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MCL-1 in human hematopoiesis
more sensitive than CD34- cells to the BCL-XL inhibitor A- 1155463 (Figure 7A). We compared colony-forming poten- tial CD34+ cells derived from cord blood or bone marrow and observed no difference between the two cell types: BCL-XL inhibition resulted in significant reduction of colony formation (Figure 7B) and cell numbers (Figure 7C), independently of the source of the human HSPC. Notably, effects on colony formation were less pronounced than those caused by MCL-1 inhibition. As documented earlier, immature CD34+ cells (Figure 7D) as well as mature ery- throid cells (Figure 7E) were more severely affected than myeloid cells (Figure 7F).
Finally, the BCL-2 inhibitor ABT-199 did not negatively affect survival or colony formation of cord blood- or bone marrow-derived CD34+ cells, even when used at the high concentration of 1 mM (Online Supplementary Figure S4). This finding is consistent with the relatively mild myelo- suppressive effects of venetoclax observed in clinical tri- als.39
Synthetic lethality of MCL-1 and BCL-XL inhibitors in human hematopoietic stem and progenitor cells
We noted a striking functional homology of MCL-1 and BCL-XL for survival of human HSPC and concluded that combined inhibition of both anti-apoptotic proteins could result in complete depletion of colony-forming stem and progenitor cells. To test this, we treated cord blood-derived CD34+ cells with increasing doses of the
Figure 5. Survival of mature hematopoietic cells is independent of MCL-1. (A-B) Freshly isolated cord blood was subjected to density gradient centrifugation and mononuclear cells were divided into CD34+ and CD34- cells using magnetic activated cell sorting technology. Both cell fractions were treated with the indicated con- centrations of the MCL-1 inhibitor S63845. After 24 h (A) and 48 h (B), percentages of living cells were determined by flow cytometry using annexin V/7-AAD. Bars represent mean ± standard error mean (SEM); n=3-6 from six independent experiments. (C-D) CD34+ cells were differentiated in MethoCult culture. After 11 days, differentiated cells were isolated and treated with S63845 for 24 h. Total cell numbers (C) were determined and erythroid and myeloid cell populations (D) were analyzed by flow cytometry (n=5 from 5 independent experiments). Bars represent mean ± SEM. Mann-Whitney test: *P<0.05, **P<0.01, ***P<0.001.
BCL-XL levels were identical in both cell types and A1 was not expressed, BCL-2 levels were higher in adult HSPC than in HSPC from neonates (Figure 6A).
Next, we performed comparative functional studies using the MCL-1 inhibitor S63845, the BCL-XL inhibitor A-1155463 and the BCL-2 inhibitor ABT-199. By treating bone marrow immature CD34+ and differentiated CD34- cells with the MCL-1 inhibitor for 24 h and 48 h, we obtained similar results as those with cord blood cells (compare Figure 6B with Figure 5A, B). Chemical inhibition of MCL-1 also confirmed our RNA interference experi- ments: when we added the inhibitor S63845 to cord blood CD34+ cells cultured in MethoCult medium, colony forma- tion was impeded in a dose-dependent manner (compare Figure 6C with Figure 2). The numbers of immature cell types, including hematopoietic stem cells, multipotent pro- genitors and mixed lymphoid progenitors, were signifi- cantly reduced (Figure 6C, right panel). Similarly, all emerg- ing erythroid and myeloid cells (Figure 6C, right panel) were depleted in a dose-dependent manner. Importantly, there was no difference in MCL-1 inhibitor sensitivity in CD34+ cells derived from cord blood (Figure 6C) or bone marrow (Figure 6D).
We have shown earlier that BCL-XL, too, is important for keeping human cord blood CD34+ cells alive,31 a finding that was unexpected considering its dispensable role for mouse HSPC.36-38 We now extended our studies to bone marrow-derived cells and showed that CD34+ cells were
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