The cancer/leukemia stem cell theory has recently been called into question (22
), based in large part on criticism of the xenograft transplantation model that has been used to lay the foundation for this area of investigation in cancer research (1
). Subsequent studies have extended the body of evidence that rare cells within the leukemic population drive tumor formation, and that these cells could in fact be transformed progenitors (8
). A caveat to these analyses is that they rely on either human to mouse xenograft or retroviral transduction of purified cell populations and subsequent transplantation into secondary recipient animals. Thus, they confer disease through non-physiologic conditions, and cannot address questions regarding the pathologic initiation and development of disease due to progressive oncogenic insults. Here we have addressed these issues by assessing the leukemia stem cell theory using a model system of disease that expresses an oncogene in the appropriate developmental compartment without the need for retroviral transduction, thus more closely mimicking the human pathologic condition.
We selected acute promyelocytic leukemia as a model to characterize leukemia stem cells for several reasons. First, we were able utilize a well-characterized knock-in mouse model of the disease in which expression of PML-RARα is targeted to the promyelocyte compartment. Second, APL represents a relatively more differentiated form of acute leukemia as compared to other subtypes, and is therefore an attractive model in which to test the possibility that more differentiated progenitors can possess leukemia-initiating activity. Of note, APL (FAB subtype M3) was the only AML subtype that was unable to transfer disease to NOD-SCID recipient mice, regardless of the fraction of cells transplanted (2
). This lends further support to the notion that the leukemia-initiating population within APL is most likely not derived from an HSC. Finally, APL is the only leukemia with a clinically proven differentiation therapy in the form of all-trans
retinoic acid. Therefore, the effects of a known successful treatment for the disease could be tested specifically on the leukemia-initiating population.
Our results demonstrate that in acute promyelocytic leukemia, a committed progenitor, the promyelocyte, has the capacity to transfer disease upon transplantation, and therefore possesses leukemia stem cell properties. This data supports the notion that leukemia stem cells need not be derived from hematopoietic stem cells, and that indeed a committed progenitor can be transformed into a cell capable of maintaining the leukemic clone. The hallmark features of a leukemia stem cell are the ability to both self-renew and to differentiate into all cell types of the primary disease. Experiments by others have indicated that PML-RARα may possess the potential to confer self-renewal, but these assays were solely conducted in vitro
and relied on retroviral transduction of PML-RARα into stem and progenitor populations (23
). Here, we have demonstrated that progenitor populations from PML-RARα expressing animals do in fact possess the capacity to self-renew. Importantly, this ability is conferred to progenitor populations in the absence of frank leukemia; the mice used in these experiments were roughly 8 weeks of age, and appeared perfectly healthy. Despite the lack of leukemic transformation, PML-RARα-expressing promyelocytes displayed properties of self-renewal both in vitro
and in vivo
, a striking finding considering promyelocytes are thought to be post-mitotic cells that are committed to terminal differentiation into granulocytes, cells that normally survive only hours to days once in circulation. This suggests that PML-RARα may confer self-renewal ability to progenitor populations normally lacking this capacity as an initiating
step in the process of leukemogenesis. Indeed, analysis of the stem and myeloid progenitor compartments of PR/+ animals revealed no differences in the frequencies of these populations in the healthy state, suggesting that despite the capacity for self-renewal, these cells do not yet possess the requisite pro-survival or proliferative advantage required for leukemogenesis.
The fact that APL is a disease of promyelocytes prompted us to carefully explore this more differentiated stage of granulocyte development. Our results demonstrate that the shift from the healthy to leukemic state is accompanied by a massive expansion of the promyelocyte (c-kit+CD34+Gr-1+) compartment. Furthermore, this compartment is capable of transferring disease to secondary recipient animals, and is highly enriched for leukemia initiating activity as determined by limiting dilution transplants. These leukemic promyelocytes possess true leukemia stem cell properties, as defined by self-renewal and differentiation. Their capacity to serially transplant disease indicates that they must possess self-renewal ability in order to perpetuate the tumor. Additionally, flow cytometric and histologic analyses of tissues from diseased animals confirm that the leukemic animals succumbed to a disease that recapitulated that of the primary donor.
These results suggest a model for the development of APL in which PML-RARα plays a key role in initiating disease by conferring self-renewal, one of the requisite features of leukemia stem cells, to committed progenitors (See Supplementary Figure 3
). Although under normal physiologic conditions of myeloid development the hematopoietic stem cell is the only cell capable of self-renewal in the pathway of differentiation leading to the formation of a mature granulocyte, upon expression of PML-RARα, promyelocytic progenitors acquire the ability to self-renew. The sustained maintenance of these progenitors “primes” them for the acquisition of additional mutations that will lead to frank leukemia. Upon acquisition of additional mutations that confer proliferative and/or pro-survival advantage within the promyelocyte compartment, cooperativity between these secondary mutations and PML-RARα leads to massive expansion of promyelocyte progenitors, and acute leukemia results.
It is important to note that our results do not formally address the target cell of transformation in APL in which the initial PML-RARα mutation occurs, a question that remains unanswered in the APL field (25
). Interesting results obtained by Lane and Ley (27
) suggest that proteolytic processing of PML-RARα by neutrophil elastase is an important event for leukemic transformation (28
) and therefore, PML-RARα may be expressed in early stem and progenitor compartments, but may not be active or oncogenic until it is cleaved by enzymes that are only present later in myeloid differentiation. Further analysis in both murine models and human patient samples will be needed to clarify this debate.
These data provide several provocative insights into the contribution of the PML-RARα oncogene to leukemogenesis. First, this experimental system enables analysis of the physiologic consequences of oncogene expression in a committed progenitor prior to the onset of leukemia, and an ability to monitor changes with disease progression. Second, these findings help to understand how a hematopoietic progenitor that is committed to differentiation and cell death may be a target for transformation. In the context of multiple mutations that are required for leukemogenesis, it would be imperative that the initiating mutation enables self-renewal potential that would allow for acquisition of secondary mutations that confer the overt leukemia phenotype. PML-RARα expression meets these criteria in conferring long-term self-renewing potential to promyelocytes that are normally short lived. Third, it is remarkable that expression of a single leukemia oncogene – PML-RARα – apparently confers such properties to a cell that is primarily distinguished by its short half-life with terminal differentiation.
The identification and characterization of cancer stem cells has been catalyzed by the notion that current chemotherapeutics fail to eradicate cancer stem cells, leading to eventual disease relapse (reviewed in (10
). Therefore, there is increasing interest in identifying biologically distinct features of cancer stem cells that distinguish them from other cell types, including normal stem cells, in order to design better therapies that specifically target cancer stem cells. Recent data from hematologic malignancies provide evidence that differences do exist between normal HSCs and leukemia-initiating cells, providing hope that this strategy will be fruitful in eventually identifying targeted therapeutics that can eradicate leukemia stem cells (29
). In the case of APL, our data suggests that the leukemia-initiating promyelocytes are susceptible to differentiation therapy mediated by all-trans
retinoic acid therapy. However, particularly for the sub-population of patients who develop eventual resistance to ATRA therapy, it is important to develop effective alternative therapies.