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W. Zhang et al.
with conventional chemotherapeutic drugs, achieved FLT3- ITD negativity as determined by the real-time polymerase chain reaction assay.
One hallmark of AML is the differentiation arrest of leukemic blasts. Promoting differentiation may therefore be beneficial for achieving and maintaining remissions in leukemias. Targeting FLT3-mutated AML cells with FLT3 inhibitors has been reported to induce cell-cycle arrest and differentiation, rather than apoptosis, which is reportedly driven by overexpression of C/EBPα and PU.1.36,38 Schepers et al. described that upregulation of C/EBPα led to growth arrest of CD34+ leukemia cells, which impaired the self- renewal capacity of the leukemic CD34+ cells, and corre- sponded with enhanced myeloid differentiation as well.39 Also, KPT-185 induced cell-cycle arrest and myeloid differ- entiation in AML cells, including patients’ samples, which increased C/EBPα.21 In fact, C/EBPα is a p53-regulated DNA damage-inducible gene,40 and p53 induction is involved in myeloid differentiation.41 Our data indicate that selinexor deactivated the nuclear export of a number of cargo proteins including p53. Furthermore, sorafenib mediated the upregulation of p21, and its combination with selinexor markedly enhanced levels of p53, p21, and C/EBPα (Figure 4D) as noted above, the last being one of the key hematopoietic-specific transcription factors medi- ating myeloid differentiation of leukemia cells. However, we did not observe an increase in another transcription fac- tor, PU.1, which is reportedly an upregulated effector of C/EBPα. The precise function of PU.1 is still unclear. Dahl et al. suggested that lower levels of PU.1 direct granulocyte differentiation, whereas higher levels are required for macrophage differentiation.37 Nevertheless, our data imply that an increase of C/EBPα levels was sufficient to induce myeloid differentiation of FLT3-ITD-mutated leukemic cells and decrease the CD34+ population, especially for the combination of sorafenib and selinexor. This treatment restores nuclear p53 level by blocking XPO1, and then upregulates C/EBPα to enhance C/EBPα/PU-1 and granulo- cyte differentiation as shown in Figure 4 and Online Supplementary Figure S8.
Signal transducer and activator of transcription (STAT)
family proteins are reportedly involved in regulation of myeloid progenitor cell differentiation.42 In fact, STAT5 plays an important role in early myeloid differentiation, and lacking expression of STAT5 reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice.43 STAT3 activation has also been reported to be a critical step in terminal differentiation of myeloid cells.44 On the other hand, upregulation of MAPK has also shown to be critical in both monocytic and granulocytic differentiation of myeloid cell lines, which can be abrogat- ed by using the MEK inhibitor U0126.45 All of these lines of evidence imply that high activation of these proteins may contribute to myeloid differentiation of leukemia cells. Of note, we observed profound upregulation of phosphorylated STAT3, STAT5 and ERK levels after com- bination treatment with low doses of sorafenib and selinexor in FLT3-mutated MOLM13 and MOLM14 cells (Figure 4D), suggesting that upregulation of STATs and/or MAPK signaling pathways may also contribute to differ- entiation induction of the combination regimen in FLT3 mutated AML cells.
In summary, our combinatorial strategy targeting FLT3 and XPO1 showed synergistic anti-leukemia effects in FLT3 inhibitor-resistant cells in vitro and in vivo. The com- bination of XPO1 and FLT3 inhibitors may also be able to eliminate leukemia-initiating cells by arresting cell growth and impairing the self-renewal capacity of leukemic CD34+ cells. These results should provide a solid basis for examining these agents further in patients with FLT3- mutated AML, including those who have acquired resist- ance to FLT3-targeted therapy.
Acknowledgments
The authors would like to thank Dr. Neil Shah for FLT3-ITD and TKD double mutant cells and Dr. Numsen Hail, Jr. for pro- viding critical review and editorial assistance in the preparation of this manuscript.
Funding
This work was supported in part by the NIH/NCI grants CA143805, CA100632, CA016672, and CA049639 (to MA).
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