Limits...
Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia

View Article: PubMed Central - PubMed

ABSTRACT

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

No MeSH data available.


Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34+AML BM (B) and CD34− AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67lowBCL2high, is observed in both AML types (B and C) and highlighted by black rectangle.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036879&req=5

pone.0163291.g005: Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34+AML BM (B) and CD34− AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67lowBCL2high, is observed in both AML types (B and C) and highlighted by black rectangle.

Mentions: Although ROS, BCL2 expression and cell-cycle status have previously been functionally linked [2,23,33,34] there are as yet no data assessing these therapeutically relevant parameters in parallel for normal or leukemic SPC subsets. We therefore developed and performed a combined assay on control BM (n = 16), AML diagnosis samples (n = 40) and MDS samples (n = 12). In control BMs CD34+CD38high cells expressed higher levels of both ki67 and BCL2 than CD34+CD38low cells (example in Fig 5A). Closer inspection (in 11 cases) showed that although there was no significant difference in ki67 between CMP, GMP and MEP subsets, BCL2 expression followed a GMP>CMP>MEP hierarchy (S3A and S3B Fig) as observed for ROS levels. Within the more immature CD34+CD38low fraction, all the subsets had low BCL2 but LMPP had high ki67 expression unlike the quiescent HSC/MPP populations (S3A and S3B Fig). CD34− cells (mixed myeloid/erythroid) in controls were as proliferative as CD34+CD38high SPCs but had low BCL2 at levels similar to CD34+CD38low SPCs and MEP (Fig 6A and 6B). The observed BCL2 upregulation in the GMP subset suggests that at this myeloid maturation stage when higher ROS levels prime for proliferation/maturation, BCL2 might be transiently expressed for protection against further oxidative stress.


Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia
Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34+AML BM (B) and CD34− AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67lowBCL2high, is observed in both AML types (B and C) and highlighted by black rectangle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036879&req=5

pone.0163291.g005: Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34+AML BM (B) and CD34− AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67lowBCL2high, is observed in both AML types (B and C) and highlighted by black rectangle.
Mentions: Although ROS, BCL2 expression and cell-cycle status have previously been functionally linked [2,23,33,34] there are as yet no data assessing these therapeutically relevant parameters in parallel for normal or leukemic SPC subsets. We therefore developed and performed a combined assay on control BM (n = 16), AML diagnosis samples (n = 40) and MDS samples (n = 12). In control BMs CD34+CD38high cells expressed higher levels of both ki67 and BCL2 than CD34+CD38low cells (example in Fig 5A). Closer inspection (in 11 cases) showed that although there was no significant difference in ki67 between CMP, GMP and MEP subsets, BCL2 expression followed a GMP>CMP>MEP hierarchy (S3A and S3B Fig) as observed for ROS levels. Within the more immature CD34+CD38low fraction, all the subsets had low BCL2 but LMPP had high ki67 expression unlike the quiescent HSC/MPP populations (S3A and S3B Fig). CD34− cells (mixed myeloid/erythroid) in controls were as proliferative as CD34+CD38high SPCs but had low BCL2 at levels similar to CD34+CD38low SPCs and MEP (Fig 6A and 6B). The observed BCL2 upregulation in the GMP subset suggests that at this myeloid maturation stage when higher ROS levels prime for proliferation/maturation, BCL2 might be transiently expressed for protection against further oxidative stress.

View Article: PubMed Central - PubMed

ABSTRACT

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

No MeSH data available.