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W. Zhang et al.
lowing the combination treatment, as was a profound increase of the CD11b+ population, which was more sig- nificant in the ITD-mutated AML sample (AML #2) than in the D835 TKD-mutated AML sample (AML #1) (Figure 4C). In addition, a decrease of the CD34+ population was observed in both tested primary AML samples (Online Supplementary Figure S8). Of note, the doses of either drug alone, or in combination, were not enough to kill the pri- mary AML cells, but were sufficient for growth arrest of the CD34+ cells, implying that the combinatorial regimen could be beneficial by impairing the self-renewal capacity of the leukemic CD34+ compartment. Mechanistically,
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marked upregulation of CCAAT/enhancer binding protein α (C/EBPα), one of the leucine zipper transcription factors that is important for normal myeloid cell differentiation, was observed in the cells following the combination treat- ment. This was accompanied by an increase in the C/EBPα/PU-1 ratio (Figure 4D), which has been reported to be associated with granulocytic myeloid cell differentia- tion.37 Interestingly, upregulation of cell proliferation-relat- ed proteins, such as phospho-ERK, -STAT5, -STAT3, and tumor suppressor proteins, including p53 and p21, was also observed after exposing cells to low doses of both drugs for 5 days (Figure 4D).
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Figure 4. Combination treatment triggers myeloid differentiation of leukemic cells. FLT3-ITD mutated cell lines MOLM13 (A), MOLM14 (B) and primary AML patients’ samples (C) were treated with the indicated concentrations of selinexor and/or sorafenib for 5 or 6 days in vitro; morphological changes of the cells were checked with Giemsa staining. Expression of the myeloid differentiation marker CD11b was determined using flow cytometry. The histograms present individual assays which measured fluorescent intensity of CD11b. Each column was generated from four individual assays. (D) Differentiation-related proteins were evaluated using immunoblotting. The expression levels and ratios of C/EBPα/PU-1 were measured (first lane which was defined as 1). Dimethylsulfoxide (DMSO) was used as a control. Seli: selinexor; Sora: sorafenib; Combi: combination.
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haematologica | 2018; 103(10)