Leukemia is a biologically heterogeneous hematological disease and has become one of the leading causes of cancer-related mortality worldwide.1 Recent studies of the pathogenesis of leukemia have revealed that mutations and/or aberrant expression of specific protein tyrosine kinases, such as FMS-like tyrosine kinase 3 (FLT3) and Abl, are responsible for the development of several common types of leukemia.2, 3, 4 For example, activating mutations in FLT3 are found in approximately 1/3 of acute myeloid leukemia (AML) patients, with the most prevalent activating mutations being ‘internal tandem duplications’ (ITD) in the juxtamembrane domain.5, 6 Several large-scale studies have demonstrated that FLT3/ITD is strongly associated with leukocytosis and a poor prognosis.7, 8, 9 Thus, FLT3 is considered to be an important molecular target in the treatment of AML.
Despite many efforts in the past few years to develop FLT3 inhibitors for the treatment of AML, no FLT3 inhibitors have been approved for clinical use. Furthermore, except for a small number of inhibitors (such as AC220), most of the known FLT3 inhibitors that are currently in clinical trials do not show impressive clinical efficacy.10, 11, 12 These facts imply a requirement to develop new-generation FLT3 inhibitors with improved clinical efficacy, especially novel multikinase inhibitors because they are thought to enhance the therapeutic effect of FLT3 inhibitors in AML.13 Under these circumstances, we recently discovered a novel multikinase inhibitor termed SKLB1028 (Figure 1a). In this letter, we report the preclinical efficacy of SKLB1028 in AML; a brief description regarding the chemical characterization of SKLB1028 is provided in the Supplementary Material. SKLB1028 also showed considerable potency in models of chronic myeloid leukemia (CML) that harbor Abl mutants, which will also be reported here.
The kinase-inhibition profile of SKLB1028 against a panel of recombinant human protein kinases was measured by the ‘gold standard’ radiometric kinase assay approach, and the results are shown in Supplementary Table S1. SKLB1028 potently inhibited the wild-type and L858R-mutant EGFR with half-maximal inhibitory concentration (IC50) values of 0.031 and 0.004 μM, respectively. The IC50 values of SKLB1028 against FLT3 (Figure 1b) and Abl were 0.055 and 0.081 μM, respectively. Of note, this compound also inhibited the Abl-T315I mutant with considerable potency (IC50: 0.071 μM); this AblT315I mutant is associated with imatinib resistance. Furthermore, SKLB1028 inhibited FYN, HCK, KDR, PDGFRβ, CSF1R, FGFR1 and FGFR2 with moderate activity (the IC50 values were 0.214, 0.487, 0.330, 0.360, 1.040, 1.20 and 0.980 μM, respectively). SKLB1028 displayed negligible inhibitory activity against the 24 additional protein kinases that were tested. These data demonstrate that SKLB1028 is a multikinase inhibitor with high potency against EGFR, FLT3 and Abl and has good kinase-spectrum selectivity.
The growth-inhibitory potencies of SKLB1028 against various leukemia cell lines as well as several other cell lines were examined using the MTT assay method, and the results are presented in Supplementary Table S2. SKLB1028 potently inhibited the growth of MV4-11 cells that express FLT3-ITD, with an IC50 value of 0.002 μM (Figure 1c), but moderately inhibited the proliferation of RS4-11 cells that express wt-FLT3, with an observed IC50 of 0.790 μM. SKLB1028 also potently inhibited the growth of Ba/F3 cells that stably express human FLT3ITD (Ba/F3-FLT3-ITD), with an IC50 of 0.01 μM, but was nontoxic toward parental Ba/F3 cells at concentrations up to 5 μM. SKLB1028 inhibited the cell growth of K562 cells that express the Bcr-Abl mutant with an IC50 of 0.190 μM. SKLB1028 exhibited only a weak inhibitory potency in the human leukemia cell lines SU-DHL-6, TF-1, Karpas299 and Jurkat. Finally, SKLB1028 at a concentration of 10 μM did not inhibit the growth of the human myeloma cell line RPMI8226, human normal cell line LO2 and Chinese hamster ovary cell line CHO (Supplementary Table S2).
The ability of SKLB1028 to inhibit the activation of FLT3 and downstream signaling proteins in intact cells was assessed using western blot analysis. As shown in Figure 1d, SKLB1028 inhibited FLT3 phosphorylation in a dose-dependent manner. Consistent with the downregulation of the phosphorylation of FLT3, the phosphorylation of the downstream signaling proteins STAT5 and ERK1/2 was also significantly inhibited at concentrations >0.003 μM (Figure 1e). We further examined the influence of SKLB1028 treatment on the cell cycle of MV4-11 cells using flow cytometry, and the results indicated that SKLB1028 could induce cell cycle arrest in G1 phase (Supplementary Figure S1). By observing the morphological changes in the nuclei using Hoechst staining, we found that the nuclei of SKLB1028-treated MV4-11 cells shrank, became rounded up, and eventually divided into several apoptotic bodies (Supplementary Figure S2), indicating cell apoptosis. The apoptosis induced by SKLB1028 was further confirmed using western blot analysis, in which we observed a dose-dependent decrease in the level of pro-caspase-3 and a dose-dependent increase in the level of the cleaved caspase-3 fragment in SKLB1028-treated MV4-11 cells at concentrations of 10–100 nM (Figure 2f).
The in vivo anti-leukemia activities of SKLB1028 were evaluated using the FLT3-ITD-positive MV4-11 and the Bcr-Abl-positive K562 xenograft models. In the MV4-11 model, when the tumor grew to a volume of 300–500 mm3, the mice were grouped and treated orally once daily with 5, 10 or 20 mg/kg SKLB1028 for 18 days. The tumor volumes were measured every 3 days. Treatment with SKLB1028 at 10 or 20 mg/kg/day resulted in rapid and complete tumor regression in all mice in both groups (Figure 2a). SKLB1028 treatment at 5 mg/kg/day stopped the tumor growth and resulted in slight tumor regression. After 7 days of additional treatment, the mice treated with 20 mg/kg/day SKLB1028 were monitored (but not treated) for the subsequent 20 days, and no tumor re-growth was observed in any of the mice in this group. Histological and immunohistochemical analyses were performed to evaluate the anti-tumor mechanism of action of SKLB1028 in the MV4-11 model. As shown in Figure 2c, SKLB1028 significantly inhibited the tumor-cell proliferation (the percentage of Ki67-expressing cells in viable tumor tissue was significantly lower following SKLB1028 treatment) and induced apoptosis in the tumor tissue (the percentage of TUNEL-positive apoptotic cells was considerably higher in SKLB1028-treated tumors than in vehicle-treated tumors).
In the K562 model, a series of doses of SKLB1028 was administered orally once daily when the tumors grew to 150–300 mm3. SKLB1028 treatment at a dose of 70 mg/kg/day significantly inhibited tumor growth compared with the vehicle group (Figure 2b). The tumor-inhibition rate of SKLB1028 at a dose of 70 mg/kg/day was 71.3%, whereas imatinib mesylate displayed a weak tumor-suppressing effect at the same dose. Again, histological and immunohistochemical analyses were performed to evaluate the mechanism of action of SKLB1028 in the K562 model. In this model, SKLB1028 considerably inhibited the tumor-cell proliferation and induced apoptosis in the tumor tissue in a manner similar to MV4-11 (Figure 2d).
We further examined the influence of SKLB1028 on survival time using a bone marrow engraftment model of MV4-11 (Supplementary Material). Supplementary Figure S3 shows that compared with vehicle and positive control (sunitinib, 10 mg/kg/day) groups, SKLB1028-treated mice demonstrated prolonged survival in a dose-dependent manner with mean survival times (MSTs) of 65, 71 and 80 days for the 5, 10 and 20 mg/kg/day groups, respectively. The MSTs for the vehicle and positive control groups were 48.5 and 61 days, respectively. An initial sub-acute toxicity test was also performed, which indicated that SKLB1028 could be continuously administered at the dose of 100 mg/kg/day in rats with no observed adverse effects (Supplementary Figure S4 and Tables S4–S6). Finally, the pharmacokinetic characteristics of SKLB1028 following per os administration to male rats were analyzed. As shown in Supplementary Figure S5, the compound was well absorbed, achieving a maximum plasma level (Cmax) of 607 μg/l within 4 h after oral administration at a dose of 60 mg/kg. Furthermore, the apparent plasma half-life was approximately 7.2 h, and the AUC(0−24h) was approximately 6.969 mg/l/h.
In summary, SKLB1028 is a novel multikinase inhibitor that displays exceptional activity in models of FLT3-driven AML and considerable potency in CML that harbors Abl mutations. SKLB1028 has the convenience of oral administration, favorable pharmacokinetic properties and low toxicity. Collectively, preclinical assessment of the pharmacodynamics, pharmacokinetics and initial toxicity of SKLB1028 supports the use of SKLB1028 as a promising candidate for clinical studies in patients with leukemia. Finally, we must mention that SKLB1028 differs from other known FLT3 inhibitors in that it is also a typical EGFR inhibitor. This characteristic of SKLB1028 is noteworthy because of the recent unexpected findings that EGFR inhibitors, such as 4,5-dianilinophthalimide (DAPH1), gefitinib and erlotinib, are able to promote differentiation and induce apoptosis in AML blasts in vitro.14, 15 Because AML cells do not express EGFR, the observed activity of EGFR inhibitors on AML cells cannot be due to inhibition of EGFR, but must have resulted from inhibition of an off-target kinase that is as yet uncharacterized. Whether SKLB1028 has the same effect on AML or acts on the same off-target kinases as other EGFR inhibitors remains to be determined.
This work was supported by the National Natural Science Foundation of China (81172987), the National S&T Major Project (2012ZX09102-101-002) and SRFDP (20100181110025).
About this article
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)