Review

Leukemia (2011) 25, 1239–1248; doi:10.1038/leu.2011.90; published online 13 May 2011

The heterogeneity of pediatric MLL-rearranged acute myeloid leukemia

B V Balgobind1, C M Zwaan1, R Pieters1 and M M Van den Heuvel-Eibrink1

1Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children's Hospital, Rotterdam, The Netherlands

Correspondence: Dr MM Van den Heuvel-Eibrink, Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children's Hospital, Room Sp 2456, Dr Molewaterplein 60, PO Box 2060, Rotterdam 3000 CB, The Netherlands. E-mail: m.vandenheuvel@erasmusmc.nl

Received 24 October 2010; Revised 10 February 2011; Accepted 18 March 2011; Published online 13 May 2011.

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Abstract

Translocations involving the mixed-lineage leukemia (MLL) gene, localized at 11q23, comprise 15 to 20% of all pediatric acute myeloid leukemia (AML) cases. This review summarizes current knowledge about the etiology, biology, clinical characteristics and differences in outcome in MLL-rearranged pediatric AML. Furthermore, we discuss the role of cooperating events in MLL-rearranged pediatric AML, and future therapeutic strategies to improve outcome. We conclude that MLL-rearranged pediatric AML is a heterogeneous disease, and prognosis depends on various factors, for example, translocation partner, age, WBC and additional cytogenetic aberrations. The relationship of outcome with specific translocation partners requires that they be searched for in the diagnostic work-up of AML. To achieve further improvements in outcome, unraveling the biology of MLL-rearranged pediatric AML is warranted.

Keywords:

MLL; pediatric AML; heterogeneity

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Introduction

Acute myeloid leukemia (AML) accounts for 15–20% of childhood leukemias.1 Despite intensive chemotherapy, only 60% of patients with AML are cured.2 The heterogeneity of AML is reflected by differences in morphology, immunophenotype as well as cytogenetic and molecular abnormalities. Recurrent (cyto)genetic aberrations and response to treatment are important prognostic factors in AML and are therefore used for risk group stratification. The cytogenetic subgroups t(8;21), inv(16) and t(15;17) have been consistently associated with favorable outcome; currently reaching 5-year overall survival (OS) rates of approximately 80%.3 Mixed-lineage leukemia (MLL) rearrangements are related to an intermediate to poor outcome.4, 5, 6

In 1991, it was discovered that the different 11q23 rearrangements involved one and the same unique locus at 11q23. This locus shows homology to sequences within the Drosophila ‘trithorax’ gene, a developmental regulator.7, 8 As these aberrations were found in AML as well as acute lymphoblastic leukemia (ALL), the gene was called mixed-lineage leukemia.9 Recent studies show that the MLL group itself is genetically and clinically heterogeneous, as more than 60 different translocation partners of the MLL gene with differences in outcome have been described to date.6, 10 The biological background of these differences remains unknown.

In addition to being involved in translocations, the MLL gene is also involved in other aberrations such as partial tandem duplications (MLL-PTD), consisting of an in-frame repetition of MLL exons.11 In pediatric AML, MLL-rearranged AML presents with a distinct, unique, expression profile as compared with MLL-PTD.12, 13 Therefore, MLL-rearranged AML and MLL-PTD are considered two different entities.

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Etiology of MLL rearrangements

A prenatal origin for MLL-rearranged AML has been shown in neonatal bloodspots taken from Guthrie cards of infants who later developed AML.14, 15 Environmental factors to which the fetus is exposed in utero may have an important role in the development of MLL-rearranged AML, which seem to occur because of inappropriate non-homologous end joining of double-strand breaks.16 The 11q23 locus is particularly sensitive to cleavage after treatment with topoisomerase-II inhibitors. As DNA topoisomerase-II seems to be highly expressed in developing fetuses,17 exposure to DNA topoisomerase-II inhibitors could induce MLL rearrangements in utero. The most abundant environmental source of DNA topoisomerase-II inhibitors is diet. For example flavonoids, such as quercetin (in some fruits and vegetables) and genistein (in soy), and catechins (in tea, cocao and red wine), inhibit DNA topoisomerase-II.18 Further evidence for a role of DNA topoisomerase-II inhibitors is provided by the fact that exposure to bioflavonoids can induce the cleavage of the MLL gene in human myeloid and lymphoid progenitor cells.19 A large case–control study of maternal diet and infant leukemia showed that the amount of maternal consumption of food containing DNA topoisomerase-II inhibitors was correlated to the risk of developing MLL-rearranged AML.20 Another case–control study found a significantly elevated risk for MLL-rearranged AML associated with maternal use of pesticide, as well DNA-damaging drugs.21

The role of topoisomerase-II inhibitors is further strengthened by the high frequency of MLL rearrangements in therapy-related AML for patients who have been treated with DNA topoisomerase-II inhibitors, for example, etoposide.22 Interestingly, identical to a short latency period to develop de novo MLL-rearranged AML in children, the latency period to develop therapy-related AML is much shorter for MLL rearrangements compared with the latency period of therapy-related AML with unbalanced aberrations, for example, monosomy-5 or monosomy-7.23

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The biology of MLL rearrangements and development of AML

The MLL gene encodes a DNA-binding protein with an N-terminal DNA-binding domain and a C-terminal SET domain. MLL was found to be part of a large chromatin-modifying complex in which the SET domain of MLL has histone methyltransferase and histone acetyltransferase activity24 (Figure 1). During the formation of this complex, MLL is cut by taspase into an N-terminal (MLLN) and a C-terminal (MLLC) fragment.25 MLLC then associates with at least four proteins, that is, the histone acetyltransferase MYST1(MOF),26 and WDR5, RBBP5 and ASH2L, to ensure histone modification and methyltransferase activity.27 The MLLN fragment has a binding site for MEN1 at the N-terminal end and both recruit PSIP1(LEDGF).28 PSIP1 then contacts the chromatin by a PWWP domain. MLLN also contains a CxxC domain, which specifically binds to unmethylated DNA.29 Transcription factors such as p53 and CTNNB1(β-catenin) are most likely to recruit the MLL complex to initiate RNA synthesis,26, 30 and specific genes, such as the HOX genes, seem to be more dependent on the MLL complex for chromatin modification during transcription than others.31, 32

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The MLL complex. MLL is part of a large chromatin-modifying complex in which the SET domain of MLL has histone methyltransferase and histone acetyltransferase activity. During the formation of this complex, MLL is cut into an N-terminal (MLLN) and a C-terminal (MLLC) fragment. MLLC associates with the histone acetyltransferase MYST1, and the WDR5, RBBP5 and ASH2L to ensure histone modification and methyltransferase activity. The MLLN fragment has a binding site for MEN1 at the N-terminal end and both recruit PSIP1. PSIP1 then contacts the chromatin by a PWWP domain. MLLN also contains a CxxC domain, which specifically binds to unmethylated DNA. MLL, mixed-lineage leukemia.

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In MLL rearrangements the breakpoint in MLL is highly conserved and all fusion partners are fused in frame, leading to a gain of function of the MLL complex. The MLL fusion most likely disrupts the MLL complex, leading to inappropriate expression of specific HOX genes, that is, HOXA4, HOXA5, HOXA9 and HOXA10, in pediatric MLL-rearranged AML.13 In addition, the HOX cofactor MEIS1 is upregulated in MLL-rearranged AML.13 In general, HOX gene expression has a key role in the regulation of hematopoietic development. Overexpression of HOXA9 results in increased numbers of self-renewing hematopoietic stem cells. Therefore, a failure to downregulate high HOX expression can inhibit maturation and trigger leukemogenesis in MLL-rearranged AML.

Although MLL rearrangements are predominantly found in AML, they are also detected in 6% of pediatric ALL cases. The association of MLL rearrangements with various hematopoietic lineages and the requirement of HOX expression in early development of hematopoietic cells suggest that MLL rearrangements occur in an early progenitor with lymphoid and myeloid potential. Although differences in translocation partner between AML and ALL suggest a role for lineage commitment, MLL–MLLT1(ENL) consistently generated AML in mice,33 whereas MLL–MLLT1 is found in both AML and ALL in humans. MLL-GAS7 induced AML, ALL and acute biphenotypic leukemia in mice,34 indicating that it is conceivable that, in addition to the MLL fusion, a secondary event or a specific microenvironment is necessary for lineage commitment.

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Epidemiology of MLL aberrations in pediatric AML

MLL rearrangements are the most common recurrent cytogenetic aberration in pediatric AML, in contrast to adult AML, where less than 3% of cases have an MLL rearrangement.35 In a large German study, the highest frequency of MLL-rearranged AML was found in children younger than 2 years.36 In a large collaborative retrospective analysis, the median age at diagnosis of MLL-rearranged pediatric AML was 2.2 years. This relation with age is probably due to the prenatal origin and short latency period. However, age differed greatly between the different translocation partners, as cases with t(6;11)(q27;q23) and t(11;17)(q23;q21) were older compared with children with other MLL rearrangements (Figure 2a). Hence, the incidence of MLL-rearranged AML decreases with age. This is in contrast to the frequency of MLL-PTD, which is equally low in pediatric AML, ranging from 1 to 10% depending on the screening method,13, 37, 38, 39 as well as in adult AML, in the range of 5–10%.37, 40, 41, 42, 43, 44, 45, 46, 47

Figure 2.
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Clinical characteristics of MLL-rearranged AML per translocation partner. Differences are identified for age (a), WBC (b) and FAB morphology (c) based on the translocation partner, but not for extra-medullary disease (d). AML, acute myeloid leukemia; FAB, French–American–British; MLL, mixed-lineage leukemia.

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The different translocation partners of the rearranged MLL gene

So far, more than 60 different fusion partners of MLL have been identified. Approximately 50% of pediatric AML cases with an MLL rearrangement consist of t(9;11)(p22;q23). The other 50% predominantly include t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3) (Figure 3; Table 1).48 This distribution is almost identical in adult AML, with the exception of t(6;11)(q27;q23), which has a higher relative frequency in adult MLL-rearranged AML.35, 49

Figure 3.
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The distribution of translocation partners of MLL in pediatric AML. The most common translocation in MLL-rearranged AML is t(9;11)(p22;q23), accounting for almost 50% of cases. In addition, t(10;11)(p12;q23), t(6;11)(q27;q23), t(11;19)(q23;p13) and t(1;11)(q21;q23) are also more frequently found accounting for 13, 5, 11 and 3% of the cases, respectively. The t(11;19)(q23;p13) group contains both t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3) as both groups could not be differentiated at time of diagnosis. The t(11;17)(q23;q21) is found to be heterogeneous, as currently 3 different translocation partners are found at 17q21 (i.e., MLLT6, ACACA and LASP4). AML, acute myeloid leukemia; MLL, mixed-lineage leukemia.

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Most of the translocation partners of MLL can be classified into cytosolic/membrane proteins and nuclear proteins.50, 51, 52, 53 Interestingly, the most frequent translocation partners of MLL encode nuclear proteins (for example, AFF1(AF4), MLLT3(AF9), MLLT1 and MLLT10(AF10)) and are most likely not randomly selected. Rather they are part of a protein network serving common functional processes capable of binding to histones. For example, direct binding interactions have already been described between AFF1, MLLT3 and MLLT1, and AFF1/MLLT10, which have a functional role in leukemogenesis.54, 55 The complex of these proteins, which is called the MLLT1/ENL-associated protein complex (EAP),55 was also linked to DOT1L, which methylates H3K79,56 and pTEFb, which is necessary to convert ‘promotor-arrested’ RNA polymerase-II into an ‘elongating’ RNA polymerase-II.57 In addition, it has been shown that MLLT10 binds to DOT1L and that binding of DOT1L was necessary for the transforming activity of MLL–MLLT10.56 These findings strongly suggest that the MLL-fusion proteins MLL–MLLT1, MLL–MLLT3 and MLL–MLLT10 recruit the EAP complex, leading to inappropriate histone methylation and transcriptional elongation.50, 51, 53, 58 Indeed, aptamere peptides that were able to disrupt the MLLT3/AFF1 complex proved to be toxic for leukemic cells with MLL–AFF1 translocations but not for blast cells of different etiology.59

The MLL fusion with translocation partners that encode for cytosolic/membrane proteins (for example, MLLT11(AF1q) and MLLT4(AF6)) seems to have different pathways leading to the oncogenic activity of the MLL-fusion gene. However, it is thought that dimerization of these proteins is contributing to the activation of target genes.60 However, it is still unknown how dimerization of these MLL-fusion proteins inappropriately activates target genes. It is possible that cytosolic proteins do enter the nucleus owing to the fusion to MLL and can have a role in the pathways found for the nuclear fusion proteins. Indeed when the ABI1 protein, a fusion partner of MLL, is imported into the nucleus, it interacts with MLLT1.61

In gene expression profiling (GEP) studies, MLL-rearranged cases clustered together as one group in AML as well as in ALL.13, 62, 63, 64 However, our laboratory showed that within MLL-rearranged infant ALL each type of MLL translocation is associated with a translocation-specific gene expression signature.65 We identified a specific gene expression signature for the total group of MLL-rearranged AML cases,12 but were also able to identify a specific signature for t(9;11)(p22;q23).66 This indicates that, in addition to the common pathways in MLL-rearranged AML, there are other pathways, which seem to be more dependent on the fusion partner of MLL.

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Clinical characteristics of MLL-rearranged pediatric AML

MLL-rearranged AML is correlated with the morphological subtypes acute myelomonoblastic leukemia and monoblastic leukemia, which represent the French–American–British (FAB) classification subtypes FAB-M4 and -M5, respectively.67 MLL-rearranged AML patients often present with high tumor load, which includes organomegaly in ~50% of cases, a high median WBC (20.9 × 109/l) and CNS involvement in 14% of cases6, 35, 48, 68 (Figures 2b–d). However, the clinical characteristics differ for the different translocation partners. For example, patients with a t(6;11)(q27;q23) had a higher median WBC than other MLL-rearranged AML. These characteristics illustrate that MLL-rearranged AML is a clinically heterogeneous disease, primarily based on the differences in translocation partner.

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Prognostic factors and outcome of MLL-rearranged pediatric AML

In general, MLL-rearranged AML is associated with a poor outcome. However, optimized intensive treatment regimens for AML have also improved outcome for MLL-rearranged AML. Table 2 shows that patients with MLL-rearranged AML have an intermediate outcome, with a 5y-pEFS (probability of EFS at 5 years from diagnosis) ranging from 32 to 54% and a 5y-pOS (probability of OS at 5 years from diagnosis) ranging from 42 to 62%.4, 5, 69, 70, 71, 72, 73, 74, 75, 76, 77 Currently, hematopoietic stem cell transplantation is no longer advised in first remission.


Recently, we identified the t(1;11)(q21;q23) subgroup as a new prognostic subgroup in pediatric AML. This type of AML has an excellent clinical outcome (5y-pEFS of 92% and 5y-pOS of 100%).6 The biological background for this favorable outcome is poorly understood and conflicting data on the overexpression of MLLT11, the translocation partner of MLL, in t(1;11)(q21;q23) have been reported. In cell lines overexpression of MLLT11 was associated with enhanced doxorubicin-induced apoptosis,78 whereas another study showed that high MLLT11 expression in AML was independently associated with poor survival.79 By contrast, the t(10;11)(p12;q23) and t(6;11)(q27;q23) subgroup have poor prognosis, with a 5y-pEFS of 31 and 11% and a 5y-pOS of 45 and 22%, respectively. Also, adult AML patients with a t(6;11)(q27;q23) have a poor outcome.80

In the past, some studies have shown that t(9;11)(p22;q23) had a favorable outcome and a higher sensitivity to drugs compared with other MLL rearrangements.68, 81, 82, 83 However, within the t(9;11)(p22;q23) subgroup, prognosis appeared to be related to morphology, as the group with acute monoblastic leukemia (FAB-M5) and t(9;11)(p22;q23) had a significantly better outcome than those with other FAB types.6 This could explain the favorable outcome for t(9;11)(p22;q23) in the study by Rubnitz et al.,68 as 21/23 cases had FAB-M5.

In addition to translocation partners, other variables such as WBC (>100 × 109/l), age (>10 years) and additional cytogenetic aberrations were of prognostic relevance in pediatric MLL-rearranged AML.6 Hence, with current intensive chemotherapy MLL-rearranged pediatric AML cases have an overall intermediate outcome. Translocation partners, WBC, age and additional chromosomal aberrations are independent prognostic factors within this disease, once again underscoring the fact that MLL-rearranged AML is a heterogeneous disease.

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The detection of MLL rearrangements

The true incidence of MLL rearrangements in pediatric AML is considered to be in the range of 15–25% according to the latest trials,4, 5 as cryptic MLL rearrangements were not always identified in the past using conventional karyotyping only. In addition, newly identified prognostic groups such as t(1;11)(q21;q23), t(6;11)(q27;q23) and t(10;11)(p12;q23) are not identified with current screening procedures.

As Southern blotting is an outdated method and has its limitations such as requirement of large amounts of DNA and a laborious procedure, other techniques are used to detect MLL rearrangements, including fluorescence in situ hybridization (FISH) and PCR.84, 85, 86 FISH screening for MLL rearrangements at diagnosis has become the standard approach in many AML protocols. However, GEP has illustrated that current diagnostic techniques do not guarantee 100% sensitivity for detection of MLL rearrangements.12 For three cases that clustered together with those with an MLL rearrangement, long-distance inverse-PCR showed that an MLL rearrangement was present, which had not been detected by split-signal FISH. This also shows the advantage of long-distance inverse-PCR, which can detect all translocation partners of MLL using only a little amount of DNA, if harvesting RNA for reverse transcription-PCR or GEP is not successful.10

As GEP is not yet feasible for diagnostic purposes and long-distance inverse-PCR for MLL rearrangements is currently performed at one center, FISH is currently the state-of-the-art method for detection of MLL-rearranged leukemia, but could lead to false-negative results. Therefore, we would suggest that FISH and long-distance inverse-PCR are the methods of choice to detect MLL rearrangements. However, as prognosis in MLL-rearranged AML depends on the translocation partner, these groups should be screened in future AML protocols for further risk stratification (Figure 4).

Figure 4.
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Future recommendations to detect MLL rearrangements in pediatric AML. AML cases are routinely screened for MLL rearrangements by conventional karyotyping and FISH. However, these techniques do not guarantee 100% sensitivity. Therefore, GEP and LDI-PCR could be used to identify cases not detected with FISH, although these techniques are currently used in research setting only. As outcome is also dependent on translocation partner, we recommend that all MLL-rearranged AML cases should be screened for the favorable prognostic groups t(1;11)(q21;q23) and t(9;11)(p22;q23) with FAB-M5 and the poor prognostic groups t(10;11)(p12;q23) and t(6;11)(q27;q23) for further risk stratification. AML, acute myeloid leukemia; FAB, French–American–British; FISH, fluorescence in situ hybridization; LDI-PCR, long-distance inverse-PCR; MLL, mixed-lineage leukemia.

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Molecular abnormalities in MLL-rearranged AML

As with other types of leukemia, the cause of MLL-rearranged AML is unknown. It has been postulated by Gilliland et al.87 that the pathogenesis of AML requires both type-I and type-II mutations. Type-II mutations are often chromosomal rearrangements of transcription factors, such as MLL rearrangements, leading to impaired differentiation of hematopoietic cells. Type-I mutations mainly reflect molecular mutation hotspots in specific genes (FLT3, KIT, NRAS, KRAS and PTPN11) involved in the proliferation of hematopoietic cells. We previously showed that only 50% of MLL-rearranged AML cases harbored a known type-I mutation, and most of these mutations were identified in genes involved in the RAS pathway, including mutations in NRAS, KRAS, PTPN11 and NF1 (ref. 88) (Figure 5). Mutations in RAF and SOS1, which are also part of the RAS pathway, are rarely found in AML.89, 90, 91

Figure 5.
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Frequency of type-I aberrations in pediatric MLL-rearranged AML. In 50% of MLL-rearranged AML cases a type-I aberration can be identified. The most common type-I aberrations are linked to the RAS pathway, that is, mutations in NRAS, KRAS, PTPN11 and NF1. The aberrations account for 37% of the MLL-rearranged AML cases. AML, acute myeloid leukemia; MLL, mixed-lineage leukemia.

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Mutations in FLT3 and KIT are rare in MLL-rearranged AML.88, 92 Recently, novel molecular aberrations were found in pediatric AML, that is, mutations in NPM1, CEBPA and WT1. Of these mutations, only WT1 mutations were found in MLL-rearranged AML, and these even at a low frequency.93, 94, 95, 96 In more than 60% of cases a molecular aberration has not been identified, which could indicate that an MLL rearrangement on its own could be sufficient to induce leukemia. However, 40% of cases do harbor a secondary aberration and therefore it is conceivable that Gilliland's hypothesis is still a valid model for at least the large subset of MLL-rearranged pediatric AML. This is supported by the fact that in MLL-AF9 transgenic mice NRAS expression contributed to acute leukemia maintenance by suppressing apoptosis and reducing the differentiation of leukemia cells.97 Also introduction of FLT3-ITD in MLL-AF9-overexpressing mice accelerated the onset of AML.98 Although FLT3-ITD is not a frequent event in MLL-rearranged AML, overexpression of FLT3 and a higher sensitivity to FLT3 inhibitors was found in MLL-rearranged ALL.99 In adult AML, highest FLT3 expression was detected in MLL-rearranged AML.100 We recently confirmed this high expression of FLT3 in a large cohort of pediatric AML cases (unpublished data).

Another gene that shows differential expression in MLL-rearranged AML cases is EVI1. In adult AML, overexpression of EVI1 is an independent poor prognostic factor, and in half of the cases an MLL rearrangement was identified.101, 102 We confirmed this in pediatric AML, where overexpression of EVI1 is mainly associated with a subset of MLL-rearranged AML.103 All cases with a t(6;11)(q27;q23), that carry a very poor outcome, showed overexpression of EVI1, consistent with adult t(6;11)(q27;q23) cases. This suggests a role for EVI1 in leukemogenesis in these specific cases. Indeed, in vivo studies with an MLL-AF9 mouse model showed overexpression of Evi1 after leukemic transformation.104 However, direct evidence showing an oncogenic effect of EVI1 in these types of leukemia is currently lacking.

Using GEP we identified specific genes involved in MLL-rearranged AML with t(9;11)(p22;q23), such as high expression of BRE, which is associated with a favorable outcome.66 In addition, overexpression of IGSF4, by epigenetic regulation, was discovered in cases with a t(9;11)(p22;q23) and FAB-M5 morphology, a subgroup with a relatively favorable outcome.105 The exact role of these genes in the leukemogenesis of MLL-rearranged AML needs to be further elucidated. It would be of importance to identify mutations or aberrant expression of specific genes or signaling pathways that are involved in those subtypes with a poor outcome, for example, t(6;11)(q27;q23).

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Toward targeted therapy

Over the last decades, outcome in pediatric AML has improved significantly, and up to 60% of children suffering from AML currently survive.3 However, improving outcome in pediatric AML using current treatment protocols is hampered by the treatment-related deaths and long-term side effects. Therefore, to improve outcome in pediatric AML, development of leukemia-specific targeting drugs is an important challenge.

MLL-rearranged AML cases are currently treated with standard treatment, where there is sufficient evidence that MLL-rearranged AML is heterogeneous in biology and outcome. New agents should target specific biological markers in MLL-rearranged AML that have a crucial role in the development of leukemia and are related to outcome. This strategy has proven to be worthwhile in other types of leukemia. For instance, using all-trans retinoic acid (ATRA) as adjuvant therapy has become the standard for acute promyelocytic leukemia. In addition, imatinib, rather than interferon and/or cytarabine, is the first choice of treatment in chronic myeloid leukemia. Moreover, imatinib is also used as adjuvant therapy in Ph+ ALL.106 As abnormal activation of tyrosine kinases predominantly leads to cell proliferation in various types of cancer, these are suitable targets for therapy. Here we will discuss the possible use of the recently developed tyrosine kinase inhibitors, other inhibitors and the possibility of targeting the MLL complex or the downstream targets.

FLT3, a tyrosine kinase, has been reported to be highly expressed in MLL-rearranged ALL,99 and co-expression of FLT3 with MLL-AF9 in AML shortened the latency period in mice significantly.98 In MLL-rearranged AML, high expression of FLT3 was found in almost all cases. FLT3 inhibitors, such as PKC412, have already been shown to induce apoptosis in MLL-rearranged ALL and FLT3-ITD/AML cells in vitro and in mouse models.107, 108, 109, 110 PKC412 showed potential in phase-I/II trials of adult AML.111 An international pediatric relapsed AML phase-II trial with PKC412 is currently ongoing.

One other possible kinase to be targeted in MLL-rearranged AML is glycogen synthase kinase-3 (GSK3), a serine/threonine kinase. MLL-rearranged leukemic cells are dependent on GSK3, as GSK3 inhibitors induce proliferation arrest in these cells.112 Inhibition of GSK3 with lithium already showed potential in these studies, but further research is warranted as GSK3 slows other malignancies, for example, colon cancer,113 and induces chromosomal instability.114

RAS pathway signaling has a significant role in leukemogenesis in AML. In fact, in MLL-rearranged AML this is currently the only pathway with known mutations. Therefore, patients harboring these mutations could benefit from RAS pathway inhibition. RAS activation depends on post-translational farnesylation, and farnesyltransferase inhibitors could be a potential targeted therapy. Unfortunately, the activity of tipfarnib, a farnesyltransferase inhibitor, did not show any correlation with RAS mutations or with pathway-dependent activation in adult AML, indicating that the antileukemic activity of tipfarnib may be because of other mechanisms than RAS inhibition.115 RAF can be inhibited by sorafenib. Sorafenib also targets FLT3-mutated AML cells,116 and current phase-I/II trials are ongoing in AML.117, 118 Inhibitors of MEK have been developed that sensitize leukemic blast cells to other drugs, for example, arsenic trioxide and demethylating agents.119, 120, 121 Current RAS pathway inhibitors probably will not fully block leukemic transformation in MLL-rearranged AML, but may have an additive effect with current treatment strategies by targeting the proliferative advantage of these leukemic cells.122

Another possibility is to directly target the MLL complex.123 The MLL complex requires the proteins MEN1 and PSIP1 to interact with chromatin. Indeed, excision of Men1 potently inhibits the proliferation of MLL–MLLT3-transformed cells and Hoxa9 expression in mice,124 and similarly, knockdown of Psip1 impaired Hoxa9 expression.28 This raises the possibility to block the function of Men1 or PSIP1 as a new targeted therapy in MLL rearrangements. The MLL protein also contains a CxxC domain, which is conserved in all MLL-fusion proteins and specifically binds to unmethylated CpG dinucleotides. Induced mutations of several residues in the CxxC domain have been shown to abolish both DNA binding and prevent myeloid transformation.125

In addition, proteins recruited by the MLL complex could be potential targets. Thus far, MLLT1 and MLLT10 have been associated with DOT1L. Compounds could be developed to inhibit the histone methyltransferase activity of DOT1L.126 However, safety studies are warranted, as genetic disruption in mice results in embryonic lethality.127

Several downstream targets of the MLL-fusion complex could be possible targets of MLL-rearranged AML. Upregulation of HOX genes is one of the most important hallmarks of MLL-rearranged leukemias. Mouse models have shown that MLL-fusion proteins are at least partially dependent on the function of Hox.128 MEIS1, a cofactor of HOX, is consistently highly expressed in MLL-related leukemias. In fact, induction and maintenance of MLL-induced leukemogenesis in a murine model required Meis1.129 Strategies based on downregulation of these genes might prove to be of benefit in the treatment of MLL-rearranged AML.

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Conclusion and future perspectives

MLL rearrangements are typically found in younger children with AML. In the past, MLL-rearranged AML has been related to poor outcome despite intensive chemotherapy. However recent studies showed that outcome in MLL-rearranged AML is dependent on different factors, for example, translocation partner, age, WBC and additional cytogenetic aberrations. Cases with a t(1;11)(q21;q23) have an excellent outcome and may benefit from less intensive treatment, whereas cases with a t(6;11)(q27;q23) or t(10;11)(p21;q23) have a poor outcome and do need adjusted and alternative treatment strategies to improve outcome. Although cooperating events are a hallmark of developing AML, additional genetic aberrations in MLL-rearranged AML are hardly identified. To achieve further improvements in outcome, unraveling the biology of MLL-rearranged AML is warranted.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Dr Anne-Sophie E Darlington for editorial assistance. We thank the different study groups (BFM, JPLSG, LAME, CPH, AIEOP, COG, St Jude, NOPHO, DCOG and MRC) for their collaboration in the MLL-rearranged pediatric AML study.