AML1-ETO driven acute leukemia: insights into pathogenesis and potential therapeutic approaches

Abstract

The AML1-ETO fusion transcription factor is generated by the t(8;21) translocation, which is present in approximately 4%–12% of adult and 12%–30% of pediatric acute myeloid leukemia (AML) patients. Both human and mouse models of AML have demonstrated that AML1-ETO is insufficient for leukemogenesis in the absence of secondary events. In this review, we discuss the pathogenetic insights that have been gained from identifying the various events that can cooperate with AML1-ETO to induce AML in vivo. We also discuss potential therapeutic strategies for t(8;21) positive AML that involve targeting the fusion protein itself, the proteins that bind to it, or the genes that it regulates. Recently published studies suggest that a targeted therapy for t(8;21) positive AML is feasible and may be coming sometime soon.

Keywords

AML1-ETO / mouse model / leukemia / t(8;21) / pathway hits / mutation / hematopoiesis / Kasumi-1; CD34⁺

Introduction

Nearly four decades have passed since Dr. Janet Rowley’s first report of the translocation between chromosomes 8 and 21 in acute myeloid leukemia (AML) [1]. In that time, significant progress has been made defining the role that the fusion transcription factor generated by this translocation plays in leukemogenesis. Clinically, the t(8;21) translocation represents the most frequent chromosomal abnormality in AML, occurring in approximately 4%-12% of adult and 12%-30% of pediatric patients [2]. t(8;21) positive leukemias exhibit some degree of myeloid differentiation, which led to their classification as the M2 subtype of AML, based on the French-American-British (FAB) Working Group Classification system [3,4]. Several groups identified the AML1 gene on chromosome 21 as being fused to the ETO gene on chromosome 8, and as a result of this work the AML1-ETO fusion cDNA was ultimately cloned. AML1-ETO contains the RUNT homology domain of AML1 and nearly the full length open reading frame of the ETO gene [5-10]. The leukemogenicity of AML1-ETO has been evaluated in multiple mouse models, and in all models AML1-ETO expression is insufficient for leukemogenesis in the absence of other secondary events [11-15]. However, within the past decade, several laboratories have identified secondary events that can cooperate with AML1-ETO to induce AML in vivo. This advance in t(8;21) leukemia modeling has not been paralleled by a similar advance in therapeutic strategies for t(8;21) positive AML patients. In this review, we provide a synopsis of the mouse models that are currently available to understand AML1-ETO driven leukemogenesis, and present several new ideas for targeting t(8;21) positive AML therapeutically.

Mouse models of t(8;21) leukemogenesis

Transgenic expression of AML1-ETO

The N-terminal component of the AML1-ETO fusion protein, AML1 (a.k.a. RUNX1 and historically CBFα or PEBP2α), is a transcription factor that regulates the expression of a variety of genes related to myeloid and lymphoid differentiation. The importance of this regulation is underscored by the fact that the lack of AML1 in mice is embryonic lethal, and these mice show no evidence of definitive (fetal liver) hematopoiesis [16,17]. The role of the C-terminal component of AML1-ETO, ETO (a.k.a. MTG8), in hematopoiesis is less well defined. There is no expression of murine ETO in early mouse hematopoietic cells, and mice lacking ETO have no obvious defects in hematopoiesis; their primary phenotype is a reduction in postnatal viability and impaired midgut formation [18]. It is generally believed that ETO and its family members ETO-2/MTG16 and MTGR1 act in transcriptional silencing, as they bind corepressors such as nuclear receptor corepressor (NCoR), silencing-mediator for retinoid/thyroid hormone receptors (SMRT), mSin3a, and various members of the histone deacetylase (HDAC) family via four evolutionarily conserved nervy homology regions (NHR) [19-21]. These domains are retained in the AML1-ETO fusion protein, along with the DNA binding domain of AML1. Initial studies of AML1-ETO identified it as a dominant inhibitor of AML1 function [22,23], and were supported by the knock-in mouse model of AML1-ETO, in which AML1-ETO is expressed from the AML1 promoter. AML1-ETO knock-in mice largely replicate the phenotype of AML1 null mice; they die at a similar embryonic stage with CNS hemorrhage, and they lack fetal liver hematopoiesis (Table 1) [24,25]. However, while AML1 null fetal liver lacks hematopoietic stem/progenitor cells (HSPCs), the fetal livers of AML1-ETO knock-in mice contain small numbers of multilineage progenitor cells with dysplastic morphology, which possess increased self-renewal capacity when cultured in vitro [24,25]. These results allude to functions of AML1-ETO that are distinct from its ability to block AML1 function, and that are likely critical to leukemogenesis.

Efforts to establish mouse models of t(8;21) positive AML, and bypass the embryonic lethality of AML1-ETO “knock-in” expression, led to the generation of several mouse models where AML1-ETO was conditionally expressed (Table 1). An elegant model, in which the translocation between the murine AML1 and ETO genes was induced via CRE/LoxP mediated recombination, showed no leukemia when CRE was expressed from the Nestin promoter. However, the use of the Nestin promoter to induce recombination in this model was not ideal, as it generated the translocation in brain, kidney, and heart tissues, but not in bone marrow cells [11]. The Zhang laboratory subsequently generated a transgenic mouse in which the expression of AML1-ETO was under the control of a tetracycline-responsive element. In the absence of tetracycline, the AML1-ETO protein was detectably expressed in bone marrow cells, and although the animals did not develop leukemia, the bone marrow cells expressing AML1-ETO did show increased self-renewal and a partial block in myeloid differentiation [12].

A similar phenotype was also seen with a conditional AML1-ETO knock-in mouse model. By placing a transcriptional stop cassette flanked by LoxP sites in the third intron of AML1, and inserting the remaining AML1-ETO cDNA in frame to exon 4, the Downing laboratory created an AML1-ETO allele that could be expressed from the endogenous AML1 promoter following CRE/LoxP mediated recombination [13]. When these mice were crossed with Mx1-CRE mice [26], and pI-pC treatment was used to delete the stop cassette, AML1-ETO expression occurred in the hematopoietic compartment. AML1-ETO increased the self renewal of the bone marrow cells without impairing their ability to form colonies in methylcellulose cultures, although there was a slight skewing toward the granulocyte/macrophage lineage. Importantly, AML1-ETO expression did not initiate leukemia in this knock-in model [13].

To induce acute leukemia in this conditional knock-in model, the AML1-ETO-Stop/ + Mx1-CRE^+/- mice were treated first with pI-pC and then N-ethyl-N-nitrosourea (ENU) to generate additional genetic events. Nearly half of these mice developed hematopoietic neoplasms, which were largely granulocytic sarcoma/acute myeloid leukemia, with features reminiscent of t(8;21) positive AML. The remaining neoplasms were thymic-derived T cell lymphoblastic lymphomas that did not express AML1-ETO [13]. A similar result was seen when ENU was administered to transgenic mice in which AML1-ETO was expressed under the human MRP8 promoter; approximately half of the treated mice succumbed to AML while the remaining mice succumbed to lymphoma or acute lymphocytic leukemia (ALL) [14]. These MRP8-AML1-ETO transgenic mice did not develop disease without ENU treatment [14], and the occurrence of lymphoma or ALL in the ENU treated mice is not surprising, as ENU induces lymphomas or ALL in wildtype mice [27]. Taken together, these models clearly show that AML1-ETO expression alone is insufficient for leukemia initiation. Ultimately, the increase in self-renewal that AML1-ETO provides must be supplemented with additional genetic or epigenetic events to initiate leukemia. This is compatible with the in utero detection of the t(8;21) AML1-ETO translocation in children who develop AML later in life, even more than 10 years after birth [28].

Mouse models of AML1-ETO driven leukemogenesis

Data emerging from the whole genome sequencing efforts of the Washington University group on primary human AML patient samples have demonstrated that the “average” AML may contain dozens of mutations [29,30]. Although they did not report on AML1-ETO positive AML specifically, their findings are consistent with the requirement for additional genetic events in AML1-ETO leukemogenesis, and with t(8;21) positive AML patients harboring other genetic abnormalities, including loss of a sex chromosome, and chromosomal deletions or trisomies, as well as mutations in receptor tyrosine kinases [31].

To better understand which secondary events can cooperate with AML1-ETO to induce leukemia, investigators have taken advantage of retroviral transduction and transplantation experiments in mice, introducing AML1-ETO together with a variety of cDNAs. When bone marrow Lin^-Sca-1⁺c-Kit⁺ (LSK) cells, a population highly enriched for HSPCs, are transduced with AML1-ETO expressing retrovirus alone, and transplanted into lethally irradiated congenic recipient mice, the mice do not develop leukemia. Instead, AML1-ETO positive myeloblasts are found in the bone marrow of recipient mice 10 months post-transplantation, and these cells, which may represent 10% of the bone marrow population, show increased self-renewal in colony forming assays [32]. However, when bone marrow cells are transduced with AML1-ETO and either the TEL/PDGFβR fusion protein [33], the FLT3 length mutation (FLT3-LM) [34], or the c-kit N822K mutation [35], recipient mice succumb to fatal AML with a median latency of 60, 233, or 178 days, respectively. Of these secondary events, the c-kit mutation is the most commonly identified mutation in t(8;21) positive leukemia patients. At euthanasia, the leukemic mice in each of these transplant models show leukocytosis, anemia, thrombocytopenia, splenomegaly, and a blast population that exceeds 20% of the bone marrow cells. These leukemias require the DNA binding ability of AML1-ETO [33-35] and they are transplantable into sub-lethally irradiated recipient mice. For instance, when TEL/PDGFβR is paired with an AML1-ETO L148D mutant that lacks the ability to bind DNA (or interact with CBFβ), the recipient mice develop a myeloproliferative disorder (MPD) similar to that seen from TEL/PDGFβR expression alone [33]. FLT3-LM and c-kit N822K also give rise to MPD when expressed individually [36]. Because all of these mutations result in the constitutive activation of a tyrosine kinase that can confer a proliferative advantage to hematopoietic cells, they are considered to be in the same class of mutations, the so-called Class I mutations. Class II mutations are those that generally involve transcription factors and result in a block in differentiation. It has been postulated that AML follows a two-hit model of pathogenesis, where the Class I and Class II mutations cooperate to initiate leukemia [37,38]. AML1-ETO is representative of a Class II mutation, and although its ability to cooperate with three different Class I mutations lends some validity to this model, this model is an oversimplification of what is required to induce AML in man. It is difficult to ignore the many additional genetic mutations and epigenetic abnormalities that have been reported in AML patients, involving genes such as TET2, IDH1/2, DNMT3A, and ASXL1, among others [39-42], which constitute additional classes of mutations.

The two-hit model is clearly biologically applicable to AML, as mutations of a given class generally do not co-occur in patients. However, the question remains as to how AML1-ETO cooperates with secondary events that are not Class I mutations, such as the transcription factor WT1, to initiate leukemia. WT1 expression is upregulated in AML patients [43], and although originally described as a tumor suppressor gene, overexpression of WT1 has been found to promote the proliferation of mouse bone marrow progenitor cells and inhibit their differentiation in response to G-CSF [44]. Mice transplanted with bone marrow cells from WT1 overexpressing transgenic (WT1-Tg) mice have splenomegaly and an increased number of mature myeloid cells and myeloid progenitor cells in the bone marrow; however, they do not develop AML. When AML1-ETO is expressed in WT1-Tg bone marrow cells, the recipient mice die of an aggressive, invasive AML 1 to 4 months post-transplant [45]. In a similar scenario, AML1-ETO cooperates with lack of the interferon consensus sequence binding protein (ICSBP, a.k.a. interferon regulatory factor 8) to induce myeloblast transformation in mice. ICSBP expression is downregulated in AML patient samples, which led to experiments where AML1-ETO was expressed in ICSBP ^{- / -} mouse bone marrow cells. While the transplantation of ICSBP ^{- / -} bone marrow cells leads to an MPD, the expression of AML1-ETO in these cells leads to the accumulation of myeloblasts in the bone marrow of the recipient mice, as well as the development of non-fatal granulocytic sarcomas [46]. This latter pathological feature, which is present in some t(8;21) positive AML patients [47], suggests that ICSBP loss mimics one of several steps in the development of overt AML. The two-hit model will need to be modified to incorporate this and other scenarios of cooperativity that involve non-Class I mutations or multiple hits. A more contemporary model of leukemogenesis is likely to be defined by cooperativity between different “pathway hits,” where genetic and epigenetic events occur within different pathways (e.g. pathways of self renewal, differentiation, cellular metabolism, DNA damage response, apoptosis, cellular polarity, etc.), which together trigger the initiation of acute leukemia. In this model, a cell becomes leukemic once a certain combination of pathways has been activated and/or disabled (Fig. 1).

Secondary or tertiary events, and the deregulation of their corresponding pathways, may cooperate with AML1-ETO to induce AML in vivo in the setting of p21 loss. Through studies in myeloid cell lines, the Zhang laboratory found that AML1-ETO could trigger growth arrest by binding to the p21 promoter and inducing its expression [48]. They hypothesized that this growth arrest and p21 upregulation must be circumvented for AML1-ETO to be fully leukemogenic, possibly through the actions of a cooperating hit. Accordingly, when AML1-ETO was expressed in p21 ^{- / -} mouse fetal liver cells, many of the recipient mice developed a lethal AML after a latency of at least 4 months. The extensive latency, the fact that the p21 gene is rarely inactivated in patients with AML [49-52], and the fact that t(8;21) positive AML patient samples generally overexpress p21 [53], makes it unlikely that the loss of p21 directly synergizes with AML1-ETO expression in this mouse AML model. Instead, this model appears to demonstrate the importance of certain p21 regulated processes in AML1-ETO driven leukemogenesis and likely the importance of acquiring multiple hits. p21 plays a role in DNA damage repair processes [54-56], and thus its loss could result in the eventual accumulation of mutations and/or epigenetic events that cooperate with AML1-ETO. By understanding what events and deregulated pathways pair with AML1-ETO in the absence of p21, we will gain a greater understanding of the requirements for AML1-ETO mediated leukemogenesis.

Aside from cooperativity studies, our understanding of the leukemogenic potential of AML1-ETO was significantly advanced by the identification of an AML1-ETO splice variant, AML1-ETO9a, which can induce AML by itself after a 5- to 6-month latency. In 2004, the Zhang laboratory transplanted mouse bone marrow cells that had been retrovirally transduced with AML1-ETO into recipient mice. One of the recipient mice developed AML 14 weeks post-transplantation without any artificially induced mutations. Sequencing of the AML1-ETO cDNA in the leukemic cells revealed a single base-pair insertion that resulted in a C-terminal truncation of approximately 180 amino acids, eliminating the NHR3 and NHR4 domains [57]. Using the point of truncation as a reference, the Zhang laboratory identified an alternatively spliced transcript of AML1-ETO in cells where the ETO portion of the fusion mRNA included a 155 base-pair exon (9a). The inclusion of exon 9a changes the reading frame of ETO and introduces a premature stop codon [58], thereby generating a truncated AML1-ETO protein that lacks the NHR3 and NHR4 domains. The AML1-ETO9a (AE9a) transcript is only ~20 base pairs longer than the truncated AML1-ETO cDNA that was found in the one mouse that developed AML. When cells are co-transduced with AE9a and AML1-ETO, leukemia occurs more rapidly than when AE9a is expressed alone. The identification of a leukemogenic isoform of AML1-ETO was made more relevant by its detection in t(8;21) positive AML patients; in one report, 27 of 37 patient samples were found to contain AE9a RNA [59]. The presence of AE9a RNA appears to correlate with the presence of c-kit mutations and a worse overall survival [60]. However, studies of AML1-ETO protein expression in AML have not really identified a C-terminally truncated protein of the correct size; the overwhelmingly detectable AML1-ETO is the full-length 94 kDa protein. At present, our understanding of why this truncated form of AML1-ETO is leukemogenic, while its full length counterpart is not, is minimal. Understanding what gain-of-function abilities are unique to AE9a, and what interactions are altered by the absence of NHR3 and NHR4, will aid in defining AML1-ETO driven leukemogenesis.

Potential therapeutic approaches to t(8;21) positive AML

Treatment for t(8;21) positive AML patients typically begins with induction chemotherapy, which for decades has been the combination of cytarabine for seven days, together with three daily doses of an anthracycline (daunorubicin or idarubicin). If a complete remission is achieved, consolidation chemotherapy is given, which for t(8;21) positive AML consists of multiple courses of high dose cytarabine [61,62]. Although AML patients harboring the t(8;21) translocation are generally given a good prognosis and the majority achieve complete remission, the 5-year survival is only ~50% [63], and the presence of a c-kit mutation decreases the prognosis significantly. Hopefully the identification of novel therapeutic targets in t(8;21) positive AML will lead to treatment options that improve patient survival.

Targeting microRNAs and their pathways in AML1-ETO leukemia

MicroRNAs are now recognized as fundamental components of gene regulation [64], contributing to the coordinate regulation of many developmental programs via effects on mRNA degradation and translation [65-67]. Given the variety of effects that AML1-ETO has on differentiation, self-renewal, and apoptosis, it is not surprising that several hematopoiesis-related microRNAs have been found to be direct AML1-ETO targets. The Nervi and Beghini laboratories have shown that miR-223 and miR-222/221 are downregulated in t(8;21) positive leukemic blasts, compared to normal bone marrow cells [68,69]. These microRNAs are expressed at low levels in primitive hematopoietic cells, and they increase in abundance with differentiation along the granulocytic lineage [70]. By binding to an AML1 consensus binding site located 5′ of the pre-miR-223 gene, AML1-ETO silences miR-223 expression and impairs differentiation. Treating AML1-ETO positive SKNO-1 cells with the DNA methyltransferase inhibitor 5-azacytidine, or knocking down AML1-ETO expression with shRNA restores miR-223 expression and induces granulocytic maturation [68].

miR-222/221 is also directly repressed by the binding of AML1-ETO to an upstream AML1 consensus binding site [69]. Interestingly, while miR-223 regulates differentiation via interactions with C/EBPα or NFI-A [70], miR-222/221 appears to regulate differentiation by targeting the 3′ untranslated region (UTR) of c-KIT mRNA [71]. The downregulation of miR-222/221 by AML1-ETO correlates with heightened c-KIT expression, providing a mechanism for the overexpression of wild type c-KIT in t(8;21) positive leukemia.

A mechanistic link between t(8;21) positive AML and mitogen-activated protein kinase (MAPK) signaling is provided by miR-24 overexpression. While miR-223 and miR-222/221 are repressed by AML1-ETO, AML1-ETO appears to activate miR-24 expression [72]. miR-24 targets the 3′ UTR of MKP-7 (a.k.a. dual-specificity phosphatase 16), lowering its expression and thereby promoting MAPK signaling via decreased dephosphorylation [73]. When miR-24 expression increases, proliferation is accelerated and granulocytic differentiation is blocked, which can contribute to the t(8;21) leukemic phenotype.

Other microRNAs may play a role in t(8;21) positive acute leukemia without being direct transcriptional targets of AML1-ETO. For instance, in a genome-wide, miRNA expression analysis of 47 primary AML samples, miR-126 was found to be specifically overexpressed in the 10 t(8;21) positive AML samples, possibly as a result of decreased methylation within a CpG island that controls its expression. Forced expression of miR-126 in mouse bone marrow cells leads to increased proliferation and enhanced serial replating ability; these effects are amplified by the co-expression of AML1-ETO [74]. Determining whether the deregulation of miR-126, or other microRNAs, is a cause or consequence of AML1-ETO positive leukemias should help determine whether targeting specific microRNAs would be useful therapeutically.

Targeting gene expression profiles in AML1-ETO leukemia

The unsupervised clustering of microarray data has allowed for the identification of leukemia subgroups, such as t(8;21) positive AML and CBFβ rearranged [inv(16) and t(16;16)] AML, based upon distinct gene expression signatures [75,76]. Many investigators have focused on the mechanisms underlying the repression of AML1-ETO target genes, however, many AML1-ETO target genes are upregulated following expression of AML1-ETO in U937 cells and human CD34⁺ cells. The relative importance of upregulated versus downregulated genes in the pathogenesis of AML1-ETO driven AML remains to be determined because these genes have generally been evaluated one at a time, an approach that is slow and painstaking. One gene, the groucho-related N-terminal enhancer of split (AES), has been identified as being consistently upregulated in t(8;21) positive AML [77,78]. Its functional importance was shown when AES knock-down in Kasumi-1 cells and AML1-ETO transduced mouse bone marrow cells decreased colony formation and methylcellulose replating ability. The self-renewal promoting effects of AES may reflect increased WNT signaling, which would be consistent with the upregulation of survivin that has also been found in t(8;21) positive AML patient samples [79]. Survivin is an inhibitor of apoptosis and a target of canonical WNT signaling; its expression is directly induced by the binding of AML1-ETO to the survivin promoter. Similar to the effects of AES knock-down, knock-down of survivin impairs the growth of AML1-ETO expressing cells, and promotes their granulocytic differentiation [80]. The therapeutic targeting of survivin is being explored in a variety of cancers [81,82] and hopefully one day will be explored in t(8;21) positive AML.

Like WNT signaling, RAS signaling has also been implicated in promoting AML1-ETO driven acute leukemia. Ras overexpression, or mutations in N-Ras that lead to its constitutive activation, are among the most frequent abnormalities found in t(8;21) positive AML [83,84], and the transduction of activated N-Ras^G12D into AML1-ETO expressing human CD34⁺ cells results in their transformation to cytokine independence with increased colony formation and replating ability. These cells also engraft better than AML1-ETO singly expressing cells in immunodeficient mice [both NOD/SCID/IL2rg ^{- / -} (NSG) mice and NOD/SCID mice transgenic for stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 (NSS)]. However, mice that are xenotransplanted with these CD34⁺ cells do not develop acute leukemia [85].

The BCL2 promoter is transcriptionally activated by AML1-ETO in leukemia cell lines [86]. This activation may be a signature of more fully transformed cells because while BCL2 is overexpressed in AML1-ETO and N-Ras^G12D coexpressing CD34⁺ cells, it is not overexpressed in AML1-ETO singly expressing CD34⁺ cells. BCL2 is potentially a clinically relevant target for the treatment of AML1-ETO and N-Ras^G12D coexpressing CD34⁺ cells, as ABT-737, an inhibitor of Bcl-2 family members, decreased their in vitro growth [85]. Though co-expression of Bcl-2 with AML1-ETO does not recapitulate all of the leukemic features of N-Ras^G12D and AML1-ETO co-expression, this sensitivity to ABT-737 suggests that BCL2 activation may play a role in the progressive transformation of AML1-ETO expressing cells, and could be an important therapeutic target for t(8;21) positive AML.

Other Bcl-2 family members, in particular Bcl-xL, may also be clinically relevant to t(8;21) positive AML. The expression of AML1-ETO in CD34⁺ cells activates signaling through the thrombopoietin (THPO)/MPL pathway, which upregulates Bcl-xL expression. Knockdown of Bcl-xL in these cells induced apoptosis, decreased in vitro colony formation, and impaired engraftment in NSG mice, while the overexpression of Bcl-xL in these cells enhanced their serial replating ability and LTC-IC frequency. MPL/Bcl-xL signaling appears to be upregulated in t(8;21) positive AML patient samples compared to leukemic blasts isolated from AML patients with normal cytogenetics. Targeting the THPO/MPL/Bcl-xL signaling axis may be another viable therapeutic option for t(8;21) positive AML patients, as withdrawal of THPO from AML1-ETO expressing CD34⁺ cultures decreased Bcl-xL expression, impaired cell growth, and increased apoptosis. Similar effects were not seen after withdrawal of stem cell factor (SCF) or Fms-like tyrosine kinase 3 ligand (FLT3L) [87].

Chemical inhibition of AML1-ETO leukemia

Chemical inhibitors targeting t(8;21) positive AML have frequently been directed against those key proteins or pathways that are required for AML1-ETO function (Table 2). One protein, calpain B, was identified based on an RNA-interference (RNAi) screen in Drosophila and shown to be required for the effects of AML1-ETO on Drosophila blood cell numbers and differentiation capability. Encouragingly, treatment of Kasumi-1 cells with calpain inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal (ALLN) and calpain inhibitor III, led to the degradation of AML1-ETO and a decrease in cell viability. In contrast, Calpain inhibitors had no effect on HL-60 cells or pediatric acute lymphoblastic leukemia patient samples [88]. Calpains represent a large family of Ca²⁺-dependent cysteine proteases; however, their role in hematopoiesis is not well understood.

In a high throughput screen, Kasumi-1 cells were treated with a total of 2 480 different FDA-approved drug compounds, with promising compounds being identified as “hits” if they could recapitulate the 28 gene-member expression profile indicating “AML1-ETO abrogation.” These hits fell primarily within two drug classes: corticosteroids and dihydrofolate reductase (DHFR) inhibitors, with methylprednisolone and methotrexate chosen to represent each class. Treatment with either drug reduced colony formation by Kasumi-1 cells, and induced myeloid differentiation and apoptosis. Methylprednisolone has been shown to specifically trigger apoptosis in AML1-ETO harboring cell lines, and to synergize with the traditional AML therapies, cytosine arabinoside (ARA-C) and daunorubicin [89]. Interestingly, a t(8;21) positive AML patient who received a short course of high-dose methylprednisolone for pneumonia had clearance of AML1-ETO positive leukemia cells [90]. Methotrexate appears to be less specific for AML1-ETO positive cells, and it did not synergize with ARA-C and daunorubicin in vitro. However, it did reduce leukemia cell burden in NOG mice transplanted with t(8;21) positive SKNO-1 cells [89].

A separate screen for chemical inhibitors of AML1-ETO function utilized transgenic zebrafish embryos heterozygous for AML1-ETO, which display hematopoietic features reminiscent of human t(8;21) positive AML [i.e. downregulation of GATA1 and SCL, and upregulation of myeloperoxidase (MPO)]. Transgenic embryos were incubated with 2 000 bioactive compounds, and the compounds were flagged as hits if at least 4 of the 5 embryos stained strongly for GATA1 expression. Of the 2 000 tested compounds, both nimesulide and dicumarol were able to restore GATA1 expression without altering AML1-ETO expression. Nimesulide is a known cyclooxygenase-2 (COX-2) inhibitor, and treatment of transgenic embryos with a similar inhibitor, NS-398, also triggered GATA1 upregulation. Investigations into the COX-2 pathway revealed that AML1-ETO upregulates COX-2 expression, and may require it and prostaglandin E2 (PGE2), a cyclooxygenase metabolite, for its effects on hematopoietic cells. PGE2 has been shown to promote the proliferation of various tumor cells by activating β-catenin-dependent signaling. Indeed, knockdown of either β-catenin-1 or β-catenin-2 in transgenic embryos was able to restore GATA1 expression. Consistent with this effect, AML1-ETO significantly increased the expression of COX-2 in human K562 cells, and treatment with NS-398 removed the AML1-ETO driven block in erythroid differentiation [91]. The importance of COX/β-catenin signaling remains to be investigated in AML1-ETO mouse models and in primary cells isolated from t(8;21) positive AML patients; however, COX and β-catenin inhibitors could have efficacy in t(8;21) positive AML.

In contrast to performing large scale screens to identify novel AML1-ETO inhibitors, some investigators have focused on using traditional Chinese herbs to treat t(8;21) positive AML [92], such as Eriocalyxin B (EriB), an ent-kaurene diterpene compound isolated from an herb of the Labiatae family, Isodon eriocalyx var. laxiflora. EriB treatment preferentially triggers the apoptosis of AML1-ETO expressing cells, possibly through the deregulation of MAPK signaling. EriB showed efficacy on five t(8;21) positive AML patients and two different mouse models of t(8;21) positive AML: C57 mice transplanted with cells expressing the C-terminally truncated AML1-ETO showed increased survival if treated with EriB (32 days for EriB at 2.5 mg/kg, compared to 25 days for control treated mice). EriB also reduced the size of subcutaneous Kasumi-1 cell tumors in a xenograft nude mouse model [93]. How EriB preferentially exerts its anti-leukemic abilities on AML1-ETO positive leukemia cells is unknown.

Another compound, Triptolide, purified from the Chinese herb Tripterygium wilfordii, also has anti-proliferative effects on SKNO-1, Kasumi-1, and t(8;21) positive AML patient cells, without significantly affecting normal blood cell growth. Triptolide may act by reducing the levels of c-KIT and NF-κB related p65, and by impairing JAK/STAT signaling [94]. These pathways are activated in AML1-ETO positive leukemia cells [95], which may explain the anti-leukemic effects of Triptolide.

Bortezomib, a proteasome inhibitor, appears to target leukemia cells with activated c-KIT. It blocked the proliferation and induced the apoptosis of Kasumi-1 and SKNO-1 cells, as well as CD34⁺ cells isolated from t(8;21) positive AML patients. Exposure to bortezomib triggered the internalization and lysosomal degradation of c-KIT. Both wildtype c-KIT and the constitutively active c-KIT mutant, N822K, phosphorylate HSP90β, which enhances the binding of HSP90β to apoptotic protease activating factor 1 (APAF-1). By inducing the degradation of c-KIT, Bortezomib indirectly decreased HSP90β phosphorylation, triggering the release of APAF-1 which leads to activation of caspase 3. Interestingly, caspase 3 is capable of cleaving AML1-ETO and AML1-ETO9a at D188, generating cleavage fragments that are deleterious to the AML1-ETO expressing Kasumi-1 cells, resulting in impaired proliferation, increased apoptosis, and decreased colony formation ability in methylcellulose. Bortezomib treatment increased the overall survival of mice transplanted with AML1-ETO9a expressing cells (34 days for Bortezomib at 2 mg/kg, compared to 19 days for control treated mice). The anti-leukemic effects of Bortezomib may relate to its ability to downregulate c-KIT and to generate AML1-ETO cleavage fragments [96].

Another study suggests that Bortezomib exerts its anti-leukemic effects by repressing the transcription factor specificity protein 1 (SP1) [97]. Using a ChIP-chip analysis of AML1-ETO expressing CD34⁺ cells, the Alvarez laboratory determined that the majority of AML1-ETO target genes did not contain an AML1 consensus binding site in their regulatory regions, but instead showed enrichment for Sp1 consensus binding sites. Many of these genes are part of the Wnt/β-catenin pathway and the canonical stem cell pluripotency pathway, according to Ingenuity Pathways Analysis. AML1-ETO can directly bind SP1, and mithramycin A, an Sp1 inhibitor, can induce the re-expression of AML1-ETO targets that possess an Sp1 consensus binding site (but that lack an AML1 consensus binding site) [98]. By repressing SP1, bortezomib may similarly alleviate AML1-ETO target gene repression. Regardless of the potential mechanism, bortezomib is being evaluated in the treatment of AML.

The ability of AML1-ETO to recruit HDAC complexes to many of its target genes has prompted the testing of HDAC inhibitors on t(8;21) positive AML cells. Trichostatin A (TSA) is a hydroxamic acid-based inhibitor of class I and II HDACs [99]; it can alleviate AML1-ETO-mediated repression of AML1 target genes in reporter assays [100] and lead to the rapid degradation of AML1-ETO in Kasumi-1 cells. Similar results have been seen with Depsipeptide/romidepsin (DEP), another HDAC inhibitor that triggers the proteasomal degradation of AML1-ETO in Kasumi-1 cells [101-103]. Because oncogenes like AML1-ETO are frequently bound to cellular chaperones like HSP90, the Hiebert laboratory investigated the effects of the HSP90 inhibitor 17-allylamino-geldanamycin (17-AAG) on Kasumi-1 cells and found that 17-AAG also triggered the degradation of AML1-ETO. This effect was not enhanced by co-treatment with DEP, suggesting that both inhibitors act on the same pathway; indeed, subsequent investigations demonstrated that treatment with DEP releases AML1-ETO from HSP90, thereby facilitating its degradation [102]. The ability of HDAC inhibitors to alleviate AML1-ETO-mediated repression and induce the degradation of AML1-ETO protein, possibly via acetylation of HSP90 [103,104], highlights their potential in treating t(8;21) positive AML.

Acetylation can be regulated by factors other than HDACs, however, and we have discovered an important role for the lysine acetyltransferase p300 in t(8;21) positive leukemia. p300 has been shown to bind to the C terminus of AML1 [105] and acetylate residues K24 and K43, thereby augmenting AML1 transcriptional activity [106]. Although the C terminus of AML1 is lost in the AML1-ETO fusion, we have found that AML1-ETO can still bind to p300 via the NHR1 domain of ETO. Binding of p300 to this region is required for the acetylation of AML1-ETO (and AML1-ETO9a) at the K24 and K43 residues. This acetylation is detectable in leukemia cells isolated from t(8;21) positive AML patients, and is essential for the ability of AML1-ETO to increase the self-renewal of human CD34⁺ cells. Acetylated AML1-ETO co-localizes with p300 at gene regulatory regions to cooperatively upregulate the expression of target genes that may be involved in self-renewal, such as Id1, p21 and Egr1. In contrast, overexpression of an AML1-ETO acetylation mutant (A-E K43R) in human or murine HSPCs had little effect on self-renewal. Compared to leukemogenic AML1-ETO9a, the AML1-ETO9a acetylation mutant (AE9a K43R) was unable to cause AML in mice, demonstrating that K43 acetylation is required for leukemogenesis in this mouse model [107]. Treatment of Kasumi-1 cells and t(8;21) positive AML patient cells with p300 inhibitors, such as Lys-CoA-TAT [108] or C646 [109], resulted in growth inhibition, without effecting normal CD34⁺ cell growth. Similarly, treatment of AML1-ETO9a expressing mouse leukemia cells ex vivo with Lys-CoA-TAT or C646 prior to transplantation resulted in a significant increase in the median survival of recipient mice, compared to the DDDD-Tat or C37 treated controls. Although AML1-ETO can be regulated by a variety of post-translational modifications [110], it appears that site-specific acetylation contributes significantly to its role as a leukemogenic oncogene. This work identifies p300 as a valid therapeutic target for t(8;21) positive AML.

Discussion

Significant progress has been made in developing mouse models that recapitulate features of t(8;21) positive AML. Though the expression of AML1-ETO alone has been found to be insufficient for leukemogenesis, subsequent retroviral transduction and transplantation experiments have identified secondary events that can cooperate with AML1-ETO to induce AML in mice. These secondary events range from constitutively activated tyrosine kinase mutations to WT1 overexpression, ICSBP loss, and p21 loss. Because the established two-hit model of AML pathogenesis only accounts for the cooperation of AML1-ETO with tyrosine kinase mutations, we believe that this model must be modified to incorporate other cooperating events. A more contemporary model where multiple genetic or epigenetic events alter critical cellular pathways to result in “pathway hits,” which ultimately cooperate to induce leukemia, may better explain how AML1-ETO cooperates with a range of secondary events to generate t(8;21) positive AML.

The importance of pathway-directed abnormalities in AML1-ETO driven leukemogenesis has been highlighted by recent expression studies identifying the mis-regulation of several microRNAs and genes whose effects are primarily on the proliferation, self renewal, protein stabilization, and apoptosis pathways. While it is currently impractical to target individual microRNAs or genes therapeutically, their fine tuning and regulation by the signaling cascades involved in these pathways makes the inhibition of signaling an appealing alternative form of therapy. In fact, c-KIT, p38 MAPK, and RAS signaling is inevitably activated in AML1-ETO positive cells, either through kinase mutations or overexpression, thereby conferring a proliferative advantage to a clonal population of cells. Self renewal is enhanced in AML1-ETO positive cells by a variety of means, including the activation of WNT signaling. Treatments that target the signaling cascades in these proliferation and self-renewal pathways, or that impair AML1-ETO protein stabilization and the evasion of apoptosis, may be particularly effective in t(8;21) positive AML.

Although the advances in modeling t(8;21) positive leukemia have not been paralleled by similar advances in effective therapeutic strategies, pathway inhibitory treatments are now being investigated in patients based on earlier studies performed in these mouse models. Hopefully, this strategy will pave the way for future clinical trials of targeted agents.

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