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Figure 5. G1blow acute lymphoblastic leukemia (ALL) cells are characterized by cell death insensitivity. (A and B) Low sensitivity for G1blow sorted ALL cells for spon- taneous (A) and drug induced (B) cell death measured by flow cytometry. Cell death upon culture or exposure (24 h) to prednisolone (PRED, 250, 500 or 1000 mg/mL) or arabinosylcytosine (ARA-C, 10, 100, 500 mg/mL) in cell cycle annotated, sorted subfractions, (mean ± Standard Deviation). Data points represent technical repli- cates within 4 individual leukemia samples, unpaired t-test with Welch correction; *P≤ 0.05; **P≤ 0.01; ***P≤ 0.001; n.s.: not significant.
Leukemia initiating cells in ALL
sic propensity of primograft ALL cells of G1blow and G2/M subfractions to undergo cell death, both upon ex vivo cul- ture and in response to prednisone and cytarabine, two drugs used in protocols to treat pediatric ALL patients. Interestingly, all 4 primograft samples analyzed showed lower rates of spontaneous and drug-induced cell death in G1blow as compared to G2/M cells (Figure 5A and B).
Leukemia-initiating activity in acute lymphoblastic leukemia is characterized by distinct cellular oxidative states
Recently, low levels of ROS were described in cellular subfractions associated with stem cell properties.30-32 We, therefore, investigated ROS activity in xenograft samples of TTLshort or TTLlong phenotypes. Lower ROS activities were observed in rapidly engrafting, TTLshort/poor progno- sis leukemia cells (Figure 6A) and in G1blow-sorted cells compared to G2/M-sorted cells (Figure 6B).
We observed that cells with low ROS activity (ROSlow) were almost exclusively allocated to G0/G1 cell cycle phases, whereas cells with high levels of ROS activity (ROShigh) included those in later S and G2/M phases (Figure 7A and B), suggesting that the ALL cell’s oxidative state is indicative of its leukemia-initiating activity. To further address this hypothesis on a functional level, we investigated 3 primograft ALL samples and sorted cellular subfractions according to high or low ROS levels (upper or lower 15% fluorescence intensity, ROShigh and ROSlow
A
cells, respectively). Upon transplantation into recipient animals, both sorted subfractions led to leukemia engraft- ment. However, in all three leukemias, ROSlow cells dis- played a higher repopulating activity and were associated with significantly shorter leukemia-free survival in con- trast to prolonged engraftment and survival in mice trans- planted with ALL cells with a high oxidative state (ROShigh) (Figure 7C). In HSC, a ROS-MAP kinase axis has been implicated in negative regulation of the life span of the cells.30,33 A high LIC potential of the ROSlow subtype was also found in T-ALL and functionally linked to expression of PKC-θ as a consequence of deregulated NOTCH signaling.34 However, the analysis of MAP kinase p38α/β-expression did not reveal significant differ- ences between the engraftment phenotypes (Online Supplementary Figure S3A and B) and, in contrast to the T- ALL data reported, we did not detect PKC-θ expression in our BCP-ALL cells (Online Supplementary Figure S3C).
Taken together, we observed that LIC are not rare in BCP-ALL, pointing to a stochastic stem cell concept in this type of leukemia. We identified distinct LIC activities in cell cycle annotated cellular subfractions, with early cycling (G1blow) cells possessing the highest LIC potential independently of the overall engraftment phenotype and high/low relapse risk. Moreover, early cycling (G1blow) cells with high LIC potential are characterized by a tran- scriptional stem cell profile, cell death resistance, and a low oxidative state, which in turn results in higher LIC
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