Short –Term Intense Bcr-Abl Kinase Inhibition Is Adequate to Trigger Cell Death in CML Cell Lines but Not in CML-CD34+ Cells Unless They Are Growth Factor Deprived.

After 5 years of imatinib treatment, only a minority of newly diagnosed chronic myeloid leukemia chronic phase (CML-CP) patients achieve complete molecular response. Imatinib has antiproliferative effects, but may not be able to eradicate CML-stem cells. Preclinical studies of imatinib suggested that sustained BCR-ABL kinase inhibition was required to block proliferation and induce apoptosis in CML cells. This formed the rationale for treatment regimens that maintain continuous kinase inhibition. Clinical studies with dasatinib suggested that daily dosing achieves equivalent response to twice daily even though ABL kinase inhibition only persists for 4–6 hours. We have demonstrated that 30 minutes of exposure to 100 nM dasatinib or 30 μ M imatinib (equipotent) inhibit p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 80 to 90% in Bcr-Abl +ve cell lines and CML-CD34+ cells (n=8). We then sought to compare antiproliferative and pro-apoptotic effects of short term (ST; cells were cultured with dasatinib/imatinib for 30 minutes and after thorough wash, were recultured without dasatinib/imatinib for 72 hours) and continuous (CT, cells were cultured with drugs continuously) dasatinib or imatinib in BCRABL +ve cell lines (K562, Meg 01) and CD34+ cells of CML-CP patients. Although Bcr- Abl kinase reactivated within 30 minutes of drug removal, ST 100 nM dasatinib (D100ST) or 30μ M of imatinib (IM30ST) induced apoptosis (~80%) and blocked cell proliferation equivalent to continuous dasatinib (10 nM; D10CT) or imatinib (2μ M, IM2CT) in Bcr-Abl +ve cell lines. The kinetics of cell death and caspase-3 activation over 72 hours of culture were similar in D100ST and D10CT. In the presence of 6-growth factors (GFs; IL-3, IL- 6, G-CSF, SCF, TPO, Flt-3) D100ST and IM30ST reduced cell viability and CFU-GM colonies of CML-CD34+ cells by only 25 to 30% of no drug control. Moreover in the presence of GFs, 30 to 40% CD34+ve cells were viable and retained CFU-GM potential in spite of continuous dasatinib 100 nM (D100CT) or 30 μ M of imatinib (IM30CT). However, in the absence of GFs, D100ST and IM30ST reduced viability by 60 to 70%, and CFU-GM by 95% of control (with GFs, no TKI control; Fig 1).

Figure 1:

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Survival of CFU-GM according to growth factor and dasatinib exposure:

Figure 1:

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Survival of CFU-GM according to growth factor and dasatinib exposure:

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Conclusion: Short term intense inhibition of BCR-ABL kinase activity triggers apoptosis in CML cell lines, which demonstrate their Bcr-Abl oncogene dependence. However, in spite of >80% kinase inhibition, D100ST and D100CT did not eliminate the majority of CML-CD34+ cells in the presence of GFs. In the absence of GFs, D100ST and IM30ST were able to inhibit cell proliferation, induce cell death and eliminate 95% of CFU-GM. This data suggests that oncogene dependence of CML CD34+ cells can be overcome by cytokines. Unlike CML cell lines where transient intense kinase inhibition leads to cell death, primary CML cells are only sensitive to this short term kinase inhibition in the absence of cytokines. Strategies that block cytokine pathways in combination with Bcr-Abl kinase inhibition may eliminate leukemic stem cells in-vivo even if only applied intermittently.

CFU-GM colonies expressed as % of control. CML-CD34+ cells (n=3) were cultured with dasatinib in the presence (With GFs) or absence (No GFs) of 6-growth factors (GF) and CFU-GM colonies were plated on D3, using Methocult 4230 (Invitrogen) along with growth factors in all cases. Colonies were read after 14 days. In each patient values were normalised to cells cultured with GFs and no dasatinib. Short term (ST) and continuous (CT), Dasatinib 10 nM (D10), 100 nM (D100).