Effective treatment of Ph-positive ALL remains a considerable challenge and dasatinib, because it is a more potent tyrosine kinase inhibitor that targets Bcr/Abl as well as SFK implicated in Ph-positive ALL, is a very promising therapeutic drug. However, most preclinical studies to date focused on the effect of dasatinib on myeloid Ph-positive leukemias. For example, Deguchi et al.
compared the growth-inhibitory effects of imatinib, nilotinib and dasatinib against six CML cell lines and reported that dasatinib showed the most potent effects. In addition, it was also effective in imatinib-resistant CML cell lines. 26
Similar results were described in Redaelli et al.
who studied the activity of dasatinib, imatinib, and nilotinib against 18 mutated forms of Bcr/Abl expressed in Ba/F3 transfected cells. 27
The first human clinical study with dasatinib 28
reported that nearly all patients with Ph-positive ALL had a relapse within 6 months, although Ottmann et al 29
reported in a phase II study, that dasatinib was safe and effective, and that, with the 8-month follow up, it seemed promising. 29
Using models for Ph-positive leukemias in which mice were transplanted with Bcr/Abl-transduced bone marrow, Hu et al.
showed that dasatinib inhibits the proliferation of both myeloid and B-lymphoid cells in vivo
However, even after treatment for 3 months with dasatinib, leukemic cells persisted in both models and gave rise to relapse when treatment was discontinued. This shows that relapse during dasatinib treatment is likely and prompted us to examine early steps in possible emergence of insensitivity to it.
In our studies, we confirm that dasatinib is “effective” against Bcr/Abl positive lymphoblastic leukemia: it clearly inhibits the proliferation of Bcr/Abl P190-expressing mouse ALL cells and, to a lesser extent, that of human Ph-positive ALL cells, causes apoptosis, and knocks down the tyrosine phosphorylation activities of both Bcr/Abl and Src-family kinases. The most critical test of the effectiveness of a drug, however, is its ability to totally eradicate leukemic cells. Using that as criterion, we find resistance against dasatinib can be evoked by providing the leukemia cells with stromal support and using suboptimal doses of dasatinib.
With a half-life of 3-5 hours and a once daily dose regimen, we would expect dasatinib to reach sub-therapeutic levels within 24 hours in patients. Shah et al 14
argue that this should not prevent dasatinib effectiveness, because leukemia cells treated with such tyrosine kinase inhibitors would be put on an irreversible path to apoptosis. We suggest that it may be very difficult to set ALL cells on the pathway to irreversible apoptosis under conditions that resemble those in vivo
. Our results show that stroma provides protection against dasatinib and allows viable cells to emerge even during long-term treatment.
The only other study to investigate dasatinib resistance in Ph-positive cells is that of Okabe et al 31
, who found decreased expression of Bcr/Abl during emerging drug resistance in K562. In our study, we did not detect changes in levels of Bcr/Abl protein and we therefore sought alternative mechanisms that could provide protection against dasatinib. Stroma is known to secrete factors that can partially protect Bcr/Abl expressing cells from the inhibitory effects of imatinib and nilotinib. 32
Using a stromal MEF co-culture system, we previously showed that SDF-1α is one of the protective factors that provide support to mouse Bcr/Abl cells treated with Imatinib. 16
Indeed, the interaction of the cytokine SDF-1α (CXCL12) with the CXCR4 receptor plays a pivotal role in the earliest stages of normal B-cell development and CXCR4 also helps retain pre-B cells in the bone marrow. 33
Because Jin et al.
demonstrated that imatinib increases the expression of CXCR4 on imatinib sensitive KBM-5 and K562 cells 23
, we considered the possibility that dasatinib could induce CXCR4 expression and by this mechanism possibly increase the longer-term survival of the ALL cells. Our results show that fully viable ALL cells that are plated de novo
on a stromal feeder layer and are stimulated by the stromal-produced SDF-1α remove the cell-surface CXCR4 over a period of 48 hours. We found that Bcr/Abl-ALL cells treated with dasatinib, similar to K562 treated with Imatinib or BV-173 cells treated with Imatinib or nilotinib, significantly increased cell surface expression of CXCR4. 23,34
Since there were no major changes in CXCR4 total cellular protein, our results are consistent with a model in which dasatinib prevents the normal internalization of the SDF-1α-stimulated CXCR4 receptor. Also, treatment with AMD3100 increased CXCR4 cell surface expression on these cells. Interestingly, the increased cell surface expression of CXCR4 did not provide a survival benefit to these cells. In contrast, cells exposed to dasatinib plus AMD3100 were killed effectively while they had amounts of CXCR4 on their surface comparable to that of non-drug treated cells at the 4-hr time point.
In spite of the importance of the CXCR4-CXCL12 axis for many cell types, surprisingly little is known about the trafficking of the CXCR4 receptor and the significance of this for the transduction of survival and other SDF-1α-associated signals. Early studies in the context of HIV research show that, depending on the cell type, there is a varying degree of spontaneous CXCR4 endocytosis. 24, 35
Internalized receptor can be degraded in the lysosome or recycled back to the cell surface. Binding of SDF-1α to CXCR4 causes rapid internalization of CXCR4 24
and activates numerous tyrosine kinases including Lyn, ZAP-70 and Tec in T-cells. 36
Based on our data, it is possible that dasatinib interferes with CXCR4 endocytosis and/or transduction of survival signals downstream of CXCR4 by blocking tyrosine kinase activity needed for this. This raises the interesting possibility that one of the mechanisms by which dasatinib kills ALL cells is by interfering with CXCR4 signal transduction.
Our in vitro
results suggest that Ph-positive ALL patients will not be able to achieve a durable response, if dasatinib is used as monotherapy. However, the combination with drugs such as AMD3100 could present an alternative strategy. More than 23 types of cancers other than leukemias express CXCR4 37
and dasatinib is in clinical trials for the treatment of, among others, prostate, pancreatic, non-small lung and metastatic breast cancer (www.clinicaltrials.gov
). The combined treatment with dasatinib and a drug that inhibits CXCR4-SDF-1α could therefore be an interesting alternative approach as well for these types of cancers.