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C. Annageldiyev et al.
AML U937 cells. Leukemia-bearing mice were random- ized into treatment groups according to body weight and treated as above, followed by the treatment regimen shown in Online Supplementary Figure S5B. Analysis of bone marrow (BM) at termination showed an approxi- mate 76% reduction of hCD45+ cells in the KS99-treated group compared to the vehicle-treated group (P<0.05) (Figure 5D). Overall, KS99 treatment at 2.5 mg/kg was well tolerated by animals, as evidenced by no significant changes in body weight, suggesting negligible drug-associ- ated toxicity (Figure 5D, see insert).
Next, KS99 was compared and combined with Ara-C in U937-Luc-bearing NOD.Cg-Rag1tm1Mom Il2rgtm1 Wjl/SzJ (NRG) mice. The treatment was initiated with vehicle control, KS99, Ara-C (50 mg/kg, IP) or combina- tion (Online Supplementary Figure S5C). Although single- agent treatments KS99 (approx. 2.5 fold) or Ara-C (approx. 8.2-fold) showed a decrease in the progression of AML, co-treatment (approx. 22.2-fold decrease) was more effec- tive at suppressing AML than either drug alone, as assessed by BLI (Figure 5E and F). Furthermore, efficacy was confirmed by flow cytometric analysis of bone mar- row cells at the time of termination (Figure 5G). Mice treated with KS99, or Ara-C monotherapy exhibited a decrease of approximately 66% or approximately 86% in hCD45+ cells, respectively (Figure 5G). Interestingly, almost complete eradication (approx. 96%) of the leukemic burden was noticed in the combined treatment (KS99 + Ara-C) group (Figure 5G).
The above observations in human AML animal models were validated in an immunocompetent syngeneic animal model of AML. As for the above results, treatment with KS99, Ara-C or combination in C1498-Luc-bearing albino C57BL/6 mice showed a reduction in leukemia progres- sion analyzed by BLI (P<0.01, P<0.0001, and P<0.0001, respectively) (Figure 5H). Moreover, KS99 improved over- all survival of mice (P=0.0295) (Figure 5I). Mice treated with both agents survived longer than single agent-treated mice (Figure 5I).
Overall these results show that KS99 reduces the leukemic burden in AML xenograft models and improves animal survival. Efficacy can be further improved by com- bining KS99 with Ara-C or other AML chemotherapeu- tics.
Discussion
Current first-line cytotoxic chemotherapy for AML shows limited success, with 50% of younger patients and 80% of older patients succumbing to the disease.36 Unfortunately, these therapies have dose-limiting normal HSPC toxicity, which is one of the major obstacles in the treatment of AML.37 It is well established that cancer stem cells have a major role in the initiation, progression, and relapse of solid tumors.38 In AML, the leukemic stem cells have a similar role, and these cells acquire resistance to conventional chemotherapeutic drugs upon the accumula- tion of molecular mutations post-primary chemotherapy.39 The LSC within AML are responsible for tumor growth and maintenance.38 Relapse is frequently observed, and is largely attributed to acquired resistance of LSC to chemotherapeutic agents.2,3,37 Therefore, eradication of the LSC is likely necessary to increase survival of AML patients.40 Our studies here focus on small molecule KS99,
a novel Isatin derivative which we show has the potential to target LSC. KS99 inhibited cell growth, induced apop- tosis, and suppressed survival in both human AML cell lines and primary human AML cells.
Studies have shown that BTK is highly expressed and remains an important target in AML.41 BTK inhibitors, ibrutinib and CG-806, are proving to be promising AML agents 27,42,43 Our previous studies showed that KS99 is a dual inhibitor of BTK/tubulin in multiple myeloma.20 However, its role in targeting the LSC which control the poor prognosis of AML have remained undiscovered until now. BTK functions by initiating a cascade of downstream transcriptional factors that increase cell proliferation in cancer and favor cell survival.20 While ibrutinib blocks the BTK and causes apoptosis of cancer cells, the fact that the LSC are functional may lead to relapse in some cases. Our data show that KS99 inhibits the BTK-driven STAT3 phos- phorylation, which is an empirical player of LSC signaling.44,45 Various reports have clarified the role of STAT3 signaling in pre-LSC and have associated it to poor prognosis in multiple cancers, including AML.22,46-48 The effect of KS99 in CD34+CD38– LSC has shown decreased levels of phosphorylated STAT3, which leads to reduced expression of MCL-1 against increased phosphorylated BCL, probably due to BTK inhibition. This observation corroborates with MM and earlier studies.20,22,46-48 In silico analysis shows binding of KS99 to STAT3 and emphatical- ly to the phosphotyrosine-containing SH2 domain of STAT3. This further proves that the interaction between KS99 and BTK has a major role in down-regulating STAT3.
Aldehyde dehydrogenase, a detoxifying enzyme, is expressed in both normal progenitor cells and AML stem cells.17,25,33-35 In colony-forming assays, KS99 illustrates the reduction in the colony-forming capacity of AML progen- itor cells but mostly spares normal HSPC. This finding, with a specific selectivity of KS99, is further supported by a reduction in ALDH activity. Our western blot and flow cytometry data showed a significant decrease in ALDH expression and activity. Furthermore, the data are sup- ported by docking studies which predict the strong bind- ing affinity of KS99 to ALDH1A1 isoform at very low pre- dictive inhibition constant. Collectively, our data suggest that KS99 induces apoptosis by inhibiting the STAT3 and ALDH activation in LSC. In silico data described here also support the effective binding of KS99 to ALDH1A1 at an inhibitory constant of 84.19 nM, suggesting that the mol- ecule has a role in directly inhibiting the activity of ALDH and further inhibiting growth and survival of AML cells. The amino acid residues involved in the probable KS99- ALDH hydrophobic interaction likely increase binding affinity, stabilizing the ligand at its binding site and further affecting ALDH activity.
We showed that KS99 is highly active in CD34+, CD34+CD38– or CD34+CD38+ expressing cells in AML patient samples. However, a number of previous studies have shown that LSC in AML can also reside within CD123+, TIM-3+ or CD96+ cells.12-16 We, therefore, extend- ed our studies to these cells and were able to show that primary human AML cells expressing or co-expressing CD123, TIM-3 or CD96 were also sensitive to KS99. These results are consistent with colony-forming and ALDH observations. Furthermore, the combination of KS99 helped to augment the pro-apoptotic efficacy of Ara- C in LSC, especially in TIM-3+ cells co-expressing CD34 or
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