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Leukemia initiating cells in ALL
activity in functional repopulation experiments, opening up ROS modulation as a perspective for treatment.
Discussion
In this study, we used engraftment phenotypes identi- fied in our NOD/SCID/huALL model to investigate char- acteristic features of LIC in BCP-ALL. First, we did not obtain any evidence of preferential accumulation of LIC in subpopulations defined by surface markers. Since high- risk leukemia was characterized by higher LIC frequencies and higher cell cycle progression in vivo, we analyzed leukemia-initiating capacities of cells in different cell cycle compartments. Using a vital DNA/RNA staining method, we found that: (i) all cells possess LIC potential in vivo independently of the cell cycle phase of origin (i.e. G1a, G1blow, G1bhigh and G2/M); (ii) early G1b cells entering into S phase (G1blow) lead to the most rapid engraftment, sug- gesting that G1blow cells are the first engrafting leukemia- initiating cells; (iii) G2/M cells possess significantly lower engraftment potential; (iv) these features were maintained independently of the leukemia cell engraftment pheno- types; and (v) G1blow LICs exhibit low metabolic activity and ROS potential, probably associated with increased cell death resistance. These findings have implications for the characterization of the putative leukemic stem cell in ALL, the heterogeneity of leukemic clones and sensitivity and/or resistance of functionally defined subpopulations for treatment approaches using targeted or conventional therapy.
Two models have been proposed for cancer stem cells or LIC. According to the hierarchical concept, a few imma- ture cells harboring stem cell properties are considered to be able to generate their progeny and give rise to leukemia. While a number of data have supported this hierarchical model in acute myeloid leukemia,2,3 for ALL and particularly for BCP-ALL a more stochastic model, in which literally every cell, including more mature and dif- ferentiated phenotypes, is considered to be able to initiate leukemia, appears to be valid. This implies that the estab- lishment of the leukemia phenotype in patients and upon transplantation in mice may be an intrinsic capacity of individual cells independently of surface phenotype and maturation stage.9-13 This concept corresponds to the find- ings of leukemia initiation by low cell numbers and by all subfractions seen in our huALL mouse model. Another issue along this line is whether or not LIC are derived from quiescent, cycling or dividing subpopulations. The data from our in vivo labeling experiments indicate that increased proliferation is associated with rapid engraft- ment and rapid development of full-blown leukemia resulting in the TTLshort phenotype defining a poor progno- sis subgroup. Thus, one may have expected that actually dividing cells (G2/M) harbor the highest leukemogenic potential upon transplantation. However, the resting/early recruitment compartment (G0, G1) contained similar reconstitution potential and the early recruitment pheno- type (G1blow) exhibited the highest leukemogenic potential in the transplantation experiments.
The potential of both G0/G1 and G2/M cells to recon- stitute the leukemia phenotype in vivo is a new finding compared to previous data on human and murine HSC. Indeed, human and murine HSC have been reported to be heterogeneous with respect to the cell cycle: while only
cells in G0/G1 sufficiently reconstituted hematopoiesis in sublethally irradiated mice,18,35-38 cells in S-G2/M did not repopulate the bone marrow at all or had only a minimal engraftment potential.35,39
The higher in vivo leukemogenic activity of G1blow cells suggests that these cells are likely the first out of the leukemic bulk to engraft in recipients. The higher “stem cellness” of this subpopulation is also supported by gene signatures previously assigned to stem cell activity. In con- trast, G2/M cells with lower LIC potential were negatively associated with stem cell-like profiles or were enriched for genes characteristic of short-term HSC or mature cells.8,25- 27,29 Importantly, the G1blow and G2/M features are main- tained irrespective of the engraftment phenotype suggest- ing that these characteristics are conserved features of BCP-ALL LIC. However, the engraftment potential of both subpopulations, irrespective of stem cell signatures, emphasizes the stochastic nature of LIC in ALL, in line with data recently reported on similar engraftment activi- ties of slowly or rapidly dividing BCP-ALL cells.40
LIC frequencies calculated in our NOD/SCID/huALL model showed variations associated with the patients’ outcome and engraftment phenotype, suggesting that speed of ALL repopulation is a measure of LIC activity, as suggested before.41 Thus, we analyzed repopulation times to evaluate LIC activities of sorted cells. In both TTLshort/poor prognosis ALL samples, engraftment was observed upon transplantation of down to 100 cells, in line with reported high LIC frequencies in studies including poor outcome BCP-ALL, even in the more immunodefi- cient NSG mouse strain.9,40,42 However, higher minimum cell numbers of up to 103 cells were required to initiate leukemia in both TTLlong samples, of which one also showed hyperdiploidy, similar to numbers observed in studies including favorable prognosis ALL.10-13,43,44 Accordingly, TTLlong phenotypes were always observed in ALL with the favorable prognostic features hyperdiploidy or ETV6/RUNX1 rearrangements,14 suggesting lower LIC frequencies in good outcome BCP-ALL.
In TTLshort leukemia, gene expression, more cells in active mitosis and the in vivo bromodeoxyuridine labeling data indicate a higher proliferation rate, including activated mTOR signaling.15 Along this line, effects of the mTOR pathway on cell cycle progression and particularly regula- tion of the G1 phase have already been described.45,46 Accordingly, in addition to a distinctive transcriptional program,14 different basal mTOR activation15 and deficient apoptosis signaling16 were found in the TTLshort versus TTLlong phenotypes.
The higher LIC capacity of G1blow appears to result from a favorable functional status. G1blow cells were less prone to undergo spontaneous and drug-induced cell death ex vivo whereas G2/M slowly engrafting cells showed a greater predisposition to both intrinsic and induced cell death. Increased intrinsic cell death resistance may be a consequence of a block in cell death pathways or alter- ation of the metabolic state. Recent work showed that dif- ferent levels of ROS and a lower mitochondrial mass dis- tinguished cells with higher LIC activity.31,32 We found that G1blow cells were characterized by lower ROS activity compared to G2/M cells. Moreover, when analyzing ROS levels in cell cycle compartments, we observed that ROSlow cells were almost exclusively found in G0/G1, while ROShigh cells were progressing not only through the G0/G1 but also the S and G2/M phases of the cell cycle.
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