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W. Zhang et al.
tions, in all human and murine AML cell lines that harbor ITD, TKD, or dual mutations of FLT3. The agent was much less effective in this regard in FLT3 wildtype Baf3/FLT3, THP-1, and Kasumi-1 cell lines (FLT3 mutant cells, regardless of whether they had single or dual muta- tions of ITD and TKD, showed 5- to 10-fold lower EC50 values than those of FLT3-wildtype ones) (Figure 1A-C, Table 1 and Online Supplementary Figures S2 and S3).
We next evaluated effects of selinexor on protein expres- sion using immunoblot analysis after 24 h of exposure to selinexor. Selinexor inhibited expression of the anti-apop- totic protein Mcl-1 and upregulated the pro-apoptotic pro- tein Bim. The tumor suppressor proteins p53, p21, and p27 were upregulated as well (Figure 1D). Unexpectedly, acti- vations of FLT3 and its downstream signaling pathways were upregulated, as evidenced by increasing levels of phosphorylated FLT3, -ERK, and -AKT after exposure to
selinexor for 24 h (Figure 1E), which were observed only in the FLT3-mutated cells. In addition, total FLT3 levels were also upregulated in FLT3-ITD and -ITD plus Y842 mutated cells, but not in ITD plus D835Y cells: this was true for both protein levels (Figure 1E) and mRNA transcriptional levels (Online Supplementary Figure S4). Kinetic analysis revealed that the upregulation of phospho-FLT3 was observable at 1 h, and phospho-ERK and -AKT at 6 h, after selinexor treatment (Online Supplementary Figure S5). These findings suggest that co-targeting FLT3 signaling, to sup- press its downstream signaling pathways, simultaneously with nuclear export may potentially trigger synergistic cytotoxic effects in these cells.
We tested this hypothesis using combinatorial treat- ment with selinexor and sorafenib. The combinatorial reg- imen did indeed trigger synergistic pro-apoptotic effects in murine FLT3-ITD mutated, ITD plus Y842C and ITD plus
AB
C
Figure 2. Combination treatment with selinexor and sorafenib triggers synergistically pro-apoptotic effects in murine cells with ITD or ITD plus TKD2 point muta- tions. (A) FLT3 mutated cells (Baf3/ITD, Baf3/ITD+Y842 and Baf3/ITD+D835Y) and wildtype cells (Baf3/FLT3-wt) were treated with either selinexor or sorafenib alone or the combination for 48 h, and examined for apoptosis induction (annexin V positivity) as described in Figure 1. (B) FLT3-mutated cells were treated with sin- gle agent(s) alone or the combination for 16 h, and levels of correlated phosphorylated proteins were measured by immunoblotting. GAPDH was used as a loading control. (C) MOLM13 cells were treated with the indicated concentrations of agent(s) for 16 h. Cytosolic and nuclear fractions were separated using a nuclear extract kit (Active Motif) following the manufacturer’s instructions. The correlated protein levels were determined with immunoblotting. Poly (ADP-ribose) polymerase (PARP) was used as a loading control of the nuclear fraction and tubulin was used as a loading control of the cytosolic fraction. Dimethylsulfoxide (DMSO) was used as a control. Seli: selinexor; Sora: sorafenib; Combi: combination. Solid line box indicates upregulation and dotted line box indicates downregulation.
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haematologica | 2018; 103(10)