Leukemia Stem Cells in CML: Characterization and Elimination
Authors: Joanne Mountford, Heather Jorgensen, Mhairi Copland, Niove Jordanides, Francesca Pellicano, Tessa Holyoake
Journal: Clinical Cancer Research (2007)

PL05-03  Chronic Myeloid Leukaemia (CML) is a myeloproliferative disease that originates in a haemopoietic stem cell (HSC) as a result of the t(9;22) translocation, giving rise to the Philadelphia chromosome and bcr-abl oncoprotein. The disease starts in chronic phase, but as a result of genomic instability, it progresses over time to accelerated phase and then to blast crisis, becoming increasingly resistant to therapy. Bcr-abl is a constitutively active tyrosine kinase that has been targeted by tyrosine kinase inhibitors (TKIs), including imatinib (IM), nilotinib and dasatinib. We have developed various flow cytometry techniques to enable us to isolate candidate CML stem cells from chronic phase patients at diagnosis. These cells efflux Hoechst dye, express CD34, lack CD38 and are cytokine non-responsive in culture over periods of up to 12 days in growth factors. These stem cells have also been shown to regenerate bcr-abl positive haemopoiesis in immuno-compromised mice upon transplantation. We previously demonstrated that IM had an anti-proliferative effect on CML stem cells but did not induce apoptosis. Clinical experience now confirms that IM may not target CML stem cells in vivo with few patients achieving complete molecular remission and relapse occurring rapidly upon drug withdrawal. Our recent efforts have focused on understanding why CML stem cells are resistant to IM and on trying to find novel ways to induce apoptosis of this population. We have shown that CML stem cells express very high levels of functional wild type bcr-abl, these findings have been confirmed by other labs. However, we did not detect gene amplification or kinase domain mutations within the stem cell population, thus the cells express very high levels of wild type bcr-abl protein.  One possible explanation for the relative insensitivity of CML stem cells to TKI-induced killing is that the level of protein is too high to be efficiently inhibited. This may be exacerbated by the presence of drug transporters on CML stem cells that may actively reduce the intracellular concentration of TKI. In order to investigate this possibility we have examined the expression and function of ATP-Binding Cassette (ABC) proteins on these cells. Members of the ABC family have previously been shown to be responsible for classic multidrug resistance in many malignancies and have been found to be highly expressed on normal HSC and in AML. We looked at the relative expression between normal CD34+ cells and CML CD34+ cells of the transporters ABCB1 (MDR1 or p-gp), ABCC1 (MRP1) and ABCG2 (BCRP or MXR). We also investigated whether TKI are effluxed by these proteins and finally whether inhibition of the transporter function enhanced TKI-induced killing of the resistant CML stem cell population. We found that ABCG2 is expressed more highly in CML whereas ABCB1 is less abundant, however, we found that both IM and nilotinib inhibited transport via these proteins but were not effluxed themselves. Additionally, when we inhibited transporter activity we did not see any increase in the effectiveness of the TKIs. Therefore, we conclude that it is unlikely that ABC transporter mediated drug resistance is solely responsible for the insensitivity of CML stem cells to TKI-induced apoptosis.  Dasatinib, a is a newer TKI which has multiple targets including src-family kinases and bcr-abl, and has greater potency than IM. We have found that dasatinib inhibits bcr-abl activity more efficiently than IM but still does not induce apoptosis of the stem cell population. Thus it seems that single agent TKI may not be the way to eradicate CML stem cells. As we do not see complete inhibition of bcr-abl activity we decided to combine TKI with agents rationally chosen to target downstream pathways that are activated by bcr-abl including PI3K, Ras and MAPK signalling. We found that most of these combinations are no more effective than TKI alone in killing CML stem cells however, farnesyltransferase inhibitors were found to have a significant effect. BMS-214662 is the most effective of these and induces apoptosis of phenotypically and functionally defined CML stem cells in vitro, as a single agent and in combination with IM or dasatinib. The effect against CML stem cells is selective with less effect on normal stem cells. The drug is also effective against blast crisis CML stem cells and equally effective against wild type and mutant bcr-abl, including the most resistant mutant T315I. In association with apoptosis there is activation of caspase-8 and caspase-3, inhibition of the MAPK pathway and other signalling pathways. Furthermore, BMS-214662 synergises with MEK1/2 inhibitors, suggesting a second mechanism other that RAS inhibition that is responsible for induction of apoptosis. This is supported by our finding that a highly related agent BMS225975 has very similar farnesyltransferase inhibitory activity but very much lower capacity to kill CML stem cells. BMS214662 is the first agent we have tested that has significant activity against CML stem cells and offers the possibility of stem cell directed therapy that we have been seeking. We are exploring further the mechanism of action of BMS-214662 and extending these findings to other cancer stem cell disorders and hope to move this pre-clinical work to a clinical trial combining dasatinib with BMS-214662 in CML.