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MLL-AF9 (MA9) is a leukemia fusion gene formed upon translocation of the AF9 gene on chromosome 9 and the MLL gene on chromosome 11. MA9 is commonly found in acute myeloid leukemia (AML) and occasionally in acute lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. We have recently described a model system whereby we expressed the MA9 fusion gene in human CD34+ Umbilical Cord Blood (UCB) cells and showed that these cells transformed to acute myeloid or lymphoid leukemia when injected into immunodeficient mice. The Mixed Lineage Leukemia (MLL) oncogenes are unique in this model system in that they promote full transformation of primary human blood cells, while all other leukemia-associated oncogenes tested thus far have induced only partial phenotypes. Here we provide an update on the use of this system for modeling human leukemia and its potential application for therapeutic testing of novel compounds to treat the disease. We focus specifically on the Rho family of small guanosine triphosphatases (GTPases) as potential therapeutic targets, which we have implicated in the pathogenesis of AML associated with MA9 expression.
Numerous attempts have been made to transform primary human hematopoietic stem and progenitor cells (HSPC), primarily by viral delivery of leukemia-associated oncogenes, but until recently these efforts have proven unsuccessful. A comprehensive review of the history of this field has recently been published.1 Nearly ten years ago, the first transformation of primary human fibroblast cells using defined genetic elements was reported.2 However, it was not until last year that this same feat was accomplished for human HSPC, using primarily the MLL-ENL oncogene and resulting in B-cell acute lymphocytic leukemia (B-ALL).3 We have recently expanded upon this model system and described the reproducible generation of immortalized myeloid and lymphoid cell lines and transformation of human HSPC to B-ALL and acute myeloid leukemia (AML) using the MLL-AF9 oncogene.4 Similar to what was described by Barabe et al., we showed that the microenvironmental influence (i.e., cytokines) was critical in the lineage decision and resultant phenotypic outcome of leukemia. We also demonstrated the unique sensitivity of these transformed MLL-AF9 (MA9) cells to inhibition of the Rac GTPase signaling pathway. We showed that treatment of MA9 cells with the specific Rac inhibitor NSC23766 or transient knockdown of Rac expression by RNAi induced cell cycle arrest and apoptosis in MA9 cells but not in control cells. We hypothesize that Rac GTPases are required for the development and progression of MLL-AF9-mediated leukemia and therapeutic targeting of Rac could be a unique and important approach to treating MLL leukemia. Here we will give an update on the dynamics of this leukemia model and a review of the Rac signaling pathway with emphasis on its potential role in MLL-AF9-associated AML.
As we have previously described, expression of MA9 in human CD34+ cells results in immortalized myeloid or B-cell lines, and the culture conditions that are used for growing the cells are the determining factors for lineage outcome.4 We have cultured these cells in vitro for two years or more with no initiation of crisis and no appreciable phenotypic change in the cells. It was recently described that the MLL fusion oncogenes are not able to immortalize human HSPC using this model system.1 However, we believe that the lack of success in promoting immortalization upon MLL fusion protein expression, as reported by Barabe et al., is likely due to the specific cytokine combination that was used in their study to propagate the cells.3 For the myeloid cell lines and for the lymphoid B-ALL cells, we have found that the use of the cytokine Flt3L is important for their long-term growth in vitro. This specific cytokine was not included when propagating transduced cells in vitro in the studies reported by Barabe et al.
As expected for immortal cells, all of the cell lines we have tested are telomerase positive.4 Whether the MLL-AF9 and MLL-ENL proteins are activating hTERT expression/activity themselves or are promoting the growth of a cell that normally expresses hTERT remains to be determined. Interestingly, despite our success, there remain additional undefined hurdles to the reproducible immortalization of HSPC upon MLL-AF9 expression. Experimentally, one-third of UCB transduction experiments have failed to produce an immortal myeloid cell line, and this rate is independent of which MLL-AF9 fusion gene construct we use4 (Table 1). The reasons for this variability are not clear and a more complete understanding of the variables involved in this phenotype await further experimentation. Thus, we conclude that it is possible to immortalize human HSPC upon expression of MLL-AF9, and the growth of the cells is highly dependent upon microenvironmental influences.
The phenotype of in vitro expansion is not common for human leukemia samples. In addition, it has recently been shown that retroviral delivery of oncogenes could result in non-physiologic outcomes due to high expression driven by strong retroviral promoters.5 These issues raise concern as to the relevance of the model system we have described. However, as we have shown, the gene expression profile of the MA9 myeloid cell lines is closely correlated with the expression signature for MLL primary AML samples.4 This data would indicate that even under very different conditions of growth, the basic pattern of gene expression initiated and maintained by the MA9 oncoprotein is preserved in the experimentally initiated leukemic cells. With regard to the difficulty that is encountered in culturing human AML patient samples, it is likely that the leukemia cells become adapted to the environment in which they reside, and the shock of in vitro culture is very difficult to overcome. Some investigators have had more success in culturing primary leukemia cells in vitro when a more supportive microenvironment, such as a stromal cell line, has been employed.6,7 In fact, we have found that approximately 40% of the experimentally-induced leukemias that are recovered from a mouse bone marrow fail to expand in vitro, even though the initiating cells that were injected into the mouse had been successfully grown in culture (Table 2). This data supports our contention that the microenvironment in vivo is likely to strongly impact on the leukemia cell, even in a xenograft situation, and the stresses and foreign conditions that are encountered in vitro variably support the continued proliferation of the leukemia stem cells (LSC). In the model system that we established, the in vitro culture conditions are the initial microenvironment to which the HSPC are exposed and adapted, and the LSC that is formed upon MLL fusion protein expression is already adapted to these conditions. This is the most likely explanation for the ease with which human HSPC are immortalized in vitro upon MA9 expression. Finally, with regard to protein expression levels of the oncogene being artificially elevated by the retroviral promoter, it has been our experience that the amount of protein expressed in the immortal and transformed MA9 cells is exceedingly low. The reagents available for detection of the fusion protein are not likely to account for this difficulty, since a FLAG epitope tag is present on the oncoprotein and the MA9 fusion protein is readily detected in transient overexpression studies (data not shown). However, in long-term cultured cells and AML or ALL samples, the fusion protein is below detectable levels by either western blot or combined immunoprecipitation/western blot analysis. Although it is still possible that the levels of oncoprotein expression or the specific modulation of expression by the retroviral promoter during hematopoietic differentiation could account for some of the effects we obtain in this model, it seems likely that the resulting LSC that develops is representative of the LSC that is present in human patients.
The variables that are important for initiation of leukemia in xenograft models are not well defined. Numerous studies have shown that 30–50 percent of primary human AML samples do not engraft immunodeficient mice.8-11 We have noticed a similar trend using the MA9 model of transduced human CD34+ cells. As described in our recent paper, the long-term cultured MA9 myeloid cell lines induce AML and the tumor cells are typically transferable to secondary mice.4 Table 3 describes the data obtained using two such cell lines, clones 3 and 6, which reproducibly induce AML upon injection into mice, with disease latency and tumor burden a function primarily of mouse strain (i.e., the presence of the three human cytokines in the NOD-SCID-SGM3) irrespective of length of time in culture or route of injection. However, we have generated two other myeloid cells lines (clones 1 and 4) with virtually identical phenotypic and morphologic characteristics that do not reproducibly induce AML when injected into mice (Table 3). While clone 1 displays impressive killing ability initially, this ability is subsequently diminished after continued in vitro culture. Clone 4 is fairly ineffective initially and became completely unable to engraft and kill mice when injected at later timepoints of in vitro culture (Table 3 and data not shown). Based on these data, it seems that in vitro immortalization and in vivo transformation are separable phenotypes. One possible explanation for these results is that the clone which predominates in vitro is the most fit under cell culture conditions, but the characteristics that are necessary for in vivo expansion are not essential components in vitro and may not be an obligate phenotype of every immortal line. These characteristics could include homing efficiency, adherence to bone marrow stroma, metastatic capability, and propensity to respond to the microenvironmental signals in the bone marrow environment rather than the Petri dish. It is also possible that the genetic background of the donor cell influences these different parameters. Additional mutations may also be required for leukemogenesis above and beyond those required for immortalization, and some in vitro clones may be more or less likely to possess or acquire these genetic alterations. Obviously each cell line or tumor that is established is derived from a unique, outbred human sample, unlike the inbred mouse models that are established using similar approaches with murine cells. Given the variability that is seen in human leukemia patients with regard to disease presentation, treatment response, relapse and overall survival, we would expect that genetic factors influence some of the parameters in our model system as well. This is in fact one of the strengths of the current model in our opinion and an advantage over the use of inbred mouse strains for leukemia modeling.
As we have previously demonstrated, the sub-strain of immunodeficient mouse that is used has a significant impact on the latency of disease.4 The NOD-SCID-SGM3 mouse, that contains the three human cytokine transgenes encoding SCF, GM-CSF and IL-3, promotes a significantly faster myeloid disease than does the NOD-SCID mouse. This is evident in the data presented for clone 6 in Table 3, and has been seen repeatedly using multiple different samples. These three cytokines have been identified as particularly important for promoting myeloid cell proliferation, and the lack of cross-reactivity of the murine cytokines for human receptors is likely to be a critical factor in the xenograft model for the cytokine dependent MA9 cell lines as well as for primary AML patient samples. We have also noticed a significant improvement in penetrance and shortened latency of disease upon direct intrafemoral injection compared to intravenous injection (Table 3). Whether this is due to a homing deficiency in the MA9 cells or is due to an increased initial “load” of tumor cells in the proper niche remains to be determined.
One interesting result that has emerged from our use of two different MLL-AF9 fusion genes is the finding that these genes reproducibly generate immortal cell lines that differ in a number of ways. As we have previously shown, the surface phenotype of these myeloid cell lines show consistent differences for some common cell surface markers, including CD13, CD117 (c-kit) and CD135 (Flt3).4 The importance of these differences is not clear, and the high variability of expression of these molecules, as well as the fact that even those myeloid lines that show essentially no Flt3 surface expression are still dependent on Flt3L for growth and survival, raises questions as to whether these are technical artifacts rather than real differences. Importantly, both constructs induce immortalization with similar success rates (Table 1). However, one highly reproducible difference between these two groups of cell lines is the in vitro clonogenic efficiency. Those myeloid cell lines that were generated using the REW-MA9 retroviral construct have an average clonogenic rate of about 40% while the cell lines generated from the second retroviral construct show a ten-fold lower frequency of approximately 5% (Fig. 1). These in vitro differences do not correlate with in vivo aggressiveness; in fact, the two cell lines that show the highest clonogenic potential, MA9.1 and MA9.4, are the two that do not efficiently initiate tumorigenesis in mice (Table 3). There are two differences between these retroviral constructs. The chromosomal breakpoints differ in the MLL portion of the fusion genes, resulting in additional MLL coding sequence in the REW construct.4 In addition, for the REW construct, we engineered it so that EGFP is expressed as a fusion protein with MLL-AF9. This fusion protein (EGFP-2A-MA9) is proteolytically cleaved after translation to generate individual proteins, as a result of the presence of the Foot and Mouth Disease Virus 2A peptide.4,12 Which of these factors is responsible for the clonogenic differences remains to be determined. However, this data clearly highlights the need for further characterization of this model system and the variables that impact on the different readouts.
In a pilot series of experiments using a single MA9 myeloid cell line, we performed limiting dilution clonal analyses and expanded some of these single cell clones for leukemogenicity testing. Interestingly, for four purified clonal lines that were generated, all four were effective in leukemia induction, with similar kinetics to the parental myeloid cell line (Fig. 2). This data indicates that the high clonogenic rate that is observed in the MA9 cell lines (approximately 30% for the MA9.3 clone used in this experiment, see Fig. 1) can be considered as a measure of the LSC frequency in these cultures. This is surprising data, given the low frequency of LSC that has been observed in experiments using AML patient samples.13 However, it appears likely that MLL leukemia is a unique disease with characteristics that are not common to other subtypes of leukemia, including the ability to transform committed murine progenitor cells and to impart self-renewal properties on a high fraction of cells within the murine tumor.14-16 The uniqueness of this class of oncogenes extends to human cells as well, given that this is the only group of oncogenes that has thus far been successful in transforming primary human HSPC. The signaling pathways downstream of MLL oncogenes that are initiating such efficient self-renewal signals remain to be identified.
The molecular pathways mediating MA9 induced transformation are likely complex. Our gene array data of the MA9 transformed CD34+ cells showed multiple clusters of gene expression changes that may collectively result in the leukemogenic phenotype.4 In murine hematopoietic stem/progenitor cells transduced with MA9, it was shown that the expression of both the Rac1 and CDC42 GTPases, members of the Rho GTPase family, were elevated.15 In the CD34+ human cord blood cells, we also found that MA9 expression caused a significant increase of Rac1 activity in the myeloid cell lines.4 Given the well established role of Rac1 GTPase in regulating cell growth, cytoskeleton organization, and transcription, we employed both pharmacological and targeted knockdown approaches to test the possibility that the Rac GTPase signaling pathway is critical for the proliferation and survival of MA9 myeloid leukemia cells.4 We found that in the MA9 transduced cells, the small molecule Rac inhibitor NSC23766 or shRNA knockdown of Rac1 expression and activity in the leukemic cell could effectively promote apoptosis and suppress proliferation, under conditions where little or no toxic or inhibitory effect could be detected on normal human CD34+ cord blood cells.4 Studies have recently been published implicating Rac as an important player in human AML cell lines from patients with 11q23 aberrations, in primary human AML leukemia samples as well as in leukemia cells from a mouse model for BCR-ABL chronic myeloid leukemia.6,17,18 These results highlight the importance of this signaling pathway in hematologic malignancy and the potential for therapeutic targeting of the Rac family of proteins.
The precise mechanism by which inhibition of Rac signaling is affecting the growth of leukemia cells is not yet clearly defined. There is significant redundancy in function among the three members of the Rac subfamily, Rac1, Rac2 and Rac3, and this is especially true in hematopoietic cells, given that Rac2 is specifically expressed only in cells of the hematopoietic system. Rac signaling is important in cytoskeleton reorganization, adhesion, motility, cell cycle progression and survival.19 In our study, we showed that interference with the Rac signaling pathway induced rapid apoptosis with concomitant loss of Bcl-2 family members.4 Effects on proliferation were also noted, similar to what was described for BCR-ABL-expressing murine cells upon inhibition of Rac GTPase activity.17 The specific roles that Rac proteins play during hematopoiesis, and in particular during HSPC engraftment, has recently been reviewed.19 In addition, the potential importance of Rac signaling in leukemia development and maintenance is the subject of a recent comprehensive review article and this information will not be duplicated here.20 However, it is clear from all of these studies that the Rac signaling pathway represents a bona fide molecular target in hematologic malignancy, and the recent availability of a small molecule inhibitor of Rac signaling has been especially useful in confirming this hypothesis.21 The particular sensitivity of specific types of leukemia, including 11q23 AML and BCR-ABL-associated CML, has served to underscore the inherent differences between cytogenetic subtypes of leukemia and the importance of mapping the signaling pathways that are critical in each subset. We believe that the use of primary human HSPC as a starting point for modeling leukemia associated with different leukemia fusion proteins adds a unique dimension to what is currently available in the field and will complement the mouse models presently used for such studies.
We wish to thank the Viral Vector Core of Cincinnati Children's Hospital for production of retrovirus, the Mouse Core of Cincinnati Children's Hospital for help with irradiation and transplantation experiments, the Translational Trials Development and Support Laboratory of Cincinnati Children's Hospital for procurement of CD34+UCB HSPC, Kirin Brewery for the cytokine TPO and Amgen for Flt3L, SCF and IL-6. This work was funded by National Institutes of Health grants CA118319 and CA90370 (JCM), University of Cincinnati Cancer Center grant (JCM), the American Society of Hematology (JPW) and by U.S.P.H.S. Grant Number MO1 RR 08084, General Clinical Research Centers Program, National Center for Research Resources, NIH.
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6951