Originally published as JCO Early Release 10.1200/JCO.2004.12.965 on January 15 2004
Journal of Clinical Oncology, Vol 22, No 4 (February 15), 2004: pp. 582-584
© 2004 American Society of Clinical Oncology.
Loss of C/EBP and Favorable Prognosis of Acute Myeloid Leukemias: A Biological Paradox
Danilo Perrotti,
Guido Marcucci,
Michael A. Caligiuri
The Human Cancer Genetics Program, The Division of Hematology/Oncology, and The Comprehensive Cancer Center, The Ohio State University, Columbus, OH
Acute myeloid leukemia (AML) is characterized by the accumulation of immature precursors arrested at different stages of myelopoiesis. Normal myeloid differentiation depends on the coordinated activity of transcription factors that control the commitment and maturation of the hematopoietic stem/progenitor cell pool by directing the expression of lineage-determining genes, including those encoding the receptor for myeloid-specific cytokines, in a time- and stage-specific manner [1]. Although it seems intuitive that genetic or functional inactivation of transcription factors regulating proliferation, survival, and differentiation of myeloid progenitors is requisite for the pathogenesis of AML, a direct link with myeloid leukemogenesis has been formally demonstrated only for a few transcription factors [2]. Among these, the CCAAT enhancer binding protein alpha (C/EBP ) of the basic region leucine zipper transcription regulators recently has been reported to be inactivated in approximately 4% to 15% of patients with AML [2].
C/EBP is an essential factor for granulocytic differentiation of common myeloid progenitors [3,4]; in fact, Cebpa-null mice lack neutrophils and eosinophils but retain monocytes [3]. Fetal liver cells from Cebpa-null mice do not form colonies in response to granulocyte colony-stimulating factor (G-CSF) but develop into immature myeloid cells in response to GM-CSF [3]. Accordingly, ectopic C/EBP expression induces granulocytic differentiation and arrests erythroid differentiation of human CD34+ cells [5]. The mechanism whereby C/EBP induces granulocytic differentiation of myeloid progenitors depends on its transcription activatory and on growth suppressive functions that require the integrity of the C/EBP basic region-leucine zipper and transcriptional activation domains that are involved in DNA binding, homodimerization and interaction with E2F, p21 and Cdk2/Cdk4 [6-8]. Notably, inhibition of E2F activity by C/EBP leads to downregulation of c-Myc, an important step required for granulocytic differentiation [9,10]. Two different groups of investigators have initially reported CEBPA mutations in AML patients [11,12]. These mutations were usually associated with the French-American-British (FAB) subtypes M1 or M2, suggesting a block at a specific stage of the myelocytic differentiation. Approximately half of the described mutations were clustered in the N-terminus, leading to either a truncated nonfunctional protein or increased expression of the dominant negative isoform p30C/EBP [11,12]. The remaining mutations were identified at the C-terminus of C/EBP and predicted mutant proteins lacking DNA binding or homodimerization activities [11,12]. Interestingly, in these and subsequent studies, some patients presented biallelic mutations at the C-terminus, whereas others were either heterozygous for distinct mutations or found to have a N-terminal mutation associated with a C-terminal mutation [13,14].
The prognostic value of these findings has only recently been recognized. Three studies, including that of Fröhling et al [15] reported in this issue of the Journal of Clinical Oncology, have linked CEBPA mutations with a favorable outcome in AML. Preudhomme et al [16] reported 22 different CEBPA mutations in 15 (11.1%) of 135 patients enrolled in the Acute Leukemia French Association (ALFA)-9000 trial. Most of these mutations were associated with FAB M1 or M2 phenotypes and intermediate cytogenetic risk by the MRC classification [17]. In this study, the presence of CEBPA mutations was a favorable predictor for overall and disease-free survival in both univariate and multivariate analyses. Similar data were subsequently reported by the Dutch-Belgium Hematology-Oncology Cooperative Group (HOVON), which identified 12 AML patients (4.3%) with CEBPA mutations at the C-terminus of the 277 patients enrolled onto the HOVON AML-4 and AML-29 trials [14]. Finally, the study by Fröhling et al has now shown that mutations of CEBPA are independent predictors for favorable clinical outcome in a homogeneously treated population of AML patients with normal cytogenetics [15].
These findings are noteworthy. First, despite differences in patient characteristics at diagnosis and treatment regimens, each of these three studies showed that CEBPA mutations favorably affect clinical outcome. This might appear to be a biologic paradox, given that loss of C/EBP itself is likely to be a critical event for maturation arrest and transformation of myeloid precursors. One could hypothesize, however, that in addition to its contribution to leukemogenesis, loss of C/EBP might uncover proapoptotic pathways that mediate response to phase-specific chemotherapeutic agents, such as cytarabine.
Second, other genomic abnormalities, such as overexpressed BAALC or tandem duplications of MLL and Flt3, have also been reported as predictors of poor clinical outcome in AML patients with normal cytogenetics [18-20]. Given that these adverse prognostic markers are not mutually exclusive in AML blasts, it becomes imperative to accurately evaluate the prognostic impact of "cross-talk" between the seemingly distinct leukemogenic pathways activated by these molecular aberrations. Activating mutations of Flt3, for example, are found in 30% of AML—with or without chromosomal abnormalities—and have been associated with a poor outcome. In particular, internal tandem duplication of Flt3 (Flt3-ITD) is the most recurrent molecular abnormality and, by virtue of its constitutive tyrosine kinase activity, is sufficient to transform, confer proliferative advantage, inhibit apoptosis, and suppress differentiation of growth factor-dependent myeloid progenitor cell lines [21,22]. Using in vitro models of Flt3-ITD–mediated leukemogenesis and Flt3-ITD+ AML patients' samples, two different groups have shown that Flt3-ITD expression induces, in a kinase-dependent manner, repression of C/EBP expression or activity. The reported mechanisms, though different, are not mutually exclusive and might both be active in Flt3-ITD+ AML. Specifically, Flt3-ITD-dependent downregulation of C/EBP differentiation-inducing activity could be achieved by either a MAPK-dependent phosphorylation of C/EBP serine 21 leading to inactivation of C/EBP [23], or by Flt3-ITD-generated signals, resulting in transcriptional suppression of Cebpa mRNA expression [22]. Preudhomme et al and Fröhling et al evaluated the prognostic impact of Flt3-ITD in patients also carrying a mutated CEBPA gene. Unfortunately, the two groups achieved discordant conclusions; therefore, the clinical impact of this association remains unsolved, underscoring the need in future trials to adopt a common strategy to prospectively assess the predictive value and prognostic interaction of these aberrant markers at diagnosis and thereafter.
Third, despite being overly represented in patients with FAB M1 or M2 phenotypes, CEBPA mutations were never reported in association with favorable cytogenetic abnormalities, such as t(8;21)(q22;q22) or inv(16)(p13q22), which disrupt the and ß subunits, respectively, of the heterodimeric transcription regulator core binding factor (CBF). This is quite surprising, in that t(8;21)(q22;q22) is one of the most common cytogenetic abnormalities associated with AML-M2 [24]. In a recent study, Pabst et al [25] reported that suppression of neutrophilic differentiation in t(8;21)(q22;q22) FAB M2 AML appears to depend on inhibition of wild type CEBPA transcription. This effect relies entirely on the ability of the oncogenic product of the t(8;21)(q22;q22) translocation, the AML1/ETO fusion protein, to physically interact with C/EBP [26] and repress C/EBP DNA binding and transactivation activity with consequent disruption of the C/EBP autoregulatory loop, a necessary step in triggering granulocytic differentiation [25]. It is noteworthy to mention that, analogous to AML1/ETO, PML/RAR, the product of the t(15;17)(q22;q12) in FAB M3 AML, post-translationally inhibits C/EBP DNA binding activity via a mechanism that involves the formation of the PML/RAR-C/EBP protein complex [27,28]. Moreover, overexpression of C/EBP in acute promyelocytic leukemia cells antagonizes the differentiation-inhibitory effect of PML/RAR [28]. Levels of C/EBP mRNA and protein are markedly reduced in FAB M4 AML patients with inv(16)(p13q22) compared with healthy donors and with FAB M4 AML patients without inv (16)p13q22) [30], suggesting that C/EBP differentiation-inducing activity may also be impaired in AML with the CBF-ß/MYH11 fusion through a mechanism that does not involve the physical interaction of CBF-ß with C/EBP [26] or direct inhibition of CEBPA transcription. Based on these data, therefore, it is not unreasonable to postulate that loss of the C/EBP either through CEBPA mutations or by the interaction with chimeric oncogenic proteins contributes to activation of common leukemogenic pathways. Thus, as high-dose cytarabine represents the backbone for the treatment of CBF AML, these data might raise the question of whether similar therapeutic approaches should also be considered for AML patients carrying CEBPA mutations or functional inactivation and normal cytogenetics or aberrant karyotypes other than t(8;21)(q22;q22) or inv (16)(p13q22) [30].
The final point relevant to the work of Fröhling et al is that although loss of C/EBP activity is associated with favorable prognosis in certain cytogenetic or molecular subgroups of AML, this might not be true for other types of leukemia. We recently reported that expression of C/EBP is also substantially reduced in the acute phase (myeloid blast crisis) of chronic myelogenous leukemia (CML-BC) [31], which has a dismal prognosis. The importance of lost C/EBP activity as a central mechanism leading to maturation arrest of CML myeloid blasts is supported primarily by two lines of evidence: (1) ectopic C/EBP expression induces granulocytic maturation of differentiation-arrested BCR/ABL-expressing myeloid precursors [31], and (2) a blast crisis-like process emerges in mice transplanted with BCR/ABL-transduced Cebpa-null, but not heterozygous or wild type fetal liver cells [32]. In BCR/ABL-expressing myeloid progenitor cells, loss of C/EBP depends on the BCR/ABL-induced activity of the RNA binding protein hnRNP E2 that, on interaction with the 5' untranslated region of CEBPA mRNA inhibits, CEBPA translation [33]. Interestingly, downregulation of C/EBP expression by the translation-inhibitory activity of another RNA binding protein, calreticulin [34], has also been observed in U937 cells expressing the fusion oncogene AML1/MDS1/EVI1 (AME) product of the t(3;21)(q26;q22) translocation, itself often found in CML blast crisis [34].
Although validation by prospective larger studies is required, the results reported by Fröhling et al are provocative and suggest that reclassification of AML patients with normal cytogenetics and CEBPA mutations into a more favorable risk category is indicated. Additionally, studies that use oligonucleotide array- or proteomic–based methodologies might prove useful to unveil similarities and differences in the complicated networks operative in blasts carrying CEBPA mutations and other cytogenetic or molecular abnormalities proven to be prognostically important. Finally, as the biologic ramifications of a mutated or modulated C/EBP become fully understood, it is not unreasonable to anticipate the use of novel therapeutic approaches that specifically target leukemogenic mechanisms activated by loss of this protein function.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
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