 22,39 In the case of B acute lymphoblastic leukemia (B-ALL), although CSCs were found in cell subpopulations expressing HSC markers or B cell markers, [40][41][42][43][44] the cells of origin for B-ALL stem cells remain unclear. We recently established a Myc-induced mouse model of pre-B-ALL based on BMT model.…”

The cell of origin and the leukemia stem cell in acute myeloid leukemia

“…Huntly et al 63 HSC from a knock-in locus does not. 60 As pointed out above, the results from the mouse models are obtained in the setting of perturbed hematopoiesis, and are confounded by retroviral insertional mutagenesis, the poorly controlled expression levels of the oncogenes and the spontaneous acquisition of additional mutations.…”

Transcriptional memory of cells of origin overrides β‐catenin requirement of MLL cancer stem cells

“…In spite of the fundamental difference between stem cells and their progenitors in self-renewal ability, we and others have shown that phenotypically and genetically indistinguishable cancers, including MLL-rearranged leukemia, can arise from not only stem cells, but also their immediate downstream short-lived progenitors with distinctive transcriptional programs So et al, 2003;Huntly et al, 2004;Krivtsov et al, 2006;Visvader, 2011;Blanpain, 2013). Consistently, gene expression signatures associated with stem cells and progenitors correlate with different clinical outcomes in AML (Eppert et al, 2011;Krivtsov et al, 2013).…”

“…Most of the current targeted therapies and their intended clinical utility are developed without considering the cancer cells of origin, which can have distinctive self-renewal and transcriptional properties. This traditional view has been challenged by the identification of phenotypically indistinguishable leukemia from different cells of origin So et al, 2003;Huntly et al, 2004), which exhibit different responses to standard chemotherapy treatment ( A, B Venn diagram showing the overlap of 33 commonly enriched up-regulated (A) and 38 commonly enriched down-regulated (B) highly significant (FDR < 0.05) gene sets between indicated comparisons (i.e., b-catenin inactivation in Hoxa9 À/À LSK-MLL-ENL, Hoxa9 inactivation in Ctnnb1 À/À LSK-MLL-ENL cells), including "Hoxa9/ Meis1_DN" (Hess et al, 2006), "LSC_mainentence_down" (Somervaille et al, 2009), "Hoxa9/Meis1_UP" (Hess et al, 2006), and "LSC_maintenance_up" (Somervaille et al, 2009) as indicated. C A small set of genes is synergistically regulated by b-catenin and Hoxa9 (the effect of knockout of both genes is smaller or larger than could have been predicted from their single knockouts).…”