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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

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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.


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ROS levels in immunophenotypic stem/progenitor cells from representative normal control, myelodysplastic and AML bone marrows.Flow cytometry histograms show intracellular ROS levels (using DCF) in HSC/MPP/LMPP and CMP/GMP/MEP of representative control BM (A), umbilical cord blood (UCB) (B), four AML BM samples at diagnosis (C), two MDS-RAEB BM samples (D), and two MDS-no-EB BM samples (E). DCF staining with normalised mean fluorescence intensity of each of these populations is shown (using DCF staining of reference lymphocyte population shown as light grey filled histogram, while for stem/progenitor subsets staining is shown as open histograms with different coloured lines as indicated on each plot). Additional examples of DCF staining of AML and MDS samples are shown in S1F–S1H Fig.
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pone.0163291.g001: ROS levels in immunophenotypic stem/progenitor cells from representative normal control, myelodysplastic and AML bone marrows.Flow cytometry histograms show intracellular ROS levels (using DCF) in HSC/MPP/LMPP and CMP/GMP/MEP of representative control BM (A), umbilical cord blood (UCB) (B), four AML BM samples at diagnosis (C), two MDS-RAEB BM samples (D), and two MDS-no-EB BM samples (E). DCF staining with normalised mean fluorescence intensity of each of these populations is shown (using DCF staining of reference lymphocyte population shown as light grey filled histogram, while for stem/progenitor subsets staining is shown as open histograms with different coloured lines as indicated on each plot). Additional examples of DCF staining of AML and MDS samples are shown in S1F–S1H Fig.

Mentions: We first asked whether low ROS levels measured with the redox-sensitive probe DCF were restricted to, and could thus differentiate HSC from, other immunophenotypic normal human hematopoietic progenitor populations (S1 Fig). In control BM samples (n = 24) total CD34+CD38high cells (fraction containing committed progenitors) had higher DCF staining than total CD34+CD38low cells (fraction containing more immature SPC) as expected. Further analysis within these two immunophenotypic compartments (representative examples in Fig 1A) confirmed that HSC had low DCF staining (median standardised-DCF-MFI = 2.05), as did the MPP population (median 1.98). The infrequent LMPP subset (<5% of CD34+CD38low) had relatively higher DCF staining (median 2.48), compared to both HSC and MPP. Interestingly there was a differential ROS profile within the CD34+CD38high fraction: GMP had highest DCF staining (median 6.64), followed by CMP (median 3.41) and then MEP (median 2.88) (complete summarised data in Fig 2A and 2B). The hierarchy in DCF staining with GMP>CMP>MEP and LMPP>HSC/MPP was also observed in umbilical cord blood (UCB) samples (n = 4) (example in Fig 1B), suggesting that this ROS profile is representative of normal non-malignant human SPC and not specific to the BM microenvironment.


Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia
ROS levels in immunophenotypic stem/progenitor cells from representative normal control, myelodysplastic and AML bone marrows.Flow cytometry histograms show intracellular ROS levels (using DCF) in HSC/MPP/LMPP and CMP/GMP/MEP of representative control BM (A), umbilical cord blood (UCB) (B), four AML BM samples at diagnosis (C), two MDS-RAEB BM samples (D), and two MDS-no-EB BM samples (E). DCF staining with normalised mean fluorescence intensity of each of these populations is shown (using DCF staining of reference lymphocyte population shown as light grey filled histogram, while for stem/progenitor subsets staining is shown as open histograms with different coloured lines as indicated on each plot). Additional examples of DCF staining of AML and MDS samples are shown in S1F–S1H Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163291.g001: ROS levels in immunophenotypic stem/progenitor cells from representative normal control, myelodysplastic and AML bone marrows.Flow cytometry histograms show intracellular ROS levels (using DCF) in HSC/MPP/LMPP and CMP/GMP/MEP of representative control BM (A), umbilical cord blood (UCB) (B), four AML BM samples at diagnosis (C), two MDS-RAEB BM samples (D), and two MDS-no-EB BM samples (E). DCF staining with normalised mean fluorescence intensity of each of these populations is shown (using DCF staining of reference lymphocyte population shown as light grey filled histogram, while for stem/progenitor subsets staining is shown as open histograms with different coloured lines as indicated on each plot). Additional examples of DCF staining of AML and MDS samples are shown in S1F–S1H Fig.
Mentions: We first asked whether low ROS levels measured with the redox-sensitive probe DCF were restricted to, and could thus differentiate HSC from, other immunophenotypic normal human hematopoietic progenitor populations (S1 Fig). In control BM samples (n = 24) total CD34+CD38high cells (fraction containing committed progenitors) had higher DCF staining than total CD34+CD38low cells (fraction containing more immature SPC) as expected. Further analysis within these two immunophenotypic compartments (representative examples in Fig 1A) confirmed that HSC had low DCF staining (median standardised-DCF-MFI = 2.05), as did the MPP population (median 1.98). The infrequent LMPP subset (<5% of CD34+CD38low) had relatively higher DCF staining (median 2.48), compared to both HSC and MPP. Interestingly there was a differential ROS profile within the CD34+CD38high fraction: GMP had highest DCF staining (median 6.64), followed by CMP (median 3.41) and then MEP (median 2.88) (complete summarised data in Fig 2A and 2B). The hierarchy in DCF staining with GMP>CMP>MEP and LMPP>HSC/MPP was also observed in umbilical cord blood (UCB) samples (n = 4) (example in Fig 1B), suggesting that this ROS profile is representative of normal non-malignant human SPC and not specific to the BM microenvironment.

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&minus; 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&minus; 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&minus; 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&minus; SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34&minus; 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