PITX2 and beta-catenin interactions regulate Lef-1 isoform expression.

Lef-1 and PITX2 function in the Wnt signaling pathway by recruiting and interacting with beta-catenin to activate target genes. Chromatin immunoprecipitation (ChIP) assays identified the Lef-1 promoter as a PITX2 downstream target. Transgenic mice expressing LacZ driven by the 2.5-kb LEF-1 promoter demonstrated expression in the tooth epithelium correlated with endogenous Lef-1 FL epithelial expression. PITX2 isoforms regulate the LEF-1 promoter, and beta-catenin synergistically enhanced activation of the LEF-1 promoter in combination with PITX2 and Lef-1 isoforms. PITX2 enhances endogenous expression of the full-length beta-catenin-dependent Lef-1 isoform (Lef-1 FL) while decreasing expression of the N-terminally truncated beta-catenin-independent isoform. Our research revealed a novel interaction between PITX2, Lef-1, and beta-catenin in which the Lef-1 beta-catenin binding domain is dispensable for its interaction with PITX2. PITX2 interacts with two sites within the Lef-1 protein. Furthermore, beta-catenin interacts with the PITX2 homeodomain and Lef-1 interacts with the PITX2 C-terminal tail. Lef-1 and beta-catenin interact simultaneously and independently with PITX2 through two different sites to regulate PITX2 transcriptional activity. These data support a role for PITX2 in cell proliferation, migration, and cell division through differential Lef-1 isoform expression and interactions with Lef-1 and beta-catenin.

Selective Targeting of Myoblast Fusogenic Signaling and Differentiation-Arrest Antagonizes Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue malignancy comprised histologically of skeletal muscle lineage precursors that fail to exit the cell cycle and fuse into differentiated syncytial muscle-for which the underlying pathogenetic mechanisms remain unclear. In contrast to myogenic transcription factor signaling, the molecular machinery that orchestrates the discrete process of myoblast fusion in mammals is poorly understood and unexplored in RMS. The fusogenic machinery in Drosophila, however, is understood in much greater detail, where myoblasts are divided into two distinct pools, founder cells (FC) and fusion competent myoblasts (fcm). Fusion is heterotypic and only occurs between FCs and fcms. Here, we interrogated a comprehensive RNA-sequencing database and found that human RMS diffusely demonstrates an FC lineage gene signature, revealing that RMS is a disease of FC lineage rhabdomyoblasts. We next exploited our Drosophila RMS-related model to isolate druggable FC-specific fusogenic elements underlying RMS, which uncovered the EGFR pathway. Using RMS cells, we showed that EGFR inhibitors successfully antagonized RMS RD cells, whereas other cell lines were resistant. EGFR inhibitor-sensitive cells exhibited decreased activation of the EGFR intracellular effector Akt, whereas Akt activity remained unchanged in inhibitor-resistant cells. We then demonstrated that Akt inhibition antagonizes RMS-including RMS resistant to EGFR inhibition-and that sustained activity of the Akt1 isoform preferentially blocks rhabdomyoblast differentiation potential in cell culture and in vivo. These findings point towards selective targeting of fusion- and differentiation-arrest via Akt as a broad RMS therapeutic vulnerability. SIGNIFICANCE: EGFR and its downstream signaling mediator AKT1 play a role in the fusion and differentiation processes of rhabdomyosarcoma cells, representing a therapeutic vulnerability of rhabdomyosarcoma.

A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets.

Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, alveolar-RMS, is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene to FOXO1. The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. Here, we report a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, we have performed a comprehensive deletion screen against the Drosophila autosomes and demonstrate that mutation of Mef2, a myogenesis lynchpin in both flies and mammals, dominantly suppresses PAX7-FOXO1 pathogenicity and acts as a PAX7-FOXO1 gene target. Additionally, we reveal that mutation of mastermind, a gene encoding a MEF2 transcriptional coactivator, similarly suppresses PAX7-FOXO1, further pointing toward MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease.

Drosophila and mammalian models uncover a role for the myoblast fusion gene TANC1 in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a malignancy of muscle myoblasts, which fail to exit the cell cycle, resist terminal differentiation, and are blocked from fusing into syncytial skeletal muscle. In some patients, RMS is caused by a translocation that generates the fusion oncoprotein PAX-FOXO1, but the underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain unclear. Using a Drosophila model of PAX-FOXO1-mediated transformation, we show here that mutation in the myoblast fusion gene rolling pebbles (rols) dominantly suppresses PAX-FOXO1 lethality. Further analysis indicated that PAX-FOXO1 expression caused upregulation of rols, which suggests that Rols acts downstream of PAX-FOXO1. In mammalian myoblasts, gene silencing of Tanc1, an ortholog of rols, revealed that it is essential for myoblast fusion, but is dispensable for terminal differentiation. Misexpression of PAX-FOXO1 in myoblasts upregulated Tanc1 and blocked differentiation, whereas subsequent reduction of Tanc1 expression to native levels by RNAi restored both fusion and differentiation. Furthermore, decreasing human TANC1 gene expression caused RMS cancer cells to lose their neoplastic state, undergo fusion, and form differentiated syncytial muscle. Taken together, these findings identify misregulated myoblast fusion caused by ectopic TANC1 expression as a RMS neoplasia mechanism and suggest fusion molecules as candidates for targeted RMS therapy.

PITX2, beta-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter.

PITX2, beta-catenin and lymphoid enhancer factor (LEF-1) are required for the inductive formation of several epithelial-derived organs, including teeth. Lef-1 is expressed in the dental epithelium after Pitx2, and both factors have overlapping expression patterns in the tooth bud and cap stages. Our analysis of Pitx2-/- mutant mice showed reduced Lef-1 expression in facial tissues by RT-PCR and quantitative RT-PCR. Consistent with these results we show that the human 2.5 kb LEF-1 promoter is activated by PITX2. Furthermore, the LEF-1 promoter is differentially activated by PITX2 isoforms, which are co-expressed in dental epithelium. The 2.5 kb LEF-1 promoter contains two regions that act to inhibit its transcription in concert with PITX2. The proximal region contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. LEF-1 cannot autoregulate LEF-1 expression; however co-transfection of PITX2 and LEF-1 result in a synergistic activation of the 2.5 kb LEF-1 promoter. LEF-1 specifically interacts with the PITX2 C-terminal tail. Deletion of a distal 800 bp segment of the LEF-1 promoter resulted in enhanced PITX2 activation, and increased synergistic activation in the presence of LEF-1. Furthermore, beta-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. beta-catenin directly interacts with PITX2 to synergistically regulate LEF-1 expression. We show a new mechanism where LEF-1 expression is regulated through PITX2, LEF-1 and beta-catenin direct physical interactions. LEF-1 and beta-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.

Protein kinase C phosphorylation modulates N- and C-terminal regulatory activities of the PITX2 homeodomain protein.

PKC phosphorylation regulates PITX2 DNA binding and transcriptional activity. Mutation of individual PKC sites demonstrates the functional regulation of PITX2 through phosphorylation. Immunoprecipitation of PITX2 and a PITX2 PKC mutant protein reveal specific in vivo phosphorylation by PKC in transfected cells. The transcriptional activity of PITX2 is negatively regulated by N-terminal phosphorylation and positively regulated by C-terminal phosphorylation. We demonstrate a mechanism of increased PITX2 transcriptional activation through protein interactions facilitated by phosphorylation of the PITX2 C-terminal tail. Phosphorylation of the PITX2 C terminus enhances the interaction with cellular factors. In corroboration with the PITX2 PKC functional studies, a newly identified C-terminal PITX2 mutation associated with Axenfeld-Rieger syndrome (ARS) demonstrates reduced phosphorylation. This mutation (PITX2 DeltaT1261) creates a frameshift mutation in codon 227 resulting in 11 novel amino acids downstream followed by premature truncation of the protein. Three PKC sites in the C-terminal tail and OAR domain are deleted, which results in decreased transcriptional activation. PITX2 DeltaT1261 is unable to interact with a cellular factor to synergistically activate transcription and demonstrates the first link of ARS with defective PITX2 protein interactions. Gene expression profiling of homozygous Pitx2 mutant mouse tissue reveals decreased Dlx2 expression as a potential molecular basis for developmental defects associated with ARS patients. Overall, phosphorylation imparts another level of regulation to the activity of the PITX2 homeodomain protein during development.