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C. Annageldiyev et al.
Figure 4. KS99 down-regulates pBTK, pSTAT3 and inhibits aldehyde dehydrogenase (ALDH) activity. (A) Immunoblot analysis of the whole MV4-11 and U937 cell lysates after the treatment either with DMSO, increasing concentrations of KS99 or ibrutinib (10 mM) for 24 hours (h). GAPDH was used as a loading control. (B) Flow cytometric detection of pSTAT3 in LSC of primary human acute myeloid leukemia (AML) samples (n=4). (C) Immunoblotting of whole-cell lysates of KS99 or ibrutinib- treated MV4-11 and U937 cells with ALDH1A1. GAPDH was used as a loading control. (D and E) Primary human AML samples were treated either with DMSO, KS99 (3 mM) or ibrutinib (10 mM) for 48 h. ALDH activity was measured by ALDEFLOUR assay kit via flow cytometry. Results are mean±standard error of the mean. n=3. *P<0.05 was assessed by unpaired t-test.
KS99 interacts with BTK, STAT3, and ALDH1A1
A molecular docking approach was used to examine the interaction of KS99 with BTK, STAT3, and ALDH1A1 (iso- form) at the atomic level. For ALDH1A1, STAT3 and BTK, the lowest binding energies were -9.65 kcal/mol, -6.76 kcal/mol, and -9.31 kcal/mol, respectively (Online Supplementary Figure S4). The inhibition constant values for the aforesaid proteins was 84.19 nM, 11.14 mM, and 150.61 nM, respectively, suggesting that KS99 interacts with them. It should be noted that the lower energy scores cor- relate with a higher binding affinity,32 i.e. better for ALDH1A1 and BTK, followed, in this case, by STAT3. KS99 binds poorly with the developed structure of STAT3 with higher binding energy and inhibition constant as compared to ALDH1A1 and BTK. The phosphotyrosine 48 amino acid binding pocket of STAT3 interacted with KS99 at comparatively higher binding energy and inhibition con- stant than other proteins docked in this study. Further details are provided in Online Supplementary Table S2.
KS99 mediated downregulation of pBTK and pSTAT3 with reduced aldehyde dehydrogenase activity in acute myeloid leukemia cell lines and primary human acute myeloid leukemia cells
To extend studies of KS99 from earlier observations, we examined BTK and STAT3 signaling in AML cell lines by western blotting and in primary AML cells by flow cytom-
etry. Western blot results confirmed that KS99 significant- ly inhibits phosphorylation of BTK, and STAT3 in a dose- dependent manner in MV4-11 and U937 cells (Figure 4A). Moreover, the degree of inhibition of pSTAT3 and pBTK with 10 mM ibrutinib was achieved with low nanomolar concentrations of KS99 in both cell lines. As reported pre- viously for MM cells,20 KS99 up-regulated BCL-2 phospho- rylation and down-regulated MCL-1 in AML cells (Figure 4A). We did not observe BCL-2 phosphorylation with ibrutinib treatment but did see MCL-1 downregulation.
We obtained comparable results in primary human AML samples using flow cytometry. KS99 or vehicle- treated (DMSO) primary human AML cells were initially gated on the 7AAD-negative population (live cells) fol- lowed by CD45+ and CD34+CD38–, respectively. pSTAT3 was reduced by KS99 treatment in 3 out of 4 of the CD34+CD38– subpopulation of tested primary human AML cases (Figure 4B). Overall, these results suggest that KS99 down-regulates pBTK and pSTAT3.
Earlier studies have shown that LSC exhibit ALDH activity and it associates with drug resistance.17 Western blot data showed downregulation of ALDH1A1, an iso- form of ALDH with KS99 treatment in a dose-dependent manner in MV4-11 cells, whereas, in U937 cells, all the concentrations showed the same degree of downregula- tion (Figure 4C). Ibrutinib also down-regulated ALDH1A1, as reported previously for ovarian cancers.33-35 ALDH activ-
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