For many patients, imatinib transforms CML from a life-threatening disease into a chronic condition. Newly diagnosed patients with chronic phase CML have an almost 90% chance of being alive at 60 months after diagnosis (2
). However, most patients continue to test positive by RT-PCR, and disease recurrence upon discontinuation of drug is the rule even in the minority of patients that become PCR undetectable (3
). This indicates that CML stem cells survive in the presence of imatinib and suggests lifelong continuation of therapy, at considerable expense and sometimes despite significant side effects. Elucidating the mechanism by which persistent CML stem cells escape the effects of imatinib will be crucial for directing strategies to eradicate residual leukemia. The central question is whether disease persistence is BCR-ABL dependent, like many cases of resistance, or BCR-ABL independent. In the first scenario, overcoming persistence will require effectively targeting BCR-ABL in the relevant (stem) cell compartment. In the second scenario, treatment directed at BCR-ABL as a biochemical target will not eliminate CML stem cells, and disease eradication will require a fundamentally different approach. This issue has been widely debated, and much of the literature to date has presented conflicting results, due in part to technical difficulties that accompany attempts to study exceedingly small, poorly defined primary cell populations combined with the use of small sample numbers. We sought to comprehensively evaluate whether BCR-ABL activity is inhibited by imatinib in several primitive CML cell populations by using multiple complimentary methods, including immunoblotting, phospho-FACS, and immunocytochemistry, evaluating large sample numbers when possible and establishing rigorous standards for negative controls to achieve reproducible results independent of the assay system. We analyzed the behavior both of populations attributed to disease persistence and of individual cells within those populations. Additionally, we examined the correlation between BCR-ABL inhibition by imatinib and survival and growth of these primitive populations to establish a model for how persistent CML cells may function in the context of treatment with imatinib and next-generation BCR-ABL inhibitors.
In a first set of experiments to determine whether CML stem cell survival is BCR-ABL dependent or independent, we used FACS sorting, intracellular staining, and immunoblot analysis to investigate phospho-CRKL and phosphotyrosine levels in CML stem and progenitor cells exposed to imatinib. In vitro imatinib treatment reduced phospho-CRKL and total phosphotyrosine levels in the CD34+CD38– and CD133+ stem cell compartments of newly diagnosed CML patients to the levels seen in normal controls. Importantly, the degree of BCR-ABL inhibition in CML stem cells was comparable to that of more mature CD34+CD38+ cells and CML-derived cell lines, which indicated that BCR-ABL in the context of CML stem cells was not resistant to inhibition by imatinib.
Our findings are consistent with those of a study in which immunoblots of CD34+
cells from a single CML patient showed a dose-dependent reduction of phospho-CRKL in response to imatinib (26
), but they differ from those of Copland et al., who used phospho-CRKL flow cytometry as their principal technique to assay BCR-ABL activity (27
). The reasons for the differing results are likely technical. Although phospho-CRKL flow cytometry presents a practical advantage over immunoblotting by permitting analysis of rare cell populations, in our hands, insufficient staining specificity in the context of primary cells posed a considerable challenge. We therefore chose to validate our results using phosphotyrosine flow cytometry and phospho-CRKL immunoblotting. Phosphotyrosine FACS analysis showed that imatinib treatment in CML stem and progenitor cells reduced signals to the levels seen in normal controls. However, by immunoblotting, we observed complete inhibition of phospho-CRKL in CML stem and progenitor cells after imatinib treatment. Thus, these data indicate that the residual signal detected by FACS is not the result of residual BCR-ABL kinase activity. We also observed the same consistent pattern of BCR-ABL inhibition by imatinib in CML stem and progenitor cells from multiple patients, consistent with adequate access of the drug to its biochemical target.
Total phosphotyrosine and phospho-CRKL as a substrate of BCR-ABL have been widely used as surrogate markers for BCR-ABL activity, largely because of the availability of high-quality antibodies allowing consistent robust detection within the context of primary cells. Ideally, however, BCR-ABL activity would be measured directly. Although we attempted to expand our assays to include phospho–BCR-ABL and additional BCR-ABL substrates and downstream signaling components, we were limited technically by the utility of available antibodies. Because our methods of detection of phospho-CRKL or total phosphotyrosine yielded reproducible results that were well supported by appropriate negative controls, we ultimately favored the use of these surrogate markers.
We also explored how the inhibition of BCR-ABL activity in primitive CML cells affected their survival and proliferation. When high concentrations of exogenous cytokines were supplied, Lin–, CD34+, and CD34+CD38– cells remained capable of in vitro proliferation despite continuous BCR-ABL inhibition. Imatinib reduced proliferation of all CML progenitor cell types to the level of normal progenitor cells over a range of cytokine concentrations, but induced only moderate apoptosis that was predominantly limited to cells with more mature immunophenotypes. Additionally, we used LTC-IC assays to confirm that cells surviving BCR-ABL inhibition retained the in vitro functional capacity of stem cells. Although it would be ideal to assay stem cells by measuring engraftment in immunocompromised mice, LTC-IC is a widely used surrogate assay to assess capacity for long-term survival. Together, the survival of CML cells with stem and progenitor cell phenotype and function and the restoration of normal homeostasis in the presence of imatinib indicate that primitive CML cells do not require BCR-ABL activity for survival when cytokines are present.
Phenotypic markers such as CD34, CD38, and CD133 are used to identify populations enriched for stem cell activity; however, it is known that these populations are not entirely homogenous in function. It is conceivable that smaller subpopulations exist within our phenotypically defined stem cell compartment that are resistant to imatinib therapy and responsible for disease persistence. The mechanisms of imatinib resistance in smaller subpopulations could be BCR-ABL independent, as we observed for the population as a whole, and thus a subgroup with reactivated BCR-ABL in the presence of imatinib could be selected by extended imatinib treatment. However, the absence of better immunological markers of stemness or markers that define the persistent leukemic population has been a major obstacle to resolving this possibility. To address this, at least in part, we used immunocytochemistry of phospho-CRKL, phospho-STAT5, and phospho-AKT to evaluate BCR-ABL activity in individual cells within the CD34+
population. We also tried to confirm our results using anti–phospho–c-ABL (Tyr245; Cell Signaling), which was used previously to detect phospho–c-ABL by FACS (42
). However, in our hands, this antibody produced unreliable FACS results due to a low signal-to-noise ratio, and on immunocytochemistry failed to detect any signal even at a very low (1:5) dilution. Therefore, we focused our analysis on other surrogate markers of BCR-ABL kinase activity, including phospho-CRKL, phospho-STAT5, and phospho-AKT. We observed that inhibition of BCR-ABL was homogeneous throughout the population, and we did not identify cells that demonstrated reactivated BCR-ABL activity in the presence of imatinib. Interestingly, phospho-AKT levels were low in CD34+
cells compared with the more mature CD34+
cells and were not influenced by imatinib (Figure D). This was in concordance with a recent report on FOXO transcription factors in the same cell populations, in which the authors demonstrate that FOXO proteins are nuclear in CD34+
CML cells and leukemia-initiating cells, despite the presence of active BCR-ABL, in contrast to their cytoplasmic localization in more mature cells (36
). Because AKT is the major kinase that phosphorylates FOXO1, 3A, and 4, this suggests that AKT activity is low in the most primitive cells. Mechanistically, this was shown to be the result of TGF-β signaling that counteracts AKT in leukemia-initiating cells, consistent with our immunocytochemistry results. Together, our data support the notion that CML cells surviving imatinib therapy do so independently of BCR-ABL activity and in a uniform fashion.
There is considerable evidence that drug transport proteins display altered expression patterns in both normal and CML stem cells relative to more committed progenitors (8
), and it has been proposed that lower intracellular imatinib concentrations may lead to resistance. It remains controversial, however, whether imatinib is a substrate of potentially relevant transport proteins (43
), and evidence that altered expression of efflux/influx proteins affects imatinib sensitivity in CML stem cells is lacking. We demonstrated that BCR-ABL activity was inhibited equivalently in CML stem and progenitor cells over a range of imatinib concentrations, which suggests that imatinib has access to the relevant target in CML stem cells, and factors other than insufficient intracellular imatinib caused by altered drug efflux/influx in CML stem cells must therefore be responsible for its limited efficacy in this population. In line with this, Copland et al. demonstrated equivalent uptake of imatinib in CD34+
stem cells versus total CD34+
), indicating that drug transport–mediated alteration of intracellular imatinib is an unlikely cause of resistance in the former population. Similarly, although BCR-ABL expression may be higher in primitive CML cells compared with progenitors, our data showed that BCR-ABL kinase activity was inhibited equally in CML CD34+
cell populations, which rules out the possibility that kinase activity is sustained by high protein levels.
Stem cell quiescence has been proposed as a potential mechanism by which primitive leukemic cells escape the cytotoxic effects of imatinib, although no mechanistic link has been provided. In particular, it was unclear whether BCR-ABL activity mediated survival of this cell population. In accord with previous reports (19
), we observed that primitive noncycling BCR-ABL–positive cells survive in the presence of imatinib upon ex vivo culture of CML cells from newly diagnosed patients. Moreover, using Ki-67 and PCNA as markers for cycling cells, we found that imatinib inhibited BCR-ABL in the quiescent population to a similar degree as in the cycling cells. This indicates that the ability of quiescent cells to survive in the presence of imatinib does not depend on BCR-ABL activity. The requirement of excessively large cell numbers for our analysis restricted us to the evaluation of BCR-ABL activity in Lin–
cells, rather than Lin–
cells, a phenotypic subgroup that would be more representative of a true quiescent stem cell population. Thus, we cannot exclude that the BCR-ABL activity and imatinib sensitivity we observed in quiescent Lin–
cells would differ in more primitive quiescent cells.
It remains unclear how the pro-proliferative effect of BCR-ABL affects quiescent CML stem cells. Expression of BCR-ABL was previously shown to increase the relative frequency of proliferating CML stem cells; however, Holyoake et al. identified a quiescent CML stem cell population that remained resistant to cycling despite expression of BCR-ABL or addition of cytokines (18
), which suggests that mechanisms governing induction of cycling of CML stem cells are complex and are, at least in part, independent of BCR-ABL signaling. Our observation of BCR-ABL activity within the quiescent progenitor population likewise suggests that the pro-proliferative signals generated by BCR-ABL are not sufficient to induce cycling in all quiescent CML cells and further supports the concept that imatinib inhibition of BCR-ABL may minimally affect this population.
Efforts to induce cycling in resistant quiescent stem cells are currently being explored as a means of sensitizing leukemic stem cells to imatinib (39
). This principle relies on the assumption that imatinib will cause cycling stem cells to either undergo apoptosis or irreversibly commit to differentiation without regenerating the leukemic stem cell pool. We found that with cytokine support, both quiescent and cycling primitive leukemic cells survived imatinib inhibition and, consistent with previous studies (38
), imatinib did not induce apoptosis. The survival of both populations despite BCR-ABL inhibition implies that driving quiescent CML stem cells into cycle as a sole means of sensitization to imatinib-induced cell death may be insufficient for their elimination. Disease persistence may therefore occur via the concurrence of 2 properties of CML stem cells: (a) the ability of primitive leukemia cells to escape apoptosis, despite BCR-ABL inhibition (i.e., lack of oncogene addiction); and (b) the ability of CML stem cells to self-renew (Figure ).
Model of CML disease persistence with imatinib treatment.
Together, our data suggest that in vivo CML stem cells use survival signals other than BCR-ABL kinase to maintain their viability in the presence of tyrosine kinase inhibitors. Most likely, these signals are provided by the microenvironment and modulate the biological outcomes of BCR-ABL inhibition, such as growth arrest and apoptosis. As there is no evidence to our knowledge that the ability of imatinib to bind to its biochemical target is under the control of extrinsic factors, our data indicate that persistence of CML stem cells must be independent of BCR-ABL activity. This notion has far-reaching implications for all therapies that biochemically target BCR-ABL, the prediction being that none of these therapies will be able to eliminate CML stem cells. Our observation that CML stem cells survived in vitro exposure to the second-line ABL kinase inhibitors nilotinib and dasatinib corroborates this notion. Previously, 2 studies of newly diagnosed chronic phase patients treated with nilotinib or dasatinib showed higher rates of CCRs and major molecular responses at 12 and 24 months compared with standard and high-dose imatinib; however, low-level disease persistence continued in almost all patients, and in those who had been on drug for 30 months, a complete molecular response (absence of detectable BCR-ABL transcripts) was seen in 2 of 30 receiving nilotinib and 0 of 23 receiving dasatinib (45
Nearly all patients undergoing imatinib treatment maintain a residual leukemic population; however, the amount of residual disease is exceedingly small relative to the restored normal hematopoietic cell populations. While this appears difficult to reconcile with our observation that CML stem cells treated with imatinib behaved similar to normal cells, this may be due to the fact that the stem cell population in many patients is predominantly Ph–, and thus prevention of expansion of the CML clone at the progenitor cell level by imatinib treatment restores this situation. Alternatively, it is possible that suppression of BCR-ABL by imatinib creates a dominant-negative situation, or that CML cells under imatinib suppression may be at a subtle disadvantage relative to normal cells that we are unable to detect in our in vitro studies but would be relevant over time in patients treated with imatinib. Additional studies to address such subtle differences in growth kinetics of normal versus imatinib-treated CML cells would be necessary to fully resolve this issue.
In conclusion, we showed that primitive CML cells are capable of BCR-ABL–independent survival, which suggests that CML stem cell elimination may require completely different strategies, such as targeting stem cell self-renewal or disrupting interactions with the microenvironment. Future research should include examination of specific cytokine or adhesion-mediated interactions within the bone marrow microenvironment that may provide critical support for leukemic stem cells, CML stem cell homing mechanisms, or pathways required for self-renewal of these cells. Alternatively, it may be possible to eliminate CML stem cells using BCR-ABL as an immunological rather than a biochemical target (47
). Nonspecific immunotherapy with interferon-α may see a revival, based on anecdotal reports that some patients with prior interferon exposure remained RT-PCR negative for prolonged periods of time after discontinuation of imatinib (5
). The demonstration of BCR-ABL inhibition by imatinib in primitive CML cells is an important step toward understanding how to approach persistent disease with the ultimate goal of leukemic stem cell eradication as a means to achieve a cure.