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Effects of XPO1 and FLT3 inhibition on AML
D835Y mutated cells (Figure 2A) and triggered additive pro-apoptotic effects in human FLT3-ITD-mutated MOLM13 and MV4-11 cells (Online Supplementary Figure S5). Of note, the combination regimen also demonstrated a synergistic pro-apoptotic effect in a primary AML sam- ple with FLT3-ITD plus D839G dual mutations (Online Supplementary Figure S6). Immunoblot analysis showed that the combinatorial treatment markedly abrogated the upregulation of phospho-FLT3, -ERK and -AKT (Figure 2B) that was observed in the experiments of selinexor treat- ment alone. In fact, all phospho-proteins were suppressed far below basal levels by the combination treatment. Interestingly, we also observed that ERK, AKT, FOXO3a, NF-kB, p53, p27, and p21 were preferentially retained in the AML cell nuclei after 16 h of treatment with selinexor and sorafenib. This treatment also completely abrogated anti-apoptotic Mcl-1 expression and decreased c-Myc lev- els (Figure 2C), suggesting greater AML cell sensitivity to apoptosis.
Co-targeting XPO1 and FLT3 partially abrogates hypoxia-mediated chemoprotection
The hypoxic bone marrow microenvironment is a reser- voir for leukemia-initiating cells, and it is associated with resistance to AML chemotherapy.30,35 We examined apop- tosis induction using the indicated concentrations of selinexor and/or sorafenib for a 68-h exposure under nor- moxic and hypoxic conditions. The combination partially abrogated hypoxia-mediated chemoprotection and induced synergistic apoptotic effects compared to those observed following treatment with either agent alone in
A
FLT3-ITD-mutated MOLM13 and MOLM14 cells (Figure 3A,B). Immunoblot analysis indicated that the combina- tion treatment profoundly suppressed the hypoxia-medi- ated upregulation of CXCR4 and HIF1α, suppressed phos- pho-FLT3, -ERK and -AKT, decreased Mcl-1, and increased the cleavage of caspase-3 (Figure 3C).
Co-targeting XPO1 and FLT3 enhances myeloid differentiation of FLT3-ITD-mutated human acute myeloid leukemia cells and human primary acute myeloid leukemia samples
Since selinexor and sorafenib treatments alone have been reported to induce differentiation of leukemic cells,21,36 we investigated if the combination could enhance the dif- ferentiation of FLT3-ITD-mutated leukemic cells in vitro. MOLM13 and MOLM14 cells were exposed to nanomolar concentrations of either agent alone or the combination for 5 days. At these doses only growth arrest was observed. Morphological changes were observed, including indenta- tion and bending of the nuclei and a decrease of nuclear/cytoplasmic ratio, with single-agent treatment in both ITD-mutated AML cell lines (Giemsa staining) sug- gesting a metamyelocyte stage of granulocytic differentia- tion. The combination treatment markedly enhanced these morphological changes. In addition, the myeloid differen- tiation marker CD11b+ significantly increased in the cells following the combination treatment (Figure 4A,B).
We used the same regimen to treat two primary, FLT3- ITD-mutated, human AML samples (Online Supplementary Table S1) in vitro for 6 days. Enhanced morphological myeloid differentiation described above was observed fol-
C
B
Figure 3. Combination treatment partially abrogates hypoxia protection and triggers synergistic apoptosis induction. MOLM13 (A) and MOLM14 (B) cells were treated with selinexor and/or sorafenib for 68 h. Apoptosis induction (annexin V positivity) was determined by flow cytometry. (C) MOLM13 cells were treated with the combination for 24 h and correlated protein levels were determined by immunoblotting.
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