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KS99 alone or in combination in AML
Figure 3. KS99 induces apoptosis in leukemic stem cells (LSC). (A) Dose-dependent apoptotic response of KS99 in primary human leukemic stem cells (LSC) iden- tified as CD34+, CD34+CD38–, CD34+CD38+, CD123+, or CD34+CD123+ cells. Error bars are mean±standard error of the mean (SEM). (B) Representative flow cyto- metric analysis of cell death in LSC. (C) Apoptosis in CD45+, TIM-3+, CD96+, or TIM-3+CD96+ cells after the treatment with KS99 and Cytarabine (Ara-C). Error bars are mean±SEM. (D) Apoptotic response of KS99, Cytarabine (Ara-C) or combination in primary human AML cells expressing or co-expressing LSC immunophenotypes; CD34, CD38, CD123, TIM-3, and CD96. Data were normalized to DMSO-treated cells. Error bars represent maximum and minimum values. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; unpaired t-test.
KS99-treated cells formed smaller AML blast colonies than the control treatment, as shown in Figure 2F and Online Supplementary Figure S3. Overall, these results showed that KS99 targets clonogenicity of leukemic cells as monother- apy while sparing normal HSPC. Since clonogenic activity is an indicator of pre-LSC, LSC and HSPC,29 these obser- vations were followed by flow cytometric analysis for cell surface markers of LSC.
KS99 induces apoptosis in primary human leukemic stem cells
To validate the anti-LSC activity of KS99, primary human AML cells were treated with increasing concentra- tions of KS99 (0.3 mM, 1 mM or 3 mM), Ara-C (5 mM), or combinations of KS99 with Ara-C for 24 h under the LSC culture conditions described by Pabst et al.27 Since there is no one perfect LSC marker, we studied multiple well reported markers.30,31 LSC were phenotypically defined by gating on CD45+ followed by CD34+C-D38–/CD38+, CD123+, TIM-3+, or CD96+. Induction of apoptosis was observed in CD123+ and CD34+CD123+ cells with KS99 treatment in a dose-dependent manner (Figure 3A). CD34+CD38– and CD34+CD38+ cells were analyzed to see whether KS99 has a pro-apoptotic activity in subpopula- tions of CD34+ cells, and we found that CD34+ cells were
sensitive to KS99 regardless of CD38 status (Figure 3A and B). We also observed that KS99 selectively targeted blast- like cells as compared to granulocyte-like or lymphocyte- like cells in causing reduction of CD34+ cells (Online Supplementary Figure S2). Next, KS99 was compared and combined with Ara-C in CD45+, TIM-3+, CD96+ or TIM-3+CD96+ human AML cells. Cells showed similar sensitivity to KS99 as observed in CD34+ and CD123+ cells (Figure 3C). When KS99 was added to Ara-C, it increased Ara-C's pro-apoptotic activity, especially in CD96+ or TIM-3+CD96+ cells (Figure 3C). Furthermore, we extend- ed our analysis by evaluating the pro-apoptotic activity of KS99 alone and in combination with Ara-C in LSC co- expressing CD34, CD38, CD123, TIM-3, or CD96 immunophenotypes (Figure 3D). Interestingly, cells coex- pressing CD123 and TIM-3 or CD96 immunophenotypes were less sensitive to Ara-C or KS99 compared to other co-expressions. However, their sensitivity was increased with combination treatment (Figure 3D, right panel). Overall, these results show that KS99 induces apoptosis not only in CD45+ or CD34+ human AML cells, but also in TIM-3+, CD96+, or cells co-expressing various LSC pheno- types. In addition, it also has the potential to enhance the activity of Ara-C in AML stem cells, given that most cells of each phenotype are sensitive to the combination.
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