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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

ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROSlowest and ROShighest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROSlowest and ROShighest subsets in (A-B) CD34+ AMLs, (C-D) in CD34− AMLs and (E-F) in CD34+ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34+ and CD34−CD117+ cells in the respective charts (grey triangles). AML patients were subdivided into Flt3ITD+/NPM1wt (simplified to F+/N-), Flt3ITD−/NPM1mut (F-/N+), Flt3ITD+/NPM1mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34+ (>6.9) or CD34− blasts (>3.3).
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pone.0163291.g007: ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROSlowest and ROShighest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROSlowest and ROShighest subsets in (A-B) CD34+ AMLs, (C-D) in CD34− AMLs and (E-F) in CD34+ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34+ and CD34−CD117+ cells in the respective charts (grey triangles). AML patients were subdivided into Flt3ITD+/NPM1wt (simplified to F+/N-), Flt3ITD−/NPM1mut (F-/N+), Flt3ITD+/NPM1mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34+ (>6.9) or CD34− blasts (>3.3).

Mentions: We hypothesised that BCL2 over-expression (with downstream effects on apoptosis, metabolic regulation and cell-cycle) might be maintained in ROS-high blasts of some AMLs to counteract further oxidative stress similar to the observed BCL2 upregulation at the GM maturation stage. We gated on blasts with 20% highest DCF fluorescence and 20% lowest DCF fluorescence to allow a comparison between ROShighest and ROSlowest blasts (AML n = 40, MDS n = 12). There was a relative enrichment of the more immature immunophenotypic SPC (hierarchy LMPP/MPP >CMP/GMP) in the ROSlowest vs ROShighest fractions for most of the CD34+AMLs (n = 24) (S5A Fig) but no SPC correlation with a specific ki67/BCL2 profile or overall high ROS levels. Interestingly ROSlowest/ROShighest gating of CD34+ SPCs in MDS-noEB revealed an aberrant relative expansion of CD34+CD38low and MEP/CMP SPCs in the ROSlowest and ROShighest fractions respectively (S5B Fig). Although some patients had lower ki67 in ROSlowest fractions as observed for normal BM and MDS-noEB (Fig 7A–7C), the majority did not have ROSlow enrichment of the LSC-like ki67lowBCL2high phenotype (S6 Fig). Similar ki67 and BCL2 levels were, however, maintained between ROSlowest and ROShighest blasts in a number of AML/MDS-RAEB patients. From available clinical data of early post-treatment outcome for CD34+ AMLs, high BCL2 over-expression (>6.9-fold above isotype) in ROShighest as well as ROSlowest fractions seemed to be most associated with initial treatment resistance (Fig 7D) although numbers were limited. A correlation between BCL2 overexpression in ROSlowest/ROShighest and early treatment response was not apparent in CD34− AMLs including the Flt3ITD+/NPM1mutated patients (Fig 7E).


Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia
ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROSlowest and ROShighest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROSlowest and ROShighest subsets in (A-B) CD34+ AMLs, (C-D) in CD34− AMLs and (E-F) in CD34+ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34+ and CD34−CD117+ cells in the respective charts (grey triangles). AML patients were subdivided into Flt3ITD+/NPM1wt (simplified to F+/N-), Flt3ITD−/NPM1mut (F-/N+), Flt3ITD+/NPM1mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34+ (>6.9) or CD34− blasts (>3.3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036879&req=5

pone.0163291.g007: ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROSlowest and ROShighest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROSlowest and ROShighest subsets in (A-B) CD34+ AMLs, (C-D) in CD34− AMLs and (E-F) in CD34+ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34+ and CD34−CD117+ cells in the respective charts (grey triangles). AML patients were subdivided into Flt3ITD+/NPM1wt (simplified to F+/N-), Flt3ITD−/NPM1mut (F-/N+), Flt3ITD+/NPM1mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34+ (>6.9) or CD34− blasts (>3.3).
Mentions: We hypothesised that BCL2 over-expression (with downstream effects on apoptosis, metabolic regulation and cell-cycle) might be maintained in ROS-high blasts of some AMLs to counteract further oxidative stress similar to the observed BCL2 upregulation at the GM maturation stage. We gated on blasts with 20% highest DCF fluorescence and 20% lowest DCF fluorescence to allow a comparison between ROShighest and ROSlowest blasts (AML n = 40, MDS n = 12). There was a relative enrichment of the more immature immunophenotypic SPC (hierarchy LMPP/MPP >CMP/GMP) in the ROSlowest vs ROShighest fractions for most of the CD34+AMLs (n = 24) (S5A Fig) but no SPC correlation with a specific ki67/BCL2 profile or overall high ROS levels. Interestingly ROSlowest/ROShighest gating of CD34+ SPCs in MDS-noEB revealed an aberrant relative expansion of CD34+CD38low and MEP/CMP SPCs in the ROSlowest and ROShighest fractions respectively (S5B Fig). Although some patients had lower ki67 in ROSlowest fractions as observed for normal BM and MDS-noEB (Fig 7A–7C), the majority did not have ROSlow enrichment of the LSC-like ki67lowBCL2high phenotype (S6 Fig). Similar ki67 and BCL2 levels were, however, maintained between ROSlowest and ROShighest blasts in a number of AML/MDS-RAEB patients. From available clinical data of early post-treatment outcome for CD34+ AMLs, high BCL2 over-expression (>6.9-fold above isotype) in ROShighest as well as ROSlowest fractions seemed to be most associated with initial treatment resistance (Fig 7D) although numbers were limited. A correlation between BCL2 overexpression in ROSlowest/ROShighest and early treatment response was not apparent in CD34− AMLs including the Flt3ITD+/NPM1mutated patients (Fig 7E).

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