Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement

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Abstract

Infant acute lymphoblastic leukemia (ALL) involving mixed-lineage leukemia (MLL) fusions has attracted a huge interest in basic and clinical research because of its prenatal origin, mixed-lineage phenotype, dismal prognosis and extremely short latency. Over 90% of infant ALLs are pro-B ALL harboring the leukemic fusion MLL-AF4. Despite the fact that major achievements have provided a better understanding about the etiology of infant MLL-AF4+ ALL over the last two decades, key questions remain unanswered. Epidemiological and genetic studies suggest that the in utero origin of MLL rearrangements in infant leukemia may be the result of prenatal exposure to genotoxic compounds. In fact, chronic exposure of human embryonic stem cells (hESCs) to etoposide induces MLL rearrangements and makes hESC more prone to acquire subsequent chromosomal abnormalities than postnatal CD34+ cells, linking embryonic exposure to topoisomerase II inhibitors to genomic instability and MLL rearrangements. Unfortunately, very little is known about the nature of the target cell for transformation. Neuron-glial antigen 2 expression was initially claimed to be specifically associated with MLL rearrangements and was recently shown to be readily expressed in CD34+CD38+, but not CD34+CD38− cells suggesting that progenitors rather than stem cells may be the target cell for transformation. Importantly, the recent findings showing that MLL-AF4 rearrangement is present and expressed in mesenchymal stem cells from infant patients with MLLAF4+ ALL challenged our current view of the etiology and cellular origin of this leukemia. It becomes therefore crucial to determine where the leukemia relapses come from and how the tumor–stroma relationship is defined at the molecular level. Finally, MLL-AF4 leukemogenesis has been particularly difficult to model and bona fide MLL-AF4 disease models do not exist so far. It is likely that the current disease models are missing some essential ingredients of leukemogenesis in the human embryo/fetus. We thus propose modeling MLL-AF4+ infant pro-B ALL using prenatal hESCs.

Etiology of the infant pro-B acute lymphoblastic leukemia harboring the MLL-AF4 fusion gene

Over 90% of the leukemias diagnosed in newborns/infants (<1 year old) are pro-B stage acute lymphoblastic leukemias (ALLs) harboring the leukemic fusion gene mixed-lineage leukemia (MLL)-AF4.1, 2 The MLL gene located in chromosome 11q23 fuses to generate chimeric genes with over 100 partners in human leukemia.3, 4 This MLL-AF4 pro-B ALL represents a very rare leukemia as compared with other pediatric ALL affecting later differentiation stages (pre-B), which are typically seen in older children (3–10 years old).5 MLL-AF4+ infant pro-B ALL is associated with dismal prognosis (5-year disease-free survival lower than 20%) and very brief latency. This raises the question of how this disease can evolve so quickly, particularly if additional secondary mutations are required. MLL-rearranged leukemias commonly have activating FLT3 mutations6, 7 and around 50% of the cases have additional chromosomal abnormalities.2, 8, 9

Over the last decades, major achievements have provided a better understanding about the etiology of infant MLL-AF4+ ALL. Elegant studies on identical twins with concordant MLL-AF4+ leukemia and retrospective analyses of the clonotypic MLL-rearranged sequences of blast cells from young patients in their neonatal blood spots revealed a in utero origin of the MLL rearrangements.10, 11 Importantly, compelling information indicates that the MLL-AF4 does not suffice to promote leukemogenesis on its own and additional secondary genetic insults are required.3, 9

Epidemiological and genetic studies support the contention that the in utero origin of MLL rearrangements in infant leukemia may be the result of exposures, during pregnancy, to genotoxic compounds present in the maternal diet intake capable of inducing breaks in the MLL locus in the fetus but not in the mother who has functional DNA repair mechanisms in place.12, 13 The MLL rearrangements may in fact be the result of transplacental exposures to substances that alter the function of DNA topoisiomerase II, a DNA repairing enzyme highly expressed during embryonic development.14, 15, 16, 17, 18, 19, 20 Among these genotoxic compounds, the etoposide (VP16) is the best studied. Etoposide is a topoisomerase II inhibitor commonly used in chemotherapy cocktails and is responsible for 5–15% of the therapy-related acute leukemias.12, 13, 21, 22 Exposure of cells to topoisomerase II inhibitors increases the frequency of illegitimate recombination events,23 a physiologic activity that may be related to both cytotoxicity and leukemogenicity of etoposide. Recent studies24, 25 suggest that high dietary intake of bioflavonoids, an abundant source of topoisomerase-II inhibitors in the diet, could cause breaks in MLL and possibly in other partner genes, therefore having an important role in the generation of the preleukemic clone in infancy and in the development of therapy-related acute leukemia.26 It has been shown that exposure to high doses of etoposide experimentally induces MLL breaks in mouse embryonic stem cells (ESCs),15 fetal liver-derived CD34+ hematopoietic stem cells (HSCs)19 and in cord blood (CB)-derived CD34+ HSCs.14, 17, 18 However, the effects of etoposide earlier during human embryonic development remain to be determined. Human ESCs (hESCs) hold the promise to become a powerful tool for drug screening and toxicity but also to determine the spatial-temporal onset of diseases that are known to arise in utero.16, 27, 28, 29

Etoposide induces MLL rearragements in hESCs and CB-CD34+ hematopoietic stem/progenitor cells

We have recently used hESCs as a model to test the effects of etoposide on human early embryonic development.16 We wanted to address whether (i) very low doses of etoposide promote MLL rearrangements in hESCs and hESC-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective proliferation/survival advantage and; (iii) whether continuous exposure to very low doses of etoposide predisposes hESCs to acquire other chromosomal abnormalities. Interestingly, exposure to a single low dose of etoposide induced a pronounced cell death in undifferentiated hESCs but not in postnatal CD34+ HSCs. The striking vulnerability of hESCs to etoposide-induced cell death is in line with previous studies confirming the crucial role of both DNA topoisomerase II-α and -β in human developing tissues.20

A single low dose of etoposide induced MLL breaks in 2–3% of the hESCs and CB-derived CD34+ HSCs (Figures 1a and b). This datum is similar to that reported in fetal and CB-derived CD34+ HSC.14, 17, 18, 19 Interestingly, however, hESCs are much more susceptible than mouse ESCs to etoposide-induced MLL breaks.15, 16 Etoposide concentrations as high as 100 μM barely induced MLL rearrangements in just one out of 62,500 mouse ESCs,15 whereas relatively low concentrations (0.2–0.5 μM) induced MLL gene fusions in an average of three out of 100 hESCs (a 1800-fold increase).16 Physiological doses of etoposide were used by Bueno et al.16 because the etoposide concentration in the plasma of cancer patients treated with this drug ranges between 1 and 2 μM,30 a concentration far below the 100 μM employed in previous studies.

Figure 1
figure1

Effects of etoposide exposure in hESCs and CB-derived CD34+ HSCs. MLL rearrangements were identified, quantified and characterized by inverse PCR (data not shown) and fluorescence in situ hybridization using a split-apart MLL probe in hESCs (a) and CD34+ HSCs (b) treated with a single dose of etoposide (0.2 or 0.5 μM). Upon continuous exposure to very low doses of etoposide (0.02 μM) chromosomal abnormalities were detected by conventional G-banding, which could be further confirmed by spectral karyotyping in hESCs (c) but not in CB-derived CD34+ cells (d).

It has been suggested that early prenatal HSCs may be the target for MLL fusions.19, 27 Therefore, using conditions previously optimized to promote hematopoietic differentiation from hESCs,16, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 Bueno et al assessed to what extent etoposide induces MLL breaks at two different developmental stages during human embryonic hematopoietic development. Early (day+15) hESC-derived hematopoietic derivatives seem to be slightly more susceptible to etoposide-induced MLL breaks than late (day+22) fully differentiated hESC-derived hematopoietic derivatives (2.9 vs 1.6%, respectively).16 This suggests that postnatal CD34+ and late hESC-derived hematopoietic cells are less vulnerable to etoposide-induced MLL rearrangements than undifferentiated hESCs or earlier hESCs-derived hematopoietic cells.

Human ESCs are slightly more vulnerable to etoposide-induced MLL rearrangements than CB-derived CD34+ HSCs. After long-term culture, the proportion of hESCs harboring MLL rearrangements diminishes and neither cell cycle variations (S-phase: 64.4 vs 63.1%, respectively) nor genomic abnormalities are observed in the etoposide-treated hESCs, suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage and that additional secondary cooperation mutations may be missing. In fact, continuous exposure to very low doses of etoposide induced MLL rearrangements and primed hESCs (Figure 1c) but not CB-derived CD34+ HSCs (Figure 1d) to acquire other major karyotypic abnormalities. Thus, chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and makes hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34+ cells, linking embryonic exposure to inhibitors of the topoisomerase II to genomic instability and MLL gene rearrangements.

Environmental exposures and delayed infection early in life as a plausible etiological mechanism for childhood leukemias

The etiology and pathogenesis of MLL-rearranged pediatric leukemias differs from other MLL-germline leukemias harboring other fusion genes such as E2A-PBX1, BCR–ABL or TEL-AML1. Much speculation and epidemiological endeavor in identifying the underlying causal mechanisms in childhood leukemia have been simplistic42 and the implicit premise that ‘the cause’ should be attributable solely to ‘an exposure’ is to ignore the complexity of biology. Leukemia, if not all cancers, is the result of a combination of crucial exposures, modifying influences, inherited susceptibility and chance.42 A plethora of candidate environmental exposures have been proposed. The only established causal exposure for childhood leukemia is ionizing radiation. This unambiguous conclusion is derived from data on Japanese atomic bomb survivors from 1945 who were acutely exposed to up to >200 mSv43 and, at a much lower dose level (10 mSv), from historical data on diagnostic exposure of the fetus from X-ray pelvimetry during pregnancy.44 Unfortunately, most of these environmental exposures lack a biological rationale or consistent epidemiological evidence. Although there might not be a single or exclusive cause, an abnormal immune response to common infection(s) has emerged as a plausible etiological mechanism for childhood leukemias.42 The most recent epidemiological surveys conclude that although conflicting data exist, the weight of evidence overall is supportive of both population mixing and delayed infection in infancy as being significant causal factors.45 Time-space clustering is also compatible with the delayed-infection hypothesis, but more-specific epidemiological predictions of this hypothesis relate to the timing of infections or opportunities for infection early in life.46, 47 The patterns of exposure, timing of infections in the first year of life and the immunological response to such challenges will have multifactorial determinants. These will include breastfeeding practices and mothers’ immune status, social factors such as hygiene conditions and interactions with other children of the same or older age, community factors such as population density, mobility, age and infectious history.46, 47 Unfortunately, we have no algorithm to compute the overall likelihood of infection and immunological response under these highly variable circumstances. The simplest prediction of the hypothesis is that patients with childhood leukemia might be expected to have fewer recorded common infections in the first year of life, and less social contact and the potential for infectious exposure outside their home—for example, through day-care attendance.46, 47

Cellular origin of the infant MLL-AF4+ pro-B ALL

In infant ALL wherein t(4;11) MLL-AF4 is very common (>90%), the fusion gene arises in utero.10, 48, 49 However, very little is known about the nature of the target cell for transformation in the embryo/fetus and the mechanisms accounting for its B-cell lineage affiliation. Unfortunately, mouse models and transformed cell lines have been used with only modest success to model the effects of MLL-AF4 and the disease phenotypes achieved do not faithfully mimic those seen in the actual infant disease. Moreover, MLL-AF4 protein seems toxic when retrovirally overexpressed in mouse or human stem cells. It could be argued that a cell in a wrong developmental or hierarchical position had been targeted in these experiments. Alternatively, MLL-AF4 might have a detrimental effect when expressed under the long terminal repeat retroviral promoter at levels much higher than required to be oncogenic. Despite the target cell where MLL-AF4 arises being undefined, HSCs and hematopoietic progenitor (HPC) cells represent likely targets for transformation: the infant MLL-AF4+ pro-B ALL displays a pro-B or pro-B/monocyte phenotype and fluorescence in situ hybridization studies in isolated cell subsets indicate that MLL-AF4 may be already present in a very primitive CD34+CD19− cell subset.50

New perspectives in the association between the expression of neuron-glial antigen 2 (NG2) and the presence of MLL rearrangements acute leukemias

The NG2 molecule and its human homolog was first reported on oligodendrocyte progenitor cells.51 NG2 is recognized by the 7.1 monoclonal antibody.52 The physiological role of this molecule remains to be elucidated.53 The expression pattern of NG2 in leukemia is controversial. NG2 expression was initially claimed to be specifically associated with MLL gene rearrangements.54 In fact, NG2 has gradually been incorporated in diagnostic panels for immunophenotyping of leukemic patients because of its potential predictive value for MLL rearrangements in childhood and adult acute myeloid leukemias.54, 55, 56, 57, 58 Moreover, commonly seen in the clinic are leukemic patients harboring MLL rearrangements but lacking NG2 expression (Table 1).55, 57, 59 Conversely, there are a proportion of cases in which expression of NG2 is clearly detected in the absence of MLL rearrangements.56, 57 More recently, it has been suggested that 7.1 expression could be specifically associated with only two specific subtypes of leukemia harboring either the translocations t(4;11)(q21;q23) or t(9;11)(p13;q23), which encode for the leukemic fusion genes MLL-AF4 and MLL-AF9, respectively, but not for other MLL rearrangements.19 Importantly, we and many others have reported the existence of acute leukemias and plasmacytoid dendritic cell (pDC) leukemias (>50%) lacking MLL rearrangements but expressing NG2 (Table 1).60, 61, 62

Table 1 Published clinical data supporting the lack of association between MLL rearrangements and NG2 expression

Based on the controversial data about the clinical relevance of NG2 expression together with the existence of NG2-expressing acute leukemias lacking MLL rearrangements, in particular pDC leukemias,60, 61 we wanted to gain further insight into the biological association between NG2 expression and MLL rearrangements. We analyzed whether the expression of NG2 may depend on the particular gene(s) paired to MLL when it is rearranged and we also explored the hypothesis that the expression of NG2 in leukemias lacking MLL rearrangements, such as NG2+ pDC leukemias, may be due to the existence of a minor subset of CD34+ hematopoietic stem/progenitor cells readily coexpressing NG2 wherein the leukomogenesis process may be initially triggered.62 Our experimental data support the clinical finding of both human leukemias with balanced MLL rearrangements coexpressing NG2 and human leukemias harboring balanced MLL gene translocations but lacking NG2 expression. Several cellular and molecular mechanisms, intrinsic molecular determinants and extrinsic signals may contribute to the controversial correlation between MLL rearrangements and NG2 regulation. Extensive in vitro data rule out the possibility that potential hits/mutations secondary to MLL translocations are required for triggering NG2 expression as we used fully transformed/immortalized cell lines derived from patients with overt disease, therefore carrying a paramount of cooperating mutations and genetic insults. The possibility that NG2 expression could be associated with non-balanced MLL rearrangements such as deletions or inversions has previously been ruled out.54, 55 Although unlikely, the possibility should not be excluded that NG2 expression could be linked to MLL internal duplications, which are not prospectively analyzed in human leukemias at diagnosis.62

We therefore hypothesized that NG2 expression may be dependent on the cell of origin where a specific leukemic abnormality initially occurs. For instance, NG2 might only be regulated when the leukemic abnormality arises either in a lineage-specific progenitor (HPC) or in a more immature, less committed stem cell (HSC). In order to confirm this hypothesis, a rare subset of the CD34+ cells is expected to coexpress NG2. When a large number of CB-derived CD34+ cells were analyzed by flow cytometry,63, 64 coexpression of NG2 was readily observed in a subset of CD34+CD38+ HPCs from CB (2.1%) (Figure 2a), bone marrow (BM) (0.83%) (Figure 2b) and mobilized-PB (1.3%)62 suggesting that HPCs rather than HSCs may be the target cell for transformation. To verify that this CD34+CD38+NG2+ cell subset truly represents HPCs, this population was enriched by fluorescence-activated cell sorting from CB and the cells plated in methylcellulose assays. Importantly, multilineage (CFU-G, CFU-M, CFU-Mix, burst-forming unit erythroid) hematopoietic colonies were obtained in in vitro colony-forming unit (CFU) assays,62 suggesting that HPCs rather than HSCs may be the target cell for transformation.

Figure 2
figure2

Flow cytometry analysis of NG2 (7.1) antigen in normal CD34+ progenitors and pDC precursors. (a) Expression of NG2 in gated CB-derived CD34+ cells. Analyses of as many as 2 × 105 CD34+ cells revealed that 2.1±2.4% of the CD34+ cells coexpress NG2+ cells. All these CD34+NG2+ cells are CD38+, therefore representing HPCs. An irrelevant isotype-matched antibody was used as a negative control (inset panel). (b) NG2 is also expressed in approximately 1% of BM-derived CD34+ HPCs. (c) The pDC precursors express NG2. Six color high-speed flow cytometry analysis showing the expression of NG2 (green dots) in a population of pDC precursors (CD45+ CD34+ CD38+ CD123++ HLADR+).

The expression of NG2 in 60% of pDC leukemias lacking MLL rearrangements have recently been reported61 (and our unpublished observations). We therefore addressed whether pDC CD34+ precursors coexpress NG2. Interestingly, we found that 12.2% of the pDC precursors (CD34+/CD45+/CD38+/CD123high/HLADR+)65, 66, 67 readily coexpress NG2 (Figure 2c). This suggests that, regardless of the status of the MLL locus, the NG2 antigen may be expressed in pDC leukemias if the leukomogenesis process is initially triggered in a pDC CD34+ precursor readily expressing NG2, which might act as a leukemic-initiating cell. In fact, based on the observation of NG2+ cell lines and NG2+ human primary leukemias lacking MLL rearrangements, our data illustrate that the leukemic abnormality underlying NG2 expression does not necessarily need to be a MLL rearrangement. It would be worthwhile to purify both NG2+ and NG2− cell subsets from acute leukemias harboring MLL rearrangements and transplant them into immunodeficient mice68, 69 in order to define which cell subset is more enriched in leukemia-initiating cells.

MLLAF4 rearrangement is present and expressed in mesenchymal stem cells (MSCs) from infant patients suffering from MLLAF4+ pro-B ALL

Of note, it is also becoming evident that the MLL-AF4+ disease may be even more complex than previously suspected. A very recent study by Stam et al.70 evaluated by whole-genome gene expression profiling a large cohort of ALL with or without MLL rearrangements. These researchers have provided convincing evidence that MLL-rearranged infants, MLL germline infants and MLL germline non-infant children can be distinguished based on gene expression profiling. MLL germline infants clustered closely to MLL-rearranged infants although they could easily be separated.70 The close clustering of infant ALL regardless of the MLL status shows gene expression similarities that are likely to be crucial for the biology of these entities. Worth noting, this study also anticipates that infants with HoxA9 negative MLL-rearranged ALL may have a much higher likelihood of relapse70 The possibility that human MLL-AF4+ leukemia has a different cellular origin than other MLL fusion leukemias is strengthened by this study showing the presence of MLL-AF4 but not other MLL fusions in BM MSCs71

There is an increasing body of evidence suggesting that balanced chromosomal translocations resulting in leukemic-specific fusion genes associated to childhood leukemias (MLL-AF4, TEL-AML1, AML1-ETO, BCR–ABL, E2A-PBX1, and so on) might be present in MSCs/stroma from the BM of these pediatric patients. The rationale for this hypothesis is based on the following:

  • There is a considerable proportion of acute leukemias in which the blasts lack the expression of the pan-hematopoietic marker CD45, indicating a potential prehematopoietic origin of the leukemia.

  • A considerable proportion of tumors secondary to the primary leukemia are non-hematopoietic mesenchymal tumors (osteosarcomas or soft-tissue sarcomas),72 suggesting that the cytotoxic treatment may be effective against the leukemic blasts inside the BM and the hematopoietic tumoral clone whereas unable to destroy the tumor-associated MSCs which seem to somehow escape from the chemotherapy-related cytotoxic effects.

  • It is well-established that upon an allogeneic transplantation MSCs are more resistant to chemotherapy treatment that the leukemic blasts as suggested by the fact that the BM stroma is commonly receptor-derived and barely donor-derived.73, 74, 75, 76

  • The presence of MSCs harboring these leukemic fusion genes would explain, at least in part, the higher sensibility of the molecular techniques such as quantitative reverse transcriptase PCR over flow cytometry methods for the detection of minimal residual disease because by flow cytometry we just analyze hematopoietic cells.77

The detection of the BCR/ABL oncogene and lymphoma-specific genetic aberrations in endothelial cells from chronic myelogenous leukemia and B-cell lymphoma patients suggests that endothelial cells may be part of the neoplastic clone78, 79, 80 and that hemangioblasts rather than HSCs appear to be target cells for the first oncogenic hit, which could occur during the first steps of ESC differentiation and/or in hemangioblasts persisting in adults.81

The cellular organization and relationships among precursors that initiate embryonic angiogenesis and hematopoiesis in humans have been characterized.82 A bipotent primitive hemangioblast derived from hESCs is uniquely responsible for endothelial and hematopoietic development.82 There is compelling evidence that several of the common chromosome translocations (that is, MLL-AF4, TEL-AML1, AML1-ETO) that are seen in pediatric leukemia often originate in utero during embryonic/fetal development,10, 48, 49 suggesting that such a rearrangement may arise in a mesodermal prehematopoietic precursor/bipotent hemangioblast capable of giving rise to both hematopoietic and endothelial lineages. Furthermore, the BM hematopoietic microenvironment has a role in the pathogenesis of a variety of hematological malignancies including acute leukemia, multiple myeloma, lymphomas or myelodysplastic syndrome80, 83, 84, 85, 86 and controversy does exist about whether the BM stroma may or may not be part of the tumoral clone.

Based on this background we wanted to ascertain whether common childhood-associated leukemic fusion genes and hyperdiploidy are present in BM MSCs from 38 children diagnosed with cytogenetically different acute leukemias.87 Fusion genes were absent in BM MSCs of childhood leukemias carrying TEL-AML1, BCR–ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected by fluorescence in situ hybridization and inverse PCR (Figure 3a, Table 2) and expressed by real-time PCR (Figure 3b) in BM MSCs from all cases of MLL-AF4+ B-ALL.87, 88 All MLL-AF4+ MSCs were consistently euploid, precluding the possibility of cell fusion between a MSC and a leukemic blast.87 Importantly, monoclonal VD(J)H immunoglobulin gene rearrangements were performed. Whereas monoclonal immunoglobulin gene rearrangements were consistently detected in MLL-AF4+ leukemic blasts, no monoclonal rearrangements could be detected in BM MSCs from any MLL-AF4+ B-ALL patient, ruling out potential contamination of the MSC cultures by leukemic cells and suggesting a close early developmental relationship between MSCs and the leukemic blasts rather than plasticity or dedifferentiation of B-ALL blasts.87

Figure 3
figure3

BM MSCs from infants with MLL-AF4+ pro-B ALL harbor and express the MLL-AF4 fusion gene. (a) Fluorescence in situ hybridization performed in patient-derived MSCs (top row) and leukemic blasts (bottom row) (n=38). Leukemia-specific fusion genes were always observed in the leukemic population. Using a split-apart probe, MLL rearrangements are identified by the presence of one red signal, one green signal and one yellow signal (germline). Using locus-specific probes, the fusions TEL-AML1, AML1-ETO and BCR–ABL are determined by the presence of yellow fusion signals (and the derivative chromosome) whereas cells without the translocation have two green (either BCR, TEL or ETO) and two red signals (either ABL or AML1). The white arrows depict the rearranged allele. Bar, 100 μm. (b) Representative quantitative reverse transcriptase PCR experiments performed in duplicate from two patients showing MLL-AF4 transcript expression in MSCs from infants with MLL-AF4+ pro-B ALL.

Table 2 Presence of leukemic fusion genes (and hyperdiploidy) in BM-MSCs from a cohort of infants/children with cytogenetically distinct acute leukemia

Some remarkable differences between these two recent studies caught our attention.87, 88 Menendez et al.87 failed to detect other chimeric mRNAs such as TEL-AML1, BCR–ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. In contrast, Shalapour et al.88 clearly demonstrated the presence of TEL-AML1 and MLL-ENL in BM MSCs. Whereas we could not detect monoclonal immunoglobulin gene rearrangements in the MLL-AF4-expressing MSCs, Shalapour et al.88 demonstrated the presence of monoclonal immunoglobulin gene rearrangements in three out of eight patients. This leukemia–stroma relationship seems therefore to be far more complex than previously described and further prospective experiments are still highly demanded.

Importantly, endogenous or ectopic expression of MLL-AF4, TEL-AML1 or MLL-AF9 exerted no effect on MSC culture homeostasis, indicating that these fusion genes themselves are not sufficient for MSC transformation and their expression in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. Together, these findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. The absence of monoclonal rearrangements in MLL-AF4+ BM MSCs precludes the possibility of cellular plasticity or dedifferentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.87, 88, 89

The above data supporting the expression of MLL-AF4 in BM-MSCs from infant ALL challenge our current view and pave the way for further experiments aimed at answering key questions such as where do the leukemia relapses come from? Should the current methods for minimal residual disease detection be revisited? Do some chromosomal abnormality aberrations prevent or facilitate the establishment of MSC cultures? How is the tumor–stroma relationship defined at the molecular level? Is it possible that genetically aberrant MSCs interfere with normal physiological immunesurveillance (that is, augmenting suppression of T-cell effector function or inhibiting DC maturation and proliferation)?

Modeling MLL-AF4+ infant pro-B ALL using hESCs

MLL-AF4 leukemogenesis has been particularly difficult to model90 and bona fide MLL-AF4 disease models do not exist as of yet. Our little understanding of transformation by MLL fusions and their mode of action come from murine models. Unfortunately, however, in vivo leukemias do not recapitulate the actual human disease. Some success has been achieved recently in the Kersey laboratory by ESC knock in,91 but the resultant disease differs significantly from that seen in infant ALL in two respects: (i) the latency is exceptionally protracted; (ii) the disease is classified as either myeloproliferative or mature/follicular B. Similarly, Rabbitts's group has developed and employed the invertor conditional technology to create a mouse model of MLL-AF4, in which a floxed AF4 complementary DNA was knocked into MLL in the opposite orientation for transcription. Cell-specific Cre expression was used to generate MLL-AF4 expression. The mice developed exclusively B-cell lineage neoplasias, but of a more mature phenotype than normally observed in childhood leukemia.92 Alternatively, it has been very recently suggested that the presence of both reciprocal MLL fusion proteins (MLL-AF4 and AF4-MLL)93 or AF4-MLL alone94 confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype. This reproducible but confusing in vitro and in vivo data suggest that these mouse models are missing some essential ingredients of leukemogenesis in the human embryo/fetus.

Potential differences may include particular stem/progenitor cell targets, level of transgene expression or the impact of etiological exposure factors or other events necessary for conversion of MLL-AF4 expression. For instance, it has been suggested that the remarkably brief latency of infant ALL might be due to the MLL-AF4-driven preleukemic cells being continuously exposed to the same putative transplacental chemical carcinogens that included the fusion gene itself.3, 16, 17, 18, 19, 21 Clearly this ‘etiological’ component is missing from the mouse models in relation to rapid acquisition of essential secondary mutations. It is plausible that MLL-AF4 specifically exerts its function in human cells, indicating that questions regarding target cells, secondary hits and latency have to be addressed using prenatally-derived human stem cells.

Leukemia is generally studied once the full transformation events have already occurred and therefore, the mechanisms by which MLL-AF4 transforms to a preleukemic state followed by rapid transition to overt ALL are not amenable to analysis with patient samples. Two fundamental aspects need to be addressed are (i) the nature of the target cell for transformation and (ii) the mechanisms accounting for the MLL-AF4-B-cell lineage affiliation. MLL-AF4 arises prenatally during embryonic/fetal hematopoiesis as evidenced by the analysis of MLL clonotypic breakpoints in leukemic cells of monozygotic twins with ALL10 and the detection of genomic MLL-AF4 fusion sequences in archived neonatal blood spots of infants who developed ALL.11 Moreover, the exceptionally high concordance rate of leukemia in monozygotic twin infants, approaching 100%,48, 49 suggests that all necessary genetic events required for leukemogenesis, are accomplished prenatally.49 The nature of the cells initially transformed by MLL-AF4 in utero is unknown. Infant ALL with MLL-AF4 fusion has a pro-B/monocyte phenotype but immunoselection and fluorescence in situ hybridization studies indicate that the fusion gene is present in a more primitive CD34+/CD19− cell subset.95 No in vivo xenogenic transplantation assay for this leukemia has been reported to date. The leukemia stem cell (LSC) may itself be distinct from the cell in which the MLL-AF4 has a preleukemic impact and this in turn, may also differ from the cell in which MLL-AF4 arises. Therefore, the hierarchical (stem cells vs progenitor cells) and ontogeny position (prenatal, neonatal vs somatic hematopoiesis), need to be considered when defining the ‘target’ cell in MLL-AF4 pathogenesis.

The hESCs were first derived by Thomson and colleagues29, 37, 96 and are envisioned to become a potentially powerful tool for modeling different aspects of human disease that cannot otherwise be addressed by patient sample analyses or mouse models. Regarding cancer biology, there are different types of childhood mesenchymal tumors, other than acute leukemias, wherein clinically significant manifestations can arise in utero. The fact that cellular transformation manifests as a blockage or altered cell differentiation suggests that hESCs differentiation could become a promising human system for characterizing the emergence of early transformation events that drive cell transformation rather than normal lineage specification.27, 28

On the basis of this hypothesis, we have recently begun to explore the developmental impact of MLL-AF4 in human embryonic and neonatal stem cell fate. Our preliminary data based on the development of transgenic MLL-AF4-expressing hESCs suggest that this fusion gene may have developmental effects not only in the hESC-derived hematopoietic cells but also on more primitive prehematopoietic precursors such as the hemangioblasts as the ectopic expression of MLL-AF4 seems to skew the hematopoietic vs the endothelial differentiation arising from these MLL-AF4-expressing hESCs (data not shown). Experiments are also underway to address whether MLL-AF4 is capable of transforming hECSs or their progeny on its own or, in contrast, it requires additional secondary cooperating mutations such as FLT3-activating mutations.6, 97, 98

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Acknowledgements

PM's group is funded by the CSJA (0029/2006 to PM) and CICE (P08-CTS-3678 to PM) de la Junta de Andalucía, the FIS/FEDER to PM (PI070026 & PI100449) and CB (CP07/00059) and the MICINN to PM (PLE-2009-0111). PM and CB have been partially supported by the International Leukemia Foundation Josep Carreras (ED-Thomas-05). RR is supported by the Spanish Association against Cancer (AECC). We are indebted to Dr Isidro Prat and Dr María del Carmen Hernandez from the Malaga Cord Blood Bank for provision of CB units and Prof Mel Greaves, Dr Gustavo J Melén, Dr Javier García-Castro, Dr Ramón García-Castro and Dr Alberto Orfao for their critical insights and fruitful discussions.

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Bueno, C., Montes, R., Catalina, P. et al. Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement. Leukemia 25, 400–410 (2011) doi:10.1038/leu.2010.284

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Keywords

  • MLL-AF4
  • infant leukemia
  • hESCs
  • mesenchymal stem cells
  • cellular origin
  • etiology

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