Gatekeeper function of the RUNX1 transcription factor in acute leukemia

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Blood Cells, Molecules, and Diseases 40 (2008) 211 – 218 www.elsevier.com/locate/ybcmd

Gatekeeper function of the RUNX1 transcription factor in acute leukemia Birte Niebuhr a , Meike Fischer a , Maike Täger a , Jörg Cammenga b , Carol Stocking a,⁎ a

b

Heinrich-Pette-Institut, Martinistr. 52, D-20251 Hamburg, Germany Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University SE-221 Lund, Sweden Submitted 24 July 2007 Available online 24 October 2007 (Communicated by M. Lichtman, M.D., 24 July 2007)

Abstract The RUNX1 gene encodes the alpha subunit of the core binding factor (CBF) and is a common target of genetic mutations in acute leukemia. We propose that RUNX1 is a gatekeeper gene, the disruption of which leads to the exodus of a subset of hematopoietic progenitors with increased self-renewal potential from the normal environmental controls of homeostasis. This pool of “escaped” cells is the target of secondary mutations, accumulating over time to induce the aggressive manifestation of acute leukemia. Evidence from patient and animal studies supports the concept that RUNX1 mutations are the initiating event in different leukemia subtypes, but also suggests that diverse mechanisms are used to subvert RUNX1 function. One common result is the inhibition of differentiation—but its effect impinges on different lineages and stages of differentiation, depending on the mutation or fusion partner. A number of different approaches have led to the identification of secondary events that lead to the overt acute phase; however, the majority is unknown. Finally, the concept of the “leukemia stem cell” and its therapeutic importance is discussed in light of the RUNX1 gatekeeper function. © 2007 Elsevier Inc. All rights reserved. Keywords: Leukemia stem cell; Core-binding factor; Differentiation; Self-renewal

Introduction It is now well established that cancers, including leukemia, develop through the accumulation of genetic and epigenetic alterations that act in concert to confer malignant phenotypes— and indeed, many of the genes and signal pathways targeted in this process have been identified [1]. Early studies led to the observation that the initiating event in solid tumors involves mutation of a specific signaling pathway for a particular tumor type, leading to the concept of “gatekeepers” [2]. It is generally postulated that gatekeeper pathways are responsible for maintaining a constant cell number in a renewing cell population and for ensuring that cells respond appropriately to situations that require increased cell growth. Inactivating mutations in this pathway are necessary to allow the full impact of mutations that perturb cell growth, leading to a selective advantage or clonal outgrowth of mutated cells. Examples of gatekeeper pathways in solid tumors include the WNT/β-catenin, ⁎ Corresponding author. Fax: +49 40 480 51 187. E-mail address: [email protected] (C. Stocking). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.07.018

hedgehog, and TGFβ/BMP pathways in colon, kidney, and skin tumors, respectively. Recently, we and others have postulated that mutations of transcription factors that promote differentiation during hematopoiesis disrupt a gatekeeper function, leading to the generation of acute myeloid leukemia (AML) [3–5]. In this review, we will expound on this hypothesis, providing evidence for a gatekeeper function of the RUNX1 transcription factor. In addition, we will extend this theory to discuss the relationship of the gatekeeper and the establishment of the leukemia (or cancer) stem cell (LSC). The existence of “cancer stem cells” was first postulated to explain the heterogeneity of cancer cells in their proliferation and cloning capacity [6,7]. The striking degree of similarity noted between somatic stem cells and cancer cells, including the fundamental abilities to self-renew and differentiate, has added fuel to a cancer stem cell hypothesis, in which the continued growth and propagation of the whole tumor depends on a small subpopulation of self-renewing cancer cells. A cancer (or leukemia)-initiating stem cell was first demonstrated conclusively for myeloid leukemia using xenograft mouse models but has more recently also been identified in breast and brain tumors

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[8–11]. Nevertheless, the frequency of the cancer stem cell within the tumor – as well as its origin – remains controversial. Whereas many lines of evidence support the concept that an early hematopoietic stem cell or progenitor (HSC/P) is the first normal cell that becomes subverted in the leukemogenic process [12], more committed progenitor cells or even mature cells may acquire mutations imparting stem-like functions in other cancers, or even certain types of leukemia [13–16]. The wide variation in the putative number of cancer stem cells in a given tumor may reflect the origin of the cancer stem cell, the assay systems used to count them, or the point during cancer progression at which it is measured. In other words, are we measuring the cancer initiating stem cell, or cells within a fully transformed cancer subpopulation? We propose that mutations affecting the gatekeeper pathway define the cancer initiating stem cell—but will not give rise to a tumor without the accumulation of secondary mutations. The function of a gatekeeper gene will be discussed using the example of the RUNX1 transcription factor—an important regulator of the HSC and a frequent target of mutations in leukemia. Runx1: a transcriptional regulator of hematopoiesis RUNX1/AML1 encodes a DNA-binding subunit that, together with the non-DNA-binding β subunit, forms a heterodimeric transcription factor, termed the core-binding factor (CBF) (Fig. 1). The conserved Runt homology domain (RHD) at the N-terminus of RUNX1 is required for binding to DNA and to its cofactor CBFβ, whereas the C-terminus contains transcriptional activation and repressor domains. Binding to CBFβ confers

both increased DNA-binding affinity and stability to RUNX1 [17,18] and is essential for many of its known functions [19,20]. CBF regulates transcription of a number of genes relevant to both myeloid and lymphoid development by associating with transcriptional cofactors, repressors, and other DNA-binding transcription factors in a promoter context-dependent fashion (Fig. 1B) [21,22]. The importance of RUNX1 in establishing and maintaining the HSC compartment in adult (murine) hematopoiesis as well as regulating T-cell and megakaryocyte differentiation has been established by the analysis of conditional knock-outs [20,23–26]. Target of genetic aberrations in leukemia In accord with its important regulatory function in hematopoiesis, disruption of the RUNX1 gene is one of the most common aberrations found in acute leukemia (Fig. 2) [27,28]. Most frequently, the RUNX1 gene is disrupted by chromosomal translocations, which are associated with distinct acute leukemias. These include the translocation t(8;21), generating the RUNX1/ CBFA2T1 fusion gene (also known as RUNX1/ETO or AML1/ ETO), associated with 40% of acute myeloid leukemia (AML) with an immature phenotype (FAB-M2), and the t(12;21), generating the ETV6/RUNX1 fusion gene (also known as TEL/ AML1), associated with 20% of pediatric proB-cell acute lymphoid leukemia (ALL). Significantly, the gene encoding CBFβ is also a target of chromosome aberrations [e.g. inv(16) and t(16;16)] in AML presenting with a myelomonocytic phenotype and an eosinophil component (FAB-M4). The fusion proteins are thought to mediate their oncogenic activity in part by dominantly repressing RUNX1-target genes [29–31]. In addition to translocations, inactivating or dominantnegative mutations in the RUNX1 gene have been identified in 15 to 25% of the relatively rare, minimally differentiated FAB-M0 AMLs, up to 25% of myelodysplastic syndromes associated with AML development, and in pedigrees of familial platelet disorder (FPD) with a propensity to develop AML [32,33]. RUNX1 mutations found in AML1-M0 and FPD-AML fall into two basic categories: (1) null mutations, in which probably no protein is generated, due to either large deletions or to the introduction of premature stop codons, which would be predicted to activate nonsense-mediated mRNA decay; and (2) Runt DNA-binding (RDB) mutations, which generate RUNX1 proteins with impaired DNA-binding, but which can still bind CBFβ [32]. Initiating event in leukemogenesis

Fig. 1. (A) The hetero-dimeric core binding factor (CBF) transcription factor is comprised of one of three RUNX family proteins (α-subunit) and a β-subunit, which is encoded by a single gene. The RUNX proteins contain two conserved and functional domains: the runt homology domain (RHD) and the transcription activation domain (TAD). Interactions between the RHD and the heterodimerization domain (HD) of CBFβ are essential for most of the known activities of CBF. (B) The synergistic activity of CBF with a number of different transcription factors is well established, leading to transcriptional activation or repression.

Several lines of evidence support the hypothesis that RUNX1 translocations or mutations are the initiating event in the hematopoietic malignancies in which they are found; establishing a preleukemic clone, but requiring secondary events for disease penetration (Fig. 3). The best support has come from patient studies, including data demonstrating the prolonged and variable latency of ALL induction in identical twin pairs, which share the initiating clone carrying the t(12;21) from birth [34,35], and studies linking RUNX1 mutations to hereditary

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Fig. 2. The RUNX1 gene is a frequent target of genetic disruption in acute leukemia. Specific chromosomal translocations or point mutations involving the RUNX1 gene are found in acute myeloid or lymphoid leukemia with a distinctive phenotype. Depicted is the lineage and differentiation stage in which the bulk of the leukemia cells with a characteristic genetic mutation accumulate. The genetic mutations result in either fusion or mutated RUNX1 proteins, as depicted.

FPD-AML syndrome [36]. The hypothesis is further supported by studies demonstrating that cells carrying the t(12;21) may be present at cell frequencies of 10− 3 in umbilical cord blood, verifying a selective expansion of a putative leukemic clone [37]. But these studies have also shown that the overall incidence of the translocation in newborn blood samples is 100times higher than the incidence of overt t(12;21) ALL, underlining the necessity of secondary events. The AML-associated t(8;21) and inv(16) have also been found at a relative high frequency in cord blood samples, suggesting a similar initiating mechanism [37,38].

techniques. Interestingly, “knock-in” mice for RUNX1/ETO [t(8;21)] and CBFB-MYH11 [inv(16)] showed almost identical phenotypes to Runx1 knock-out mice, arguing for a dominantnegative function of the translocation proteins involving RUNX family members [28]. However, as these mice were embryonic

Experimental models to study RUNX1 function Although the high incidence in acute leukemia of translocations and mutations involving the RUNX1 gene, coupled with their early detection in a postulated “pre-leukemic” stage, is strong evidence that these are initiating events in leukemogenesis, data supporting the causal role is necessary. An important approach has been the use of animal models in which the altered RUNX1 protein is expressed. Several mouse models for CBFassociated AML have been generated using different molecular

Fig. 3. Mutations in the RUNX1 gene precede the onset of clinical symptoms. Translocations or point mutations are detectable before birth in cases of pediatric acute leukemia, and at an incidence 100-fold higher than that of the leukemia incidence. The accumulation of secondary mutations is probably necessary for the overt disease—but for the most part the critical mutations are unknown. Adapted from [81].

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lethal, the effect on adult hematopoiesis and leukemia induction could not be evaluated. Strikingly, a retroviral-mediated expression of the fusion protein in bone marrow cells followed by transplantation in conditioned mice has proven to be a powerful approach to monitor the role of mutant RUNX1 forms in leukemia induction [39,40]. This is due to two advantages that this approach has in contrast to conditional “knock-in” mouse strains (theoretically but not practically more attractive): (1) establishment of a chimeric system, in which normal and “abnormal” hematopoietic cells coexist (thus mimicking the situation in man); and (2) marking of the cell population expressing the fusion oncogene by coexpression of a fluorescent protein in the retroviral vector. This approach has also recently been expanded to human hematopoietic cells [41,42]. After transduction with the RUNX1 fusion protein, these cells are monitored either in vitro or transplanted in immunodeficient mice. These types of studies have lead to three main conclusions, which are summarized below. (1) RUNX1 mutants or fusion proteins establish a preleukemic clone The foremost conclusion of studies designed to evaluate the effect of the three most common fusion proteins involving RUNX1, or its cofactor CBFβ, is that these mutant forms establish a covert pre-leukemic cell population. This is evidenced by the accumulation of early progenitors with abnormal differentiation capacity but not overt leukemia (e.g. b 30% blast in bone marrow; no blasts in blood). In the case of the gene products t(8;21) (RUNX1/ETO) and inv(16) (CBFβ-SMMHC), an accumulation of committed myeloid progenitors with impaired differentiation but increased self-renewal capacity was observed in mouse models [40,43–45]. However, in contrast to CBFβ-SMMHC, RUNX1/ETO expression also led to an increase in the number of HSCs, suggesting distinct mechanisms and/or target cells [39,46,47]. Studies using human cells have confirmed the ability of both RUNX1/ETO and CBFβ-SMMHC to impart a selective advantage and increased long-term self-renewal capacity on early myeloid progenitors [41,42,48,49]. Although at least some of this activity can be attributed to inhibition of wild-type RUNX1 activity [29,50], other gain-of-function mechanisms must be at play. We have recently shown that a DNA-binding independent function of RUNX1 is important in conferring self-renewal capacity to early progenitors—underlining the importance of mutations found in AML that selectively target the DNA-binding domain [51]. Possible mechanisms for the increased self-renewal capacity is the down-regulation of transcription factors that promote differentiation (e.g. PU.1 and C/EBPα) [4] or the upregulation of cytokine receptors (e.g. cKIT and TRKA) that maintain tight contact with the stroma cells, important regulators of self-renewal and proliferation in the HSC niche [52,53]. Mouse models have also revealed an expansion of a preleukemic clone for the RUNX1 fusion protein associated with t(12;21) ALL, generating the ETV6-RUNX1 fusion protein [54,55]. Interestingly, both an expansion of an early pro-B cell population as well as an HSC-like fraction (Kit+Sca+Lin−) was

observed. At present the importance of the Kit+Sca+Lin− population is unclear, as these cells were unable to differentiate into B-cell lineage [55]. In a xenotransplantation model, a striking increase in a pro-B cell population was also observed within the transduced cells of human origin (B. Niebuhr and C. Stocking, unpublished results). These cells carry the same markers (CD34+CD10+), as that observed in the leukemia, and support the hypothesis that ETV6-RUNX1 specifically imparts selfrenewal capacity to cells of the early B-cell compartment. In support of this hypothesis, Morrow and coworkers [56] have demonstrated the striking ability of ETV6-RUNX1 to transform B-cells derived from fetal liver in vitro. (2) Leukemia phenotype is influenced by RUNX1 fusion protein The second striking conclusion from mouse models of RUNX1/CBFβ fusion proteins is that the fusion protein itself is a strong determinant of the leukemia phenotype. Early work revealed the strong correlation between the leukemia phenotype (i.e. lineage and differentiation stage) and specific chromosomal translocations; however, a long-standing debate has been whether this is due to the target cell of the translocation or inherent function of the fusion protein. Several studies have clearly demonstrated that the t(8;21) and inv(16) are present in an HSC/P compartment [38,57], thus supporting the importance of the fusion protein itself in determining the leukemia phenotype. This idea has also been underlined in mouse models, where the leukemia phenotype induced by gene transfer mirrors the human leukemia with the corresponding chromosome abnormality, i.e. a blastic leukemia for RUNX1-ETO [40] and a myelomonocytic leukemia for CBFβ-SMMHC [43]. The case for the t(12;21) associated with preB-cell ALL is less clear. Contradictory evidence as to whether the translocation occurs in the HSC or in cells committed to the B-cell lineage has been reported [58]. The inability to detect the translocation in earlier cells or cells of different lineages may reflect the strong expansion pressure that the ETV6-RUNX1 fusion protein has on the B-cell compartment, coupled with the fact that its expression is incompatible with differentiation along specific lineages. Such an incompatibility has been observed in the analysis of bone marrow samples from AML patients carrying the t(8;21), which seldom contain T-cells carrying the translocation—although the translocation is present in B-cells and the HSC compartment [57]. Similarly, cells expressing either ETV6/RUNX1 or RUNX1/ETO in mouse models were almost never found in the T-cell compartment—most likely reflecting functional inhibition of wild-type Runx1 and its importance in T-cell differentiation and proliferation [40,55]. Current mouse models, in which an expanded B-cell population is observed after ETV6/ RUNX1 transduction, cannot discern if the expanded population arose from an infected long-term repopulating HSC or a committed B-cell. Further work is needed to resolve this question. (3) Secondary mutations are necessary for overt leukemia Finally, mouse studies have confirmed the hypothesis that RUNX1 mutations are initiating events—but require secondary

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mutations for acute leukemia induction. A major emphasis of current work is to determine the type of genetic mutations that collaborate with RUNX1 aberrations to induce an acute leukemia. Screening of patient samples has underlined the importance of mutations in receptor tyrosine kinases (RTK) in these leukemias. Interestingly, mutations in the KIT receptor are relative rare in AML but are found at an incidence of circa 45% in AML with CBF mutations, perhaps reflecting high levels of KIT expression in these leukemia types [53]. Mutations affecting the tryosine kinase domain are more often found in t(8;21) AML, whereas in inv(16) AML a high incidence of mutations in the extracellular domain surrounding the D419 have been described, and shown to be constitutively active [59–61]. In contrast, FLT3 mutations are found at a rather lower incidence in CBF leukemia (ca 7%) as compared to the high incidence (up to 45%) of mutations in either acute promyelocytic leukemia (APL), associated with translocations involving the gene encoding retinoic acid receptor-alpha [62], or in AML with a undifferentiated phenotype (FAB-M0) coupled with RUNX1 mutations [63,64]. It is tempting to speculate that these distinctive patterns of RTK mutations reflect the importance of the specific kinase in the transformed cells—although a specific synergistic activity cannot be ruled out. Interestingly, mutations in RAS, a downstream signaling module of RTKs, occurs at incidence of circa 10% AMLs, but up to 38% in inv(16) AML [65,66]. Direct mutations in the PI3K-pathway appear to be a rare event in AML [67]. Despite the overall high incidence of RAS and KIT mutations in RUNX1/CBFβ AML, the secondary mutations occurring in the majority of patients are not known. Similarly, the secondary mutations cooperating with t(12;21) preB-ALL have also remained elusive. Mutations in RTK occur rarely in t(12;21) ALL, and indeed deletion on chromosome 12p, including the non-translocated ETV6 allele, is the only consistent alteration in these patients observed to date [68,69]. Much promise has been made for gene expression analysis using large number of patient samples to identify underlining mutations— and although this work has provided prognostic expression signatures, it is just beginning to provide insight into causality [70,71]. A more recent genome-wide analysis of genetic alterations using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing has demonstrated the power of this approach, identifying a high incidence of deletions in genes encoding B-cell transcription factors in ALL samples, including those carrying t(12;21) [72]. Several approaches have been taken to identify mutations that cooperate with RUNX1/CBFβ fusion proteins using mouse models. One approach has been to test cooperating mutations by retroviral coexpression or by taking advantage of transgenic or knock-out mouse lines, in which candidate genes are activated or inactivated [40,47,73,74]. Another successful approach is the use of retroviral–insertional mutagenesis in mouse models expressing the fusion protein. The power of this approach has been shown by the identification of the PLAG family as cooperating partners CBFβ/SMMHC transformation [75,76]. Clearly, many questions remain unanswered with regard to the activating pathways that cooperate with RUNX1 alterations

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to induce acute leukemia. Is constitutive activation of KIT or FLT3 sufficient to induce leukemia, as proposed by animal models? Or are several independent genetic alterations required, as suggested in colon carcinoma? Interestingly, recent evidence suggests that alternatively spliced forms of the fusion gene itself may generate more “oncogenic” proteins, contributing to the progression of the leukemia phenotype [77]. Finally, it is important to ask: Do animal models duly reflect the situation in humans? RUNX1, leukemia stem cells, and therapeutic implications As summarized above, overwhelming evidence from patient samples and animal models support the concept that mutations affecting the RUNX1 gene are the initiating event in several different AMLs and ALLs. In all cases studied, these mutations lead to the accumulation of progenitor cells (either myeloid or lymphoid) with increased “self-renewal” capacity, the hallmarks of which are impaired differentiation and increased replication potential (as reflected in the frequency of immortalization in vitro.) We propose that these mutations provide the needed environment for the accumulation of secondary mutations, which inhibit apoptosis, maintain cell-cycle progression, and stimulate growth. Notably, without disruption of the RUNX1 specific “gatekeeper” functions, cells incurring the “proliferation” mutations would be lost through normal cell differentiation (Fig. 4). RUNX1 is a scaffolding protein that integrates cellular signals through the formation of gene promoter regulatory complexes; thus, the disruption of normal RUNX1 activity can impinge on a wide spectrum of signaling pathways. Future studies are necessary to identify the mechanisms involved, which surely differ between lineages and differentiation status of the target cell.

Fig. 4. Model to demonstrate the importance of a potential RUNX1 gatekeeper function. (A) Mutations that exclusively confer proliferation or protect from apoptosis will be lost during differentiation. (B) In contrast, mutations that confer self-renewal properties, such as RUNX1 mutations, allow the accumulation of early progenitors, which are then the target of secondary events.

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The corollary to the statement that RUNX1 mutations initiate the leukemogenic process is that RUNX1 mutations define the leukemia initiating cell; but are these cells equivalent to the leukemia stem cells that sustain the leukemia? Analysis of patient samples have clearly shown the emergence of subclones carrying independent secondary mutations, e.g. FLT3 activation [78]. Are these subclones thus the leukemia stem cells that drive the leukemia—or will elimination of these cells be insufficient to eradicate the leukemia? Are the initiating RUNX1 mutations still required for the maintenance of the leukemia?—Or has the accumulation of secondary mutations made them obsolete? Clearly inducible mouse models, such as described for MYC and BCR/ABL [79,80], are important to answer these questions, in order to design therapeutic interventions that target pathways that are crucial for tumor survival. Acknowledgments We thank all many members of the Stocking lab for creating a fruitful environment for work and discussion. We are also grateful for the continued support of the Deutsche Krebshilfe, the Deutsche José Carreras Leukämie Stiftung, and the FritzThyssen Foundation. The Heinrich-Pette-Institut is supported by the Freie und Hansestadt Hamburg and the German Ministry of Health and Social Safety. This paper is based on a presentation at a Focused Workshop on “Molecular Aspects of Myeloid Stem Cell Development and Leukemia” sponsored by The Leukemia & Lymphoma Society (in Annapolis, MD, May 13–16, 2007). References [1] B. Vogelstein, K. Kinzler, Cancer genes and the pathways they control, Nat. Med. 10 (2004) 789–799. [2] K.W. Kinzler, B. Vogelstein, Cancer-susceptibility genes. Gatekeepers and caretakers, Nature 386 (1997) 761–763. [3] J. Cammenga, Gatekeeper pathways and cellular background in the pathogenesis and therapy of AML, Leukemia 19 (2005) 1719–1728. [4] F. Rosenbauer, S. Koschmieder, U. Steidl, D.G. Tenen, Effect of transcriptionfactor concentrations on leukemic stem cells, Blood 106 (2005) 1519–1524. [5] D.G. Tenen, Disruption of differentiation in human cancer: AML shows the way, Nat. Rev., Cancer 3 (2003) 89–101. [6] A. Hamburger, S. Salmon, Primary bioassay of human tumor stem cells, Science 197 (1977) 461–463. [7] B. Huntly, D. Gilliland, Leukaemia stem cells and the evolution of cancerstem-cell research, Nat. Rev., Cancer 5 (2005) 311–321. [8] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 3983–3988. [9] H. Hemmati, I. Nakano, J. Lazareff, M. Masterman-Smith, D. Geschwind, M. Bronner-Fraser, H. Kornblum, Cancerous stem cells aris from pediatric brain tumors, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 15178–15183. [10] S. Singh, C. Hawkins, I.D. Clarke, J.A. Squire, J. Bayani, T. Hide, R.M. Henkelman, M.D. Cusimano, P.B. Dirks, Identification of human brain tumour initiating cells, Nature 432 (2004) 396–401. [11] D. Bonnet, J. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nat. Med. 3 (1997) 730–737. [12] J. Warner, J. Wang, K. Hope, L. Jin, J. Dick, Concepts of human leukemic development, Oncogene 23 (2004) 7164–7177. [13] T. Somervaille, M. Cleary, Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia, Cancer Cell 10 (2006) 257–268.

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