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A. Gao et al.
Statistics
Unless otherwise stated, data are expressed as mean or mean ± standard error of the mean as indicated. P values were gener- ated using an unpaired Student t-test and analysis of variance. GraphPad Prism and IBM SPSS Statistics software were used for the statistical analyses.
Results
Megakaryocytic differentiation from hematopoietic stem cells via both canonical and non-canonical routes was markedly inhibited in acute myeloid leukemia bone marrow
To elucidate the perturbation of the orchestrated process of thrombopoiesis in AML, we used a non-irradi- ated MLL-AF9-induced AML mouse model (Online Supplementary Figure S1A).10 AML mice exhibited a pro- gressive decrease of platelets during leukemia develop- ment. On day 14, when leukemic infiltration exceeded 80% of the whole BM, platelet counts were reduced to ~14% of the normal level (Online Supplementary Figure S1B). Meanwhile, we detected simultaneous loss of BM MK using a flow cytometry gating strategy introduced by Heazlewood et al.28 (Online Supplementary Figure S1C, D). These results suggested that our AML mouse model could mimic the thrombocytopenic process that occurs in patients with AML.
The developmental landscape of BM MK has not been fully understood. It was estimated that approximately half of all MK progenitors (MkP) were from long-term (LT)-HSC which contributed little to other lineages29 in native hematopoiesis, and the other half were from mul- tipotent progenitors (MPP), especially MPP2.21 Thus, we decided to measure the numbers and proportions of these MK-primed subpopulations during the progression of AML (Figure 1A).
For ease of comparison, we normalized the values of the AML group at indicated time points to that of healthy controls (day 0). All of these subpopulations decreased significantly as the leukemia developed, but to different extents. The changes of pre-megakaryocytic-erythroid (PreMegE) and MkP were much more dramatic: at the end stage of AML, the total number of residual PreMegE was less than 1% of normal level, and ~4.9% of MkP were left in leukemic BM (Figure 1B and Online Supplementary Figure S2). In contrast, ~17.2% of MPP2 and ~29.6% of LT-HSC were preserved in AML mice (Figure 1B and Online Supplementary Figure S2). Regarding their proportions in CD45.2+ normal hematopoietic cells, the proportion of PreMegE dropped substantially and continuously, to about 1/10 of normal level; MkP declined at a moderate rate, to approximately 56% of that in the healthy state (Figure 1C). In contrast, the proportions of MPP2 and LT-HSC increased progressively ~3.4-fold and ~5.9-fold, respectively, as compared to the proportions in healthy controls at day 14 (Figure 1C).
We then assessed the apoptosis and cell cycle state of PreMegE and MkP to determine if the marked decrease of both subsets resulted from excessive cell death or growth arrest. We did not find significant changes in the rate of apoptosis of PreMegE during leukemia progression (Online Supplementary Figure S3A). In addition, their cycling was not inhibited in leukemia BM, and even dis- played slightly active cycling in the mid-phase of disease
(Online Supplementary Figure S3B), suggesting their active production of downstream populations. Correspondingly, PreMegE isolated from AML mice at the late stage yielded equal numbers of CD41+ MK as the PreMegE isolated from healthy controls (Online Supplementary Figure S3C, D). As far as concerns MkP, their rate of apoptosis even declined in AML (Online Supplementary Figure S3E, F), and no significant alteration was detected in their cycling state (Online Supplementary Figure S3G). Additionally, the MK colony-forming capac- ity of MkP from AML hosts was comparable to that of MkP from healthy controls when cultured in vitro (Online Supplementary Figure S3H). Since MkP derived from the non-canonical pathway, or shortcut, comprised nearly 70% of the total MkP pool,21 the severe reduction of their number (~95% lost) Figure 1B) and proportion in normal hematopoietic cells (~44% lost) (Figure 1C) suggests a major defect with the shortcut, or LT-HSC and MPP2.
We then sought to evaluate the MK differentiation potential of LT-HSC and MPP2 in AML BM, which was likely to be reflected by the expression of MK-specific proteins in these subsets. vWF is a protein involved in platelet aggregation and is abundant in MK and endothe- lial cells.30 It has been reported that vWF+ HSC have strik- ingly higher platelet reconstitution potential than vWF- HSC.31 We, therefore, evaluated intracellular vWF protein levels in these subsets by flow cytometry using a verified antibody. The proportions of vWF+ cells were significant- ly reduced in both LT-HSC (from ~21.1% to ~13.2%) and MPP2 (from ~19.1% to ~5.9%) in the late stage of AML (Figure 2A, B), indicating reduced MK differentiation from both subsets. Taken together, these data suggest that the scarcity of residual MkP in AML BM was due to severe blockade of MK differentiation in both the canon- ical and non-canonical routes.
We then investigated whether the decline of vWF expression indicated a prolonged impairment of the MK potential of MPP2 and LT-HSC. To do this, we used actin- eGFP mice as AML recipients and isolated eGFP+ MPP2 and LT-HSC from AML (day 14) or healthy control BM to transplant into sub-lethally irradiated mice. We tracked the reconstitution rates of MPP2 and LT-HSC from day 10 to day 20 for maximum exhibition of donor-derived platelet potential. We observed lower levels of platelet reconstitution of both LT-HSC and MPP2 subsets from AML hosts as early as 14 days after transplantation (Figure 2C). However, the reconstitution of peripheral blood nucleated cells from AML and control groups at that time point showed no difference (Figure 2D). These results suggest that the specific impairment of MK differ- entiation induced by the AML BM microenvironment would not easily recover in a leukemia-free niche.
Megakaryocyte maturation was severely impaired in acute myeloid leukemia bone marrow
MkP should experience several cycles of endomitosis and cytoplasmic maturation before they become giant, multinucleated, platelet-producing MK.32 Endomitosis leads to polyploidization of DNA content, which has been extensively demonstrated to be of vital importance for efficient platelet production.33 Of note, it was reported that apoptosis and polyploidization are synchronous and intimately linked events during MK maturation; inhibi- tion of apoptosis delays polyploidization and proplatelet formation.34,35 We questioned whether the decreased
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haematologica | 2019; 104(10)