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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.


Related in: MedlinePlus

Summarised ki67 and BCL2 expression in control, AML and MDS subsets.CD34+CD38low, CD34+CD38high and CD34− (CD117+) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67lowBCL2high phenotype (C), which is defined using isotype control staining (see S3E Fig). Data includes 16 control BM, 29 CD34+ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34− plots include data from 11 CD34− diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34+ and open for CD34− AMLs) and genetic subgroups. F+N- denotes ITD+/NPM1wt patients (red filled squares, all CD34+), F+N+ denotes ITD+/NPM1mut patients (red open squares, all CD34−) and F-N+ denotes ITD−/NPM1mut (blue filled open/blue open squares for CD34+/CD34− respectively). CBF-AMLs are shown as green squares.
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pone.0163291.g006: Summarised ki67 and BCL2 expression in control, AML and MDS subsets.CD34+CD38low, CD34+CD38high and CD34− (CD117+) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67lowBCL2high phenotype (C), which is defined using isotype control staining (see S3E Fig). Data includes 16 control BM, 29 CD34+ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34− plots include data from 11 CD34− diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34+ and open for CD34− AMLs) and genetic subgroups. F+N- denotes ITD+/NPM1wt patients (red filled squares, all CD34+), F+N+ denotes ITD+/NPM1mut patients (red open squares, all CD34−) and F-N+ denotes ITD−/NPM1mut (blue filled open/blue open squares for CD34+/CD34− respectively). CBF-AMLs are shown as green squares.

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
Summarised ki67 and BCL2 expression in control, AML and MDS subsets.CD34+CD38low, CD34+CD38high and CD34− (CD117+) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67lowBCL2high phenotype (C), which is defined using isotype control staining (see S3E Fig). Data includes 16 control BM, 29 CD34+ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34− plots include data from 11 CD34− diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34+ and open for CD34− AMLs) and genetic subgroups. F+N- denotes ITD+/NPM1wt patients (red filled squares, all CD34+), F+N+ denotes ITD+/NPM1mut patients (red open squares, all CD34−) and F-N+ denotes ITD−/NPM1mut (blue filled open/blue open squares for CD34+/CD34− respectively). CBF-AMLs are shown as green squares.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163291.g006: Summarised ki67 and BCL2 expression in control, AML and MDS subsets.CD34+CD38low, CD34+CD38high and CD34− (CD117+) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67lowBCL2high phenotype (C), which is defined using isotype control staining (see S3E Fig). Data includes 16 control BM, 29 CD34+ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34− plots include data from 11 CD34− diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34+ and open for CD34− AMLs) and genetic subgroups. F+N- denotes ITD+/NPM1wt patients (red filled squares, all CD34+), F+N+ denotes ITD+/NPM1mut patients (red open squares, all CD34−) and F-N+ denotes ITD−/NPM1mut (blue filled open/blue open squares for CD34+/CD34− respectively). CBF-AMLs are shown as green squares.
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.


Related in: MedlinePlus