Leukemia Initiating Cells In Acute Lymphoblastic Leukemia Are Characterized By Low Metabolic Activity and Enriched In Early Cell Cycle

Leukemia initiating cells (LICs) in acute lymphoblastic leukemia (ALL) have been considered to be organized in a similar hierarchical fashion as in hematopoietic differentiation and acute myeloid leukemia (AML). Following this hierarchical concept, only few immature leukemia cells at the apex of a differentiation tree possess self renewal and differentiation potential and are capable of propagating leukemia upon transplantation onto immunodeficient mice. This view however has been challenged by recent findings showing that LICs in ALL are not restricted to few phenotypically defined cellular subsets but that both immature and mature B-cell precursor (BCP)-ALL cells are equally able to reconstitute leukemia in vivo, thus indicating a stochastic model for LICs in ALL. However, identification and characterization of a distinct leukemia initiating cell population in ALL remains still elusive although of key clinical importance since these cells giving rise to the disease need to be eradicated to cure the patient.

To further characterize LICs in BCP-ALL, we assessed leukemia initiating activities of cellular ALL subpopulations corresponding to distinct cell cycle phases using our NOD/SCID/huALL xenotransplantation model. Patient derived xenograft B-cell precursor ALL samples were stained, sorted according to cell cycle phases (i.e. G0/G1 and G2/M) and transplanted onto recipient animals. Interestingly, transplantation of all four sorted cellular leukemia sub-fractions led to engraftment and development of leukemia showing leukemia initiating capacity irrespective of cell cycle stage. However, cells isolated from early phases of the cell cycle (G0/G1) led to earlier leukemia engraftment in the recipients in contrast to cells from later cell cycle phases with cellular G2/M sub-fractions constantly showing the longest time from transplantation until leukemia engraftment.

In order to define additional biological features characteristic for leukemia initiating cells in ALL, we investigated the metabolic activity of cellular sub-fractions which had been functionally characterized with respect to their NOD/SCID mouse repopulating activities. For this purpose, levels of reactive oxygen species (ROS) were analyzed by detection of oxidation-specific fluorescence of chloromethyl-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) in patient derived xenograft ALL samples. Most interestingly, low ROS levels indicating inferior metabolic activity were identified in leukemia cell fractions of early G0/G1 cell cycle phases, which also showed increased NOD/SCID repopulating activity in functional assays. In contrast, leukemic cell populations of late G2/M cell cycle phases characterized by prolonged engraftment and lower leukemia initiating capacity, displayed increased metabolic activity as reflected by high ROS levels. Vice versa, analysis of cell cycle distribution with respect to metabolic activity revealed that leukemia cells characterized by low ROS levels (10% lowest) are allocated to early cell cycle whereas the majority of cells with high levels of ROS (10% highest) are shifted towards later cell cycle phases, denoting that the ALL cell’s oxidative state is indicative for its leukemia initiating activity.

Taken together, in ALL cells of all cell cycle phases possess the ability to engraft and reconstitute leukemia upon transplantation into recipient mice. Thus, all ALL cells harbor leukemia initiating capacities irrespective of cell cycle phases, a finding which is in line with recent data showing that LICs in ALL are frequently and ubiquitously found. Most importantly, despite LIC activity in all cellular sub-fractions of the cell cycle, a higher leukemia repopulating activity was identified in ALL cells which display low ROS levels and originate from G0/G1 subpopulations indicating that leukemia initiating cells in ALL are enriched in early cell cycle phases and characterized by low metabolic activity.

In conclusion, our data indicate that leukemia initiating potential in BCP-ALL is not restricted to distinct cellular subpopulations but that all cells show LIC activity with cells in early cell cycle and low metabolic activity representing the driving leukemia initiating cell compartment.