Review

Oncogene (2008) 27, 5759–5773; doi:10.1038/onc.2008.196; published online 7 July 2008

Complementing mutations in core binding factor leukemias: from mouse models to clinical applications

A M S Müller1,2, J Duque1, J A Shizuru2 and M Lübbert1

  1. 1Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany
  2. 2Stanford University Medical Center, Department of Medicine, Division of Blood and Marrow Transplantation, Stanford, CA, USA

Correspondence: Professor Dr M Lübbert, Department of Hematology/Oncology, University Medical Center Freiburg, Hugstetterstr. 55, Baden Wuerttemberg, Freiburg D-79106, Germany. E-mail: michael.luebbert@uniklinik-freiburg.de

Received 23 July 2007; Revised 10 March 2008; Accepted 15 May 2008; Published online 7 July 2008.

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Abstract

A great proportion of acute myeloid leukemias (AMLs) display cytogenetic abnormalities including chromosomal aberrations and/or submicroscopic mutations. These abnormalities significantly influence the prognosis of the disease. Hence, a thorough genetic work-up is an essential constituent of standard diagnostic procedures. Core binding factor (CBF) leukemias denote AMLs with chromosomal aberrations disrupting one of the CBF transcription factor genes; the most common examples are translocation t(8;21) and inversion inv(16), which result in the generation of the AML1-ETO and CBFβ-MYH11 fusion proteins, respectively. However, in murine models, these alterations alone do not suffice to generate full-blown leukemia, but rather, complementary events are required. In fact, a substantial proportion of primary CBF leukemias display additional activating mutations, mostly of the receptor tyrosine kinase (RTK) c-KIT. The awareness of the impact and prognostic relevance of these ‘second hits’ is increasing with a wider range of mutations tested in clinical trials. Furthermore, novel agents targeting RTKs are emanating rapidly and entering therapeutic regimens. Here, we present a concise review on complementing mutations in CBF leukemias including pathophysiology, mouse models, and clinical implications.

Keywords:

core binding factor leukemias, AML1-ETO, CBFβ-MYH11, receptor tyrosine kinase, c-KIT mutation

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Introduction

Acute myeloid leukemias (AMLs) comprise a heterogeneous group of hematopoietic neoplasias. Approximately 55% of adult patients with de novo AML display recurrent chromosomal aberrations and/or gene rearrangements (Byrd et al., 2002; Mrozek et al., 2004; Marcucci et al., 2005), which often can be correlated with the morphology-based AML subgroups of the French–American–British (FAB) Working Group classification (Bennett et al., 1985). The increasing knowledge on cytogenetic characteristics of acute leukemias and their association to distinct biological and clinical features demand the designation of novel prognostic categories. Moreover, cytogenetic changes provide important insights into the pathogenesis of the disease: although transcription factors are common targets of chromosomal translocations, it has been hypothesized that only the collaboration with additional genetic events, such as mutations of receptor tyrosine kinases (RTKs), provides the selective advantage required for full leukemogenesis (Kelly and Gilliland, 2002).

Leukemias with mutations affecting the core binding factor (CBF) transcription factor genes present an ostensive example for this molecular interplay. Chromosomal translocations that disrupt CBF frequently coincide with specific RTK mutations. This observation has attracted a great deal of attention, not only because of a prognostic impact of certain mutations, but also because RTKs are appealing targets for novel therapeutic agents such as small-molecule tyrosine kinase inhibitors (TKIs).

Here, we give a synopsis of recent studies on complementing mutations in CBF leukemias. Pathophysiology, mouse models, state of clinical data and trials, and novel therapeutic implications are discussed.

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Core binding factor leukemia: chromosomal translocations generate fusion genes

Genes encoding the subunits of CBF are common targets of chromosomal aberrations, and alterations are present in up to 20% of human AMLs (Slovak et al., 2000) and 22% of pediatric acute lymphoid leukemias (Shurtleff et al., 1995) (Table 1). The most common examples are translocation t(8;21) and inversion inv(16), which result in the AML1-ETO and CBFβ-MYH11 (myosin heavy chain protein) fusion proteins, respectively. AML with disruptions of the CBF are collectively referred to as ‘CBF leukemias,’ as they share many clinical features, including a generally favorable prognosis (Byrd et al., 2002).


The term CBF comprises a small family of heterodimeric transcription factors, composed of an alpha (α) and a common beta (β) subunit. The CBF transcription factor complex transactivates the expression of a broad spectrum of genes that are critical for the establishment and development of normal hematopoiesis, such as interleukin-3 (Cameron et al., 1994), granulocytic differentiation factor C/EBPα (Pabst et al., 2001), M-CSF receptor (Zhang et al., 1996) and myeloperoxidase (Nuchprayoon et al., 1994).

CBFα

The α subunit AML1 (also known as RUNX1, PEBP2αB or CBFA2) is the target of a number of translocations both in myeloid and lymphatic leukemias (Table 1).

With an occurrence of 4–12% in adult (Look, 1997; Grimwade et al., 1998; Kottaridis et al., 2001; Byrd et al., 2002; Thiede et al., 2002; Mrozek et al., 2004; Schnittger et al., 2006) and 12–30% of pediatric patients (Grimwade et al., 1998; Goemans et al., 2005; Shimada et al., 2006), the translocation t(8;21)(q22;q22) (henceforth referred to as t(8;21)) represents one of the most frequent cytogenetic events in de novo AML. Typically, t(8;21) is associated with the French–American–British AML M2 subtype, of which ~30–40% demonstrate this chromosomal abnormality (Erickson et al., 1992; Downing, 1999).

AML 1 is a member of the RUNX family of transcription factors, which are characterized by a Runt homology domain at the amino terminus. The Runt homology domain is required for heterodimerization with CBFβ and for DNA binding. AML1 activates transcription from enhancer core motifs (TGT/cGGT), which are present in a number of genes relevant to myeloid and lymphoid development (Meyers et al., 1993; Peterson and Zhang, 2004). Its key regulatory role in hematopoiesis has been confirmed in knockout studies, where AML1/ mice displayed a complete lack of definitive hematopoiesis (Okuda et al., 1996; Wang et al., 1996a).

ETO (for 8;21; also called MTG8 and CDR) encodes a zinc-finger-containing protein that belongs to a protein family characterized by four evolutionarily conserved Nervy homology regions (Peterson and Zhang, 2004). Besides its molecular role as an inhibitor of C/EBPβ and a regulator of early adipogenesis (Rochford et al., 2004), ETO appears to be involved in early gastrointestinal development as shown by ETO knockout experiments in mice (Calabi et al., 2001). The function of ETO in normal physiology has not yet been fully clarified.

The t(8;21) generates the AML1-ETO fusion gene by juxtaposing coding sequences of the AML1 gene on chromosome 21 with sequences of the ETO gene on chromosome 8. The translated chimeric protein consists of the NH2-terminal portion of wild-type AML1, fused in-frame to the nearly full-length ETO protein (Miyoshi et al., 1991; Erickson et al., 1992). Although the Runt homology domain at the amino terminus of AML1 is included in the fusion protein, the transactivation domain at the COOH terminus is absent and replaced by ETO (Miyoshi et al., 1991; Erickson et al., 1992; Kitabayashi et al., 1998). The AML1-ETO fusion protein exerts a dominant-negative effect on AML1-dependent transcriptional activation. This suppression occurs mostly through interaction of the ETO moiety with the nuclear receptor corepressor N-CoR-mSin3-HDAC1 complex that normally recruits histone deacetylase activity. This interaction is believed to result in a lower level of histone acetylation, less accessible chromatin, and thereby repression of the transactivation activity of wild-type AML1 (Gelmetti et al., 1998; Lutterbach et al., 1998; Wang et al., 1998). Furthermore, AML1-ETO alters the expression of a number of specific intranuclear target genes that are normally not regulated by AML1 (Shimada et al., 2000).

CBFβ

Inversion inv(16)(p13q22) (hereafter termed inv(16)) constitutes another common chromosomal aberration. Inv(16) is detectable in up to 12% of AML patients and is usually accompanied by monocytic and eosinophilic differentiation, correlating to the French–American–British AML M4Eo subtype (Look, 1997; Gari et al., 1999; Kottaridis et al., 2001; Thiede et al., 2002; Bacher et al., 2006; Schnittger et al., 2006). Inv(16), and less commonly translocation t(16;16), disrupt CBFβ and result in the fusion protein CBFβ-MYH11. Both inv(16) and t(16;16) may be cryptic or masked by a concomitant deletion del(16)(q22). Although isolated del(16)(q22) is a recurrent abnormality reported in patients with AML M4Eo, the deletion by itself does not create the fusion protein (Mrozek et al., 2001; Merchant et al., 2004). CBFβ-MYH11 contains amino acids 1–165 of the CBFβ protein, including the heterodimerization domain for the α-subunit, fused to various lengths of the C-terminal α-helical rod domain of the MYH11 (Liu et al., 1993). Physiologically, the β-component increases the transcriptional activity of CBFα by stabilizing the flexible C-terminal loop of the Runt homology domain (Tahirov et al., 2001) and protects it from proteolytic degradation through ubiquitination (Huang et al., 2001). Apparently, CBFβ-MYH11 dominantly inhibits CBF in its transactivating function and its capability to regulate gene expression by sequestering the CBFα subunit into cytoplasm (Kanno et al., 1998) and/or into cytoskeletal filaments and aggregates (Adya et al., 1998).

The dominant inhibitory effect of fusion proteins targeting CBF is considered a critical step in the development of hematopoietic malignancies. However, there is clear evidence from in vitro and in vivo assays that chromosomal CBF aberrations alone are not sufficient to induce leukemia.

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Mouse models of CBF leukemia

A comprehensive list of mouse models involving CBF is given in Tables 2a and b. Murine embryos lacking either AML1 (AML1/) (Okuda et al., 1996; Wang et al., 1996a) or CBFβ (CBFβ/) (Sasaki et al., 1996; Wang et al., 1996b; Niki et al., 1997) showed a complete lack of definitive hematopoiesis and died at mid-gestation due to lethal hemorrhage. Mice carrying a monoallelic inactivation of one AML1 allele (AML1+/) were phenotypically healthy. Myeloid cell differentiation and proliferation appeared normal in vitro, except for some minor abnormalities in hematopoietic stem cell proliferation (Sun and Downing, 2004).


Mouse models of t(8;21) AML

Various approaches have been used to create a chimeric murine/human hybrid AML1-ETO gene mimicking t(8;21) in the mouse germline. For example, the human AML1-ETO sequence has been fused in-frame to the murine AML1 exon 4 (Okuda et al., 1998), or exon 5 of the murine AML1 gene (the t(8;21) break point) has been replaced by cDNA containing exon 5 sequences of the human AML1 gene linked to the entire coding region of ETO (Yergeau et al., 1997). With a phenotype similar to that resulting from homozygous disruption of the AML1 or CFBβ gene (AML1/ or CBFβ/), knock-in mice heterozygous for the AML1-ETO alleles succumbed to lethal hemorrhage into central nervous sysytem, pericardial sac and soft tissue at embryonic days 11.5–13.5. Fetal livers lacked definitive hematopoiesis, but displayed dysplastic multilineage hematopoietic progenitors with an increased self-renewal capacity in vitro (Okuda et al., 1998). Primitive hematopoiesis in the yolk sac appeared to be unaffected, although in CFU assays these cells gave rise to macrophages only. This selective pattern suggests that AML1-ETO interferes with the differentiation of certain hematopoietic lineages and presumably promotes development down the monocyte/macrophage pathway (Yergeau et al., 1997).

To bypass embryonic lethality and define the contribution of AML1-ETO to leukemogenesis in adults, transgenic mice with conditional expression of the fusion protein in hematopoietic stem/progenitor cells or bone marrow (BM) have been generated. Inducible promoter systems, such as the murine mammary tumor virus-tet-controlled transcriptional activator, contain a tetracycline-responsive element that allows the control of the knocked-in AML1-ETO. This approach resulted in a high expression of the fusion gene in BM and peritoneal macrophages. However, despite this, double-positive adult mice disclosed no leukemic phenotype (Rhoades et al., 2000).

Similar results were obtained with the alternative promoter MRP-8, a small calcium-binding protein specifically expressed in myeloid cells of the neutrophil and monocyte lineage. The hMRP8-AML1-ETO transgenic mice were healthy during their lifespans, unless additional mutations were induced (see below) (Yuan et al., 2001).

When a loxP-bracketed transcriptional stop cassette was inserted into the AML1-ETO fusion gene, it was transcriptionally silent in its intact state, but could be activated following birth through Cre9-mediated deletion of this stop cassette (Buchholz et al., 2000). The activation of this allele in vivo was insufficient to induce leukemia despite an enhanced replating efficiency of myeloid progenitors in vitro. Again, only the induction of cooperating mutations resulted in the development of an AML (Higuchi et al., 2002).

Several models reconstituted lethally irradiated mice with BM or hematopoietic stem/progenitor cells that were retrovirally transduced to express AML1-ETO (for references see Table 2). Some degree of hematopoietic disturbance, manifesting as long-latency myeloproliferative or myelodysplastic syndromes in vivo (Grisolano et al., 2003; Fenske et al., 2004; Nishida et al., 2006) or an increased replating capacity in vitro, was observed. However, despite a high expression of AML1-ETO, none of these models achieved the spontaneous development of leukemia.

The idea that AML1-ETO requires secondary mutagenic events to promote AML development was supported by studies treating AML1-ETO-positive mice with N-ethyl-N-nitrosurea (ENU), an efficient DNA-alkylating inducer of single base mutations. In contrast to untreated AML1-ETO mice, 30–55% of those additionally receiving ENU developed AML and granulocytic sarcomas mimicking many of the features of human t(8;21) AML (Yuan et al., 2001; Higuchi et al., 2002). These leukemic cells expressed AML1-ETO, grew efficiently in vitro, readily established immortalized immature myeloid cell lines, and were transplantable into secondary recipients (Higuchi et al., 2002). In the same studies, a substantial proportion of animals came down with T-acute lymphoid leukemia/T-cell lymphoblastic lymphomas, but these leukemic cells did not express AML1-ETO (Yuan et al., 2001; Higuchi et al., 2002). Evidently, AML1-ETO transgenic mice develop both myeloid and lymphoid malignancies after treatment with ENU, whereas animals without AML1-ETO expression, including wild-type controls and AML1-deficient chimeric mice, only develop lymphoid malignancies (Kundu et al., 2005). These findings imply on the one hand that AML1-ETO, although not solely sufficient, is required for myeloid leukemogenesis, and on the other that development of T-cell neoplasias appears to be independent from the presence of CBF mutations.

Advanced CBF leukemia models have combined AML1-ETO expression with defined additional mutational events, such as activating mutations of the RTKs TEL-PDGFRβ (Grisolano et al., 2003) or FLT3 (Schessl et al., 2005), overexpression of Wilms’ tumor gene (Nishida et al., 2006), or deficiencies of ICSBP (interferon consensus sequence-binding protein) (Schwieger et al., 2002), or p21/waf1 (Peterson et al., 2007). Coexpression of the TEL-PDGFRβR and AML1-ETO fusion genes in primary hematopoietic cells rapidly induced an acute leukemia resembling AML1-ETO-positive AML M2. Blasts transplanted into secondary recipients readily established the same disease (Grisolano et al., 2003).

To study the functional correlation between AML1-ETO and the FLT3-length mutation, retrovirally transduced hematopoietic cells expressing AML1-ETO and/or FLT3-length mutation were used for BM transplantation. No disease developed in recipients of BM singly transduced with AML1-ETO or FLT3-length mutation, whereas all recipients of doubly transduced BM succumbed to an aggressive acute leukemia (Schessl et al., 2005).

AML1-ETO-transduced BM from transgenic mice that overexpress the Wilms’ tumor gene rapidly induced AML in all recipients. Myeloid differentiation was inhibited at an immature stage, and the in vitro colony-forming ability was higher as compared to AML1-ETO-transduced BM cells from wild-type mice (Nishida et al., 2006). Similarly, collaboration between AML1-ETO and inactivating mutations, such as deficiency of ICSBP, appeared to advance myeloid tumorigenesis. Accumulation of morphologically altered myeloblasts (20–30%) in the BM was observed as well as generation of granulocytic sarcoma-like tumors. Both of these findings resemble the pathognomonic features of t(8;21) AML (Schwieger et al., 2002). Transplanted AML1-ETO-transduced p21/waf1-deficient hematopoietic cells facilitated leukemogenesis in the recipients, which provides another example of the necessity of collaborative interplay of AML1-ETO with additional cytogenetic events (Peterson et al., 2007).

In contrast to studies of full-length AML1-ETO, no additional cytogenetic events were required for leukemogenesis in recent mouse studies on a truncated AML1-ETO protein: a single-nucleotide insertion mutation in the AML1-ETO DNA sequence was found to result in a truncated form due to a frame shift with introduction of a stop codon downstream of the insertion. The truncated AML1-ETO protein of 575 amino acids lacked a critical domain for NCoR/SMRT and ETO interactions, and, unlike its full-length counterpart of 752 amino acids, resulted in a rapid onset of leukemia in transplant recipients (Yan et al., 2004). More importantly, the coexpression of the full-length AML1-ETO and its truncated form resulted in a substantially earlier onset of AML and blocked myeloid cell differentiation at a more immature stage (Yan et al., 2006).

Mouse models of inv(16)

Fewer models disrupting the CBFβ subunit exist, but the pathologic findings observed resemble models involving AML1. CBFβ-MYH11 germline knock-in strategies with F1 embryos heterozygous for the CBFβ-MYH11 allele fail to generate definitive hematopoiesis and die around embryonic day 12.5 owing to central nervous sysytem hemorrhage and a lack of definitive hematopoiesis in the fetal liver. The resemblance to the AML1/- and CBFβ/-phenotype suggests that CBFβ-MYH11, similar to AML1-ETO, abrogates CBF function (Castilla et al., 1996). Chimeric mice containing only one CBFβ-MYH11 allele appeared healthy aside from some alterations in adult multilineage hematopoietic differentiation, and did not develop any spontaneous tumors (Castilla et al., 1999). Similarly, conditional knock-in of CBFβ-MYH11 controlled by an hMRP8 promoter element resulted in impaired neutrophil maturation but no overt leukemia (Kogan et al., 1998). In contrast, a mouse model utilizing Cre-mediated expression of CBFβ-MYH11 generated an abnormal myeloid progenitor compartment with leukemic predisposition, block of megakaryocytic maturation, and delayed but spontaneous development of AML (after 11–25 weeks). Latency and efficacy correlated with the number of polyinosinic-polycytidylic acid injections given (1–3) as well as the number of transplanted BM cells. This interval implies that the penetrance depends upon the size of the progenitor population at risk to develop full-blown disease after acquiring cooperating random mutations (Kuo et al., 2006).

Models combining the expression of the CBFβ-MYH11 fusion protein with additional genetic events applied ENU mutagenesis (Castilla et al., 1999), retroviral insertional mutagenesis (Castilla et al., 2004), removal of tumor suppressors (Yang et al., 2002) or the coexpression of oncogenes (Kogan et al., 1998; Yang et al., 2002). Only random mutagenesis approaches, in addition to chimeric inv(16), resulted in AML. Mostly, myelomonocytic leukemias developed after ENU treatment (84%) (Castilla et al., 1999) and after neonatal application of the retrovirus 4070A (63%) (Castilla et al., 2004).

Regarding the potential collaboration of several mutagenic events, the disturbance of neutrophilic maturation was more severe in transgenic mice coexpressing CBFβ-MYH11 and activated RAS as compared to those without activated RAS, although none of them displayed a full AML phenotype (Kogan et al., 1998).

When the CBFβ-MYH11 vector was inserted into BM cells lacking the genes for the cell cycle inhibitor p16INK4a and the tumor suppressor p19ARF (alternative reading frame), or alternatively BM cells expressing the HPV16 oncogene E7 (which is known to decrease p53 activity), a higher rate of clonal acute leukemia could be observed, presumably due to cooperation between an accelerated G1 cell cycle phase and CBF mutations. However, penetrance was incomplete. Exposure to ENU further increased the rate of disease, and leukemias were lymphoid rather than myeloid, regardless of whether CBFβ-MYH11 was coexpressed with E7, lack of p16INK4a and p19ARF or random mutations. Hypothetically, these specific secondary mutations may have selected for lymphoid leukemias (Yang et al., 2002).

A different outcome was observed in another BM transplant model transducing CBFβ-MYH11 into either wild-type, p19ARF+/ or p19ARF−/ BM. Expression of the fusion gene was sufficient to induce myelomonocytic leukemia even when expressed in wild-type BM, yet removal of a single allele of p19ARF+/ accelerated the disease (Moreno-Miralles et al., 2005).

Similarly, the coexpression of Plag1 or Plagl2 with CBFβ-MYH11 in BM and transplantation efficiently resulted in AML in 100% of recipients after 3–12 weeks. In vitro, Plag1 and Plagl2 expanded hematopoietic progenitors and increased proliferation by increasing the transition of G1 to the S phase of the cell cycle (Landrette et al., 2005).

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2-Hit hypothesis

The evidence from mouse models suggests that neither AML1-ETO nor CBFβ-MYH11 alone is sufficient to induce or sustain leukemia. This conclusion is supported by the observation that patients with t(8;21) AML often retain low numbers of AML1-ETO-positive cells in BM and peripheral blood persisting during many years of clinical remission (Nucifora et al., 1993). Moreover, in children with t(8;21) AML, clonotypic AML1-ETO sequences were identified in DNA retrospectively extracted from stored neonatal blood samples that preceded the development of AML by 5–10 years (Wiemels et al., 2002). As studies have detected AML1-ETO transcripts in fractions of stem cells, monocytes and B cells, but not in T lymphocytes of remission BM, it appears that the acquisition of the t(8;21) occurs at the level of stem cells or progenitors capable of differentiating into B cells as well as all myeloid lineages (Miyamoto et al., 2000; Passegue et al., 2003).

The so-called ‘2-hit-hypothesis’ proposes that translocations involving the CBF genes constitute a required but not solely sufficient precondition for transformation and that only collaboration with additional oncogenic events can ultimately result in transformation into a leukemic (stem) cell (Miyamoto et al., 2000). Class II mutations are usually chromosomal translocations (such as AML1-ETO, HOX fusion genes and PML-RARA) that cause loss of function of hematopoietic transcription factors. Consequently, they impair hematopoietic differentiation, promote growth arrest and provoke apoptosis (Burel et al., 2001; Elsässer et al., 2003; Li et al., 2006; Lu et al., 2006). Class I mutations often affect and activate genes involved in signal-transduction pathways, particularly RTKs, such as FLT3, c-KIT, N-RAS and K-RAS. Thus, they confer a proliferative and/or survival advantage to hematopoietic progenitors (Alcalay et al., 2001; Kelly and Gilliland, 2002; Passegue et al., 2003).

With emerging evidence of the widespread existence of alternatively spliced t(8;21) transcripts, this hypothesis needs to be regarded with restrictions. As mentioned above, mouse models transplanting retrovirally transduced BM revealed that in contrast to full-length AML1-ETO, truncated forms such as AML1-ETO9a do not require additional genetic events to induce spontaneous leukemia. Notably, recent studies were able to detect alternatively spliced transcripts in human leukemic cells and cell lines (Yan et al., 2006). Alternatively spliced transcripts such as AML1-ETO9a also lead to the production of a truncated AML-ETO protein, which is very similar to the variant found in mouse models.

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Receptor tyrosine kinase mutations in core binding factor leukemias

In a considerable proportion of acute leukemias, secondary activating class I mutations have been identified in coexistence with (class II) translocations. Overall, FLT3 represents the single most common mutated gene in AML, and it is affected by length mutations (internal tandem duplications) in the juxtamembrane domain in 24% (Kottaridis et al., 2001) and by activating loop mutations in 7% of cases (Thiede et al., 2002). Although normal karyotype AMLs display FLT3 mutations in 26–39%, their frequency is even higher in those with a PML/RARA fusion (30–43%) (Kottaridis et al., 2001; Stirewalt et al., 2001; Schnittger et al., 2002; Thiede et al., 2002; Kuchenbauer et al., 2005).

In contrast, t(8;21) and inv(16) AMLs feature FLT3 mutations at a comparatively low rate (<10%) (for references, see Table 3), which led to the expectation that the prevalence of RTK mutations in CBF leukemias is low. It has only recently become clear, that c-KIT instead of FLT3 represents a common target for class I mutations in CBF leukemias. In fact, c-KIT mutations cluster within CBF AMLs; 9–48% of AML M2 patients with t(8;21) and 10–45% with inv(16) carry activating mutations in the c-KIT receptor, whereas the total incidence of c-KIT mutations in AML in general is ~5% (references listed in Table 3).


The c-KIT belongs to the type III RTK family, which consists of five immunoglobulin-like repeats in the extracellular domain, a single transmembrane a juxtamembrane and a cytoplasmic kinase domain. The latter comprises the first and second catalytic domains (TK1 and TK2), which carry an adenosine triphosphate (ATP)-binding region, and the kinase-activating loop, respectively. TK1 and TK2 are separated by the kinase insert sequence (Figure 1). Autoinhibitory function to maintain the kinase in an inactive conformation is exerted by the juxtamembrane and the activating loop domain (Roskoski, 2005).

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 c-KIT receptor—structure and reported mutations. The human c-KIT gene belongs to the type III receptor tyrosine kinase (RTK) family with five immunoglobulin (Ig)-like repeats in the extracellular (EC) domain, a single transmembrane (TM) domain, a juxtamembrane (JM) domain and a cytoplasmic kinase domain that is split by a kinase insert sequence (KIS) into the adenosine triphosphate (ATP)-binding and phosphotransferase regions. In core binding factor (CBF) acute myeloid leukemia (AML), c-KIT mutations cluster most frequently within exons 17 and 8. Exon 8 mutations are usually represented by a single amino acid substitution at locus D419, which presumably alters conformation of the fourth and fifth Ig-like repeats with consecutive receptor activation or altered ligand affinity. The substitution of a variety of amino acids for the normal c-KIT D816 residue results in kinase activation. Conversely, the deletion of codon 816 severely impairs RTK activity in response to ligand binding. c-KIT D816 mutations are predicted to be in the activating loop (between the ATP binding and substrate phosphotransferase loops).

Full figure and legend (106K)

In CBF leukemias, c-KIT mutations accumulate within exons 8 and 17 (Figure 1). These exons are uncommon targets in other neoplasias, as mutations typically involve exon 11 or 9. Exon 17 translates into the activating loop of the second TK domain and corresponds to the D835 location of FLT3. In particular, the c-KIT D816 residue of exon 17 represents a mutational ‘hot spot’ harboring ~30% of c-KIT mutations (for example, Beghini et al., 2004; Nanri et al., 2005a). Remarkably, in inv(16) AML, exon 17 mutations seem to occur exclusively at codon c-KIT D816. In t(8;21) also mutations at c-KIT N822 have been described (Goemans et al., 2005; Wang et al., 2005; Nanri et al., 2005a; Paschka et al., 2006). Exon 8 encodes the fifth immunoglobulin-like unit and comprises an evolutionarily highly conserved region in the extracellular domain. It is believed to play a role in dimerization. Exon 8 mutations usually result in single amino acid substitutions at codon D419 and cause hyperactivation of the receptor in response to the stem cell factor (Kohl et al., 2005). Exon 8 mutations can be detected in ~10% of CBF AML patients. According to some investigators, they appear to be more common in patients with inv(16) than in those with t(8;21), with incidences of 20–25 versus ~6%, respectively (Care et al., 2003; Boissel et al., 2006). However, others have reported equal distributions (Cairoli et al., 2006).

Determination of the frequency at which association between specific c-KIT mutations and distinct cytogenetic AML subtypes occurs remains hampered by the fact that in most studies, only selected known mutations were probed. A multitude of isolated cases of ‘novel’ c-KIT mutations have been described as single cases but have not been systematically evaluated (Gari et al., 1999). As more mutations continue to be discovered, it is likely that the overall incidence of c-KIT mutations may have been underestimated.

Other common mutations in CBF leukemias, predominantly in those with inv(16), involve the oncogenes N-RAS (26–38%) (Valk et al., 2004; Bacher et al., 2006) and K-RAS (7–17%) (Valk et al., 2004; Bowen et al., 2005). Moreover, the novel mutation V617F of the Janus kinase JAK2 has been found primarily in therapy-related or secondary but also in some rare cases of de novo t(8;21) positive leukemias (Döhner et al., 2006; Lee et al., 2006; Illmer et al., 2007; Schnittger et al., 2007a, 2007b). In contrast, no expression of the gain-of-function fusion gene FIP1L1/PDGFRα was detectable in 22 patients tested (Monma et al., 2006).

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Prognostic and therapeutic implications of mutated c-KIT

Acute myeloid leukemia patients with CBF leukemia present at a lower median age, have a higher rate of complete remissions, prolonged complete remission duration and a better prognosis as compared to those with a normal karyotype or other chromosomal aberrations (Byrd et al., 2002). However, certain patients appear to suffer a worse outcome, and only ~50% are alive at 5 years after diagnosis (Marcucci et al., 2005). Studies that have attempted to identify those at risk suggest that a high white blood cell (WBC) index (expressed as WBC × (% BM blasts/100)) at diagnosis is the most important adverse clinical indicator. Thrombocytopenia, CD56 expression, extramedullary disease and/or the existence of additional cytogenetic abnormalities also appear to be of prognostic relevance (Baer et al., 1997; Nguyen et al., 2002; Schlenk et al., 2004).

Given the heterogeneity of mutations and the lack of comprehensive standardized clinical trials, it is not surprising that the predictive value of (activating) c-KIT mutations in CBF leukemias has not yet been fully clarified. Although available data from previous trials are not completely consistent, they provide evidence that there are correlations between outcome and RTK mutations. These studies report a significantly higher cumulative incidence of relapse (80 versus 13.5%) and a lower 6-year relapse-free survival of 18 versus 60% in patients with c-KIT-mutated as compared to unmutated t(8;21) AML (Nanri et al., 2005a). Similarly, patients with t(8;21), but not those with inv(16), appear to have a shorter event-free survival and relapse-free survival when carrying an additional c-KIT mutation (Boissel et al., 2006). Subgroup analyses have revealed that exon 17 activating-loop mutations, which often present with a high WBC count at diagnosis (Beghini et al., 2004), predict an adverse outcome with regard to event-free survival, overall survival, relapse risk and salvage after relapse in t(8;21) AML (Cairoli et al., 2006; Paschka et al., 2006; Schnittger et al., 2006).

In inv(16) AML the prognostic impact of c-KIT mutations remains controversial. Although some studies were unable to establish an association between c-KIT mutations and prognosis in inv(16) AML (Boissel et al., 2006; Cairoli et al., 2006), others found that exon 8 mutations increased the relapse rate but did not affect overall survival (Care et al., 2003). Of note, the adverse effect of mutated c-KIT on the cumulative incidence of relapse in inv(16) AML observed by Paschka et al. was due mainly to the presence of exon 17 mutations (Paschka et al., 2006).

These discrepancies indicate that larger trials performing multivariable analyses are warranted to evaluate the predictive value of c-KIT mutations in the context of other prognostic factors and different treatments received. The differentiation of specific c-KIT mutations is particularly crucial with the availability of novel TKIs, which are important emerging treatment options for patients with RTK mutations. Overall, 59% of c-KIT exon 8 (Goemans et al., 2005) and exon 17 c-KIT N822, but not D816, mutations were sensitive to the TKI imatinib in vitro. Despite their resistance to imatinib or the TKI SU5614, c-KIT D816 mutations were reported to be successfully targeted by other TKI compounds such as PKC412 or dasatinib (Schittenhelm et al., 2006; Schnittger et al., 2006). This difference in the sensitivity of a mutated c-KIT D816 to different PTK inhibitors and their dephosphorylation efficiency quite likely relies on the binding mode of these compounds. Exon 17 c-KIT D816 mutations with autophosphorylation of the activating loop confer constitutive activation of the receptor. Their resistance to imatinib may be due to imatinib binding the TK domain in the inactive state. In contrast, PKC412 is thought to bind within the ATP-binding pocket of the active conformation of the RTK.

Hence, assessment of the exact c-KIT mutational status in CBF leukemias may have direct therapeutic consequences and will presumably guide treatment strategies in the future, as patients with unfavorable c-KIT mutation might benefit from early hematopoietic stem cell transplantation or they may be considered for regimens containing novel RTK inhibitors.

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Conclusions and outlook

Over the past years, cytogenetic and molecular studies have substantially changed the classification system of acute leukemias. Morphology of the leukemic cell has become less relevant for clinical treatment decisions, which at the present time largely rely on cytogenetic and molecular features. Besides the well-established metaphase cytogenetics, including the assessment of specific translocations, mutational status has become important, and in a great proportion of leukemias, additional genetic events (point mutations, deletions or insertions) can be detected. These cytogenetic findings increase our understanding of the pathogenesis of acute leukemias and appear to be of major clinical and prognostic value. Furthermore, emerging new therapeutic agents targeting specific (mutated) receptors are opening novel treatment strategies. Therefore, clinical trials that include screening methods for assessment of the mutational status of RTKs in specific subgroups of leukemias are warranted and will provide a better understanding of the pathogenesis and treatment of acute leukemias.

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Acknowledgements

We are grateful for outstanding support by the comments and remarks of both reviewers of this paper. This study was supported by the Deutsche Forschungsgemeinschaft (DFG), Germany (AM), the La Caixa Stiftung–Deutscher Akademischer Austauschdienst (DAAD), Germany (JD), and the German Jose-Carreras Leukemia Foundation (ML), respectively.