Phosphorylation Regulates Transcriptional Activity of PAX3/FKHR and Reveals Novel Therapeutic Possibilities

Abstract

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy. [Cancer Res 2008;68(10):3767-76]

p21 Downregulation is an important component of PAX3/FKHR oncogenicity and its reactivation by HDAC inhibitors enhances combination treatment

“…We recently identified PKC412 as a potent antitumorigenic agent for aRMS treatment in vitro and in vivo (Amstutz et al, 2008). To further increase treatment efficacy, we performed in vitro combination studies with chemotherapeutic agents used in RMS therapy or HDAC inhibitors which have been shown to act synergistically with PKC412 against acute myelogenous leukaemia (AML) (Bali et al, 2004).…”

“…As PKC412 induced cell death is due to caspase-3-dependent apoptosis (Amstutz et al, 2008), we tested whether VPA might further enhance apoptosis by an activated caspase-3 assay. This revealed an at least 2-fold increase in the number of apoptotic cells in combined treatment compared to PKC412 single treatment (Figures 2a and b).…”

“…Recently, we identified the kinase inhibitor PKC412 to exert a potent antitumor effect in aRMS in vitro and in vivo by reducing the transcriptional activity of the PAX3/FKHR oncogene (Amstutz et al, 2008).…”

“…Because inhibition of DNA-binding of PAX3/FKHR by PKC412 (Amstutz et al, 2008) was not able to induce p21 expression ( Figure 2d) we reasoned that PAX3/FKHR might regulate p21 gene expression at levels other than transcription. Recently, it has been demonstrated that PAX3/FKHR can interact with and mediate destabilization of the transcription factor EGR1 ( (Roeb et al, 2007) and Supplementary Figures S3a and b) which itself can directly modulate gene expression of p21 ( (Choi et al, 2008;Ragione et al, 2003) and Supplementary Figure S3c)).…”

“…Along these lines, we recently reported that the kinase inhibitor PKC412 (midostaurin) can influence the oncogenic activity of PAX3/FKHR through modulation of specific phosphorylation sites within the PAX3 domain (Amstutz et al, 2008). Phosphorylation at these sites was shown to perturb efficient DNA-binding.…”