Abstract
Imatinib mesylate (IM) is highly effective in the treatment of CML. However resistance to IM can develop in a subset of patients. In addition CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK or other growth stimulatory pathways, or Bcr-Abl kinase mutations resulting in IM resistance. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models, and is currently being investigated in phase1/2 clinical trials. Here, we investigated whether SKI-606 could effectively target CML primary progenitors. Flow cytometry selected CD34+CD38− primitive progenitor (PPC) and CD34+CD38+ committed progenitor cells (CPC) from untreated CML patients were cultured for 96h in growth factor (GF) supplemented medium in a range of concentrations of SKI-606 (0–0.5μM), and with 5μM IM for comparison. Cells were labelled with CFSE-prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells were similarly incubated with SKI-606 and subsequently plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. CFSE assays indicated significant dose dependent antiproliferative activity of SKI-606 with IC50-values of 0.2μM for CML PPC. SKI-606 resulted in moderate induction of apoptosis of CML PPC (from 22±7.2% [control] to 49±16.4% [0.5μM], n=3, ns). CFC-assays consistently revealed significant dose dependent growth inhibitory effects of SKI-606 with IC50-values of 0.1μM SKI-606. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1μM and 0.5μM SKI-606 significantly suppressed phospho-CrkL levels (from 94.7±3.5% [control] to 14.9±4.8%, n=4, p=.0001, and to 6.8±2.5%, n=4, p=.00003, respectively), whereas a higher concentration of IM (5μM) was needed to achieve a similar degree (13.1±5.0%, n=4, p=.0000007). Whereas treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity (n=3, p=.038), a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606 (n=3, ns). In conclusion, SKI-606 significantly inhibits CML progenitor proliferation and moderately induces apoptosis at lower concentrations than previously observed with IM, although the maximum growth suppression effects observed were not greater than those observed with high concentrations of IM. SKI-606 is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further studies investigating the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds are warranted.
Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI-606