A chemical and genetic approach to the mode of action of fumagillin

Abstract

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/beta-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.

Figure 1. Schematic of Canonical and Non-canonical Wnt Signaling

Although not all Wnt signaling effectors/transducers are included, key components relevant to this work are depicted.

Figure 2

Structure of the Antiangiogenic Natural Product Fumagillin, 1, the Clinical Trial Drug Candidate, 2, and the Nonspecific MetAP-1/MetAP-2 Inhibitory Natural Product Bengamide E, 3

Figure 3. Genetic Interactions between MetAP-2 and Wnt5 in Zebrafish

(A) Wild-type zebrafish at 24 hr postfertilization denoting normal anterior-posterior length. (B) Homozygous ppt embryo. (C) Coinjection of Wnt5 MO and a missense MetAP-2 MO as control. (D) MetAP-2 MO injection phenocopies Wnt5 MO-injected zebrafish, as shown in (C). (E) Coinjection of MetAP-2 and Wnt5 MOs in zebrafish embryos at minimal concentrations resulted in a strong synergistic effect rather than an additive effect (also see Table 1). (F) Coinjection of activated CamKIItr RNA with MetAP-2 MO partially rescues the MetAP-2 MO phenotype (also see Table 1).

Figure 4. Wnt5a/Fz2-Induced F9 Differentiation Was Attenuated by TNP-470 or siRNA-Mediated MetAP-2 Downregulation

(A) F9 cells expressing wild-type rat Frizzled 2 (Rfz2) were cocultured with HEK293 cells expressing Wnt5a in the presence or absence of 10 nM TNP-470 for 4 days. The primitive endoderm (PE) marker tPA was measured by ELISA. Retinoic acid-induced F9 cell differentiation was unaffected by TNP-470 (insert). Means + SD are shown (n = 6). (B) F9 cells with siRNA-mediated MAP2 knockdown showed a similar inhibitory effect as TNP-470. Means + SD are shown (n = 6). (C) Wnt5a-induced F9 cell differentiation was monitored by immunofluorescence staining with TROMA-1 antibody against PE-specific cytokeratin Endo-A. Identical fields are depicted showing phase contrast (left), HEK293 cells coexpressing GFP with Wnt5a (center), and TROMA induction in Rfz2-expressing F9 cells (right).

Figure 5. MetAP-2 Is Essential in Noncanonical Wnt Signaling but Has a Negligible Effect on the Canonical Wnt/β-Catenin Pathway

(A) F9 cell differentiation induced by 10 µM isoproterenol in cells expressing chimeric β₂-AR/Rfz2 and β₂-AR/Rfz1 was monitored by immunofluorescence staining with TROMA-1 antibody. (B) MetAP-2 protein levels in F9 cells expressing β₂-AR/Rfz2 or β₂-AR/Rfz1 with MAP2-targeting siRNA. (C) F9 cells expressing chimeric β₂-AR/Rfz1 transfected with the pTOPflash reporter plasmid and treated with 10 µM isoproterenol for 6 hr. Relative LEF/TCF-luciferase activities of triplicate samples are presented as fold induction. Means + SD are shown (n = 6).

Figure 6. TNP-470 Regulates Noncanonical Wnt Signaling

(A) F9 cells expressing chimeric β₂-AR/Rfz2 with or without siRNA targeting MAP2 were treated with 100 µM isoproterenol for 10 min with or without pretreatment with 10 nM TNP-470 for 24 hr. CamKII activity was determined by the [γ-³²P]ATP in vitro kinase assay with biotin-linked peptide substrate. Means + SD are shown (n = 3). (B) Wnt5a-stimulated JNK activation in RFz2-F9 cells in the presence or absence of 10 nM TNP-470 for 4 hr, 8 hr, and 16 hr, as measured by phospho-63-c-jun. (C) Wnt5a-stimulated RhoA activity with or without pretreatment with 10 nM TNP-470 for 16 hr. (D) Proliferation of hTERT MPE cells expressing an empty vector (control) or an activator of noncanonical Wnt signaling, ΔDIX-Dvl2, as measured by [³H]thymidine incorporation. Means + SD are shown (n = 6).