Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Abstract

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34(+)CD38(-) stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34(+)CD38(+) progenitors were reduced. Residual normal CD34(+) cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.

Keywords: anemia; neutropenia; thrombocytopenia; xenotransplant.

Fig. 1.

Effect of AML on mouse hematopoietic populations in a xenograft model. (A and B) The gating strategy for identifying mouse hematopoietic progenitors and HSCs in BM is shown for a control mouse (A) and a mouse transplanted with AML (B). (C) The early (blue bars), mid- (pink bars), and late (gray bars) phases are seen following transplant of AML sample 1 (Left) whereas only early and midphases are seen following transplant of sample 3 (Right). (D) Summary of data from all experiments showing numbers of mouse CD45⁺ cells, progenitors, and HSCs in mice with AML as a percentage of values in controls in the three phases. The gray line represents the controls. (E) The mean AML percentage is shown for each of the time points and its relation to the phase. (F) The cellularity of BM in the ilium is not reduced in midphase following transplant of AML samples (three examples, Right) compared with control (Left) (20× objective magnification). *The HSCs and progenitors are expressed as a percentage of total CD45⁺ cells (AML plus mouse). ^†P < 0.05 and ^‡P < 0.05, comparing mean percentages in mice with AML to control values at each time-point prenormalization, using a paired T test.

Fig. 2.

Mouse CD45⁺ cells from mice with AML in midphase are enriched in HSCs. (A) HSCs from mice transplanted with AML show reduced cycling as assessed by BrdU incorporation. (B) The percentage of apoptotic mouse CD45⁺ cells was not increased in mice transplanted with AML in midphase. (C) Similar numbers of colonies were obtained from mouse CD45⁺ cells derived from mice with AML and controls on initial culture (P > 0.4) but on replating more colonies were derived from mouse CD45⁺ cells from mice transplanted with AML (P < 0.0007). (D) The frequency of repopulating cells within mouse CD45⁺ cells from mice transplanted with AML sample 10 in midphase was significantly higher than in controls at each time point. Error bars indicate 95% confidence intervals. *P < 0.05.

Fig. 3.

Assessment of normal residual hematopoietic populations in humans with AML. (A) The gating strategy displayed was used to define normal HSPCs and progenitors in the BM of a control (Upper) and a patient with subtype A phenotype AML (Lower). (B) Numbers of phenotypically defined HSPCs are preserved (P = 0.6) in the BM of patients with subtype A AML (n = 16) whereas progenitors are reduced compared with controls (n = 42) (P < 0.0001). (C) A significantly greater proportion of BM CD34⁺CD38⁻ cells from patients with subtype A AML (n = 7) were CD49f⁺ CD45RA⁻ Rhodamine123low compared with controls (n = 7) (P = 0.003). (D) The percentage of HSPCs in cell cycle was lower in subtype A AML (n = 7) BM than controls (n = 9) (P = 0.002). (E) Similar numbers of colonies were observed after culture of CD34⁺ cells in methylcellulose from three subtype A AML BMs and three controls. On replating, increased numbers of colonies were seen from CD34⁺ cells derived from AML marrow. (F) More LTC-ICs were present in the CD34⁺ cells from two subtype A AML BMs than in controls. Error bars indicate 95% confidence intervals. (G) More LTC-ICs were present in the CD34⁺CD38⁻ cells from three subtype A AML BMs than in controls. Error bars indicate 95% confidence intervals. (H) More human CD45⁺ cells were detected in mice transplanted with CD34⁺ from one subtype A AML sample (n = 3) than in controls (n = 4) whereas similar numbers were seen following transplant of another sample (n = 4 for each arm). (I) More CD45⁺ cells were seen in the BM of secondary recipients (n = 3 per arm) transplanted with 4 million human CD45⁺ cells derived from subtype A AML than in controls in both experiments. (J) The plots shows human grafts with predominant B lineage populations and smaller myeloid populations in secondary recipients injected with human CD45⁺ cells derived from CD34⁺ BM cells from a subtype A AML sample (Upper) and from control CD34⁺ BM cells (Lower). *P < 0.05.

Fig. 4.

Normal CD34⁺ cells are not displaced from the paratrabecular region by AML. (A) Normal stem-progenitor cells, expressing dual CD45 and CD34, were identified in BM biopsy sections from patients with subtype A AML (n = 4) and controls (n = 8). CD45⁺ and CD34⁺ cells were visualized using Fluorescein and AlexaFluor 546, respectively. An example from a patient with AML is shown. Two CD45⁺CD34⁺ cells are indicated by white arrowheads. (B) The distance of normal stem-progenitor cells (CD45⁺CD34⁺ cells) from the nearest trabecular bone was measured. Six stem-progenitor cells are seen with distance from the trabecular bone indicated in microns. (C) There was no significant difference in the distribution of stem-progenitor cells between AML and controls. Error bars indicate SD.