The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RAR␣), and PLZF-RAR␣ encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RAR␣, or PLZF-RAR␣. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (␥-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF-and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. ␤-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

IntroductionReceptor tyrosine kinases (RTKs) bind extracellular growth factors and activate intracellular signaling networks. The magnitude and kinetics of RTKs activation are tightly regulated, since they determine the quality and extent of the biologic response. 1 Attenuation of RTK signaling occurs by endocytosis and subsequent protein degradation. [2][3][4] Cbl proteins have been shown to be central players in these processes. [5][6][7] The members of the Cbl family, c-Cbl, Cbl-b, and Cbl-3, contain an N-terminal phosphotyrosine-binding (PTB) domain that allows direct interaction with activated RTKs, and a RING finger domain that classifies Cbl proteins as E3 ubiquitin ligases. 5,[8][9][10] Ubiquitylation of RTKs is important for their internalization, endocytic sorting, and targeting for degradation. 2 In addition to their ubiquitin E3 ligase activity, Cbl proteins also associate with the endocytic machinery via their C-terminus by recruiting proteins such as CIN85 and endophilins. 11,12 However, not only are Cbl proteins important for RTK signal termination, but they also mediate positive RTK signaling events to downstream effectors. Upon phosphorylation, Cbl molecules bind signaling molecules including SHP-2, Gab2, and PI3-kinase. 13 In animal models, but not in human cancers, oncogenic forms of Cbl have been described that are characterized by loss of the E3 ubiquitin ligase activity. 14,15 It has been reported that these oncogenic mutants of Cbl interact with activated RTKs and function in a dominant-negative fashion. 16,17 Aberrant signaling by the type III RTK Flt3 is an important event in the pathogenesis of acute myeloid leukemia (AML). Flt3 strongly influences hematopoietic progenitor cell homeostasis and is highly expressed in AML. [18][19][20][21] Also, about one third of AML cases harbor somatic, activating Flt3 mutations that cause myeloid transformation. [21][22][23][24] In contrast to activating mutations, little is known about the potential functions of Flt3 in AML cases lacking Flt3 mutations. Also, the mechanisms of Flt3 signal regulation and the role of Cbl proteins in these processes remain undetermined, although it has been shown that Flt3 activation is followed by Cbl phosphorylation. 25 Here, we analyzed the role of c-Cbl in the internalization, ubiquitylation, and biologic functions of wild-type Flt3 (Flt3-WT) and the most commonly described Flt3 mutations in AML, internal tandem duplication (Flt3-ITD). We found that the inhibition of Cbl function severely disturbed Flt3 signal transduction kinetics by blocking Flt3 internalization and ubiquitylation. As a consequence, interference with Cbl function induced ligand-independent, autoactive biologic effects of Flt3. Also, we describe a novel E3-ligase inactivating c-Cbl mutation isolated from the blasts of one AML patient. This mutant Cbl protein interfered with the function of endogenous c-Cbl and displayed in vitro transforming activity in myeloid cells that was dependent on the presence of Flt3. To our knowledge, this represents the ...

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein α (c/EBPα) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPα and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.