? ? The availability of therapies directed against specific receptor tyrosine kinases (RTKs) active in cancer offers a unique opportunity to address how to optimally treat complex cancers using targeted therapies. We hypothesize that treatment of cancers with agents targeted against multiple pathways critical to tumorigenesis will diminish the emergence of therapeutic resistance, thereby increasing the likelihood of effective treatment. In this application, we will derive chimeric alleles of receptor tyrosine kinases implicated in the pathogenesis of glioma, and coupled to activation of firefly or Renilla luciferase respectively. We will derive chimeric alleles of EGFR fused to split firefly luciferase (flue) and of PDGFRa fused to split Renilla luciferase (rluc). We will verify that activation of EGFR reconstitutes active flue, that activation of PDGFRa reconstitutes active rluc, and that flue and rluc activities can be measured independently. We will test selective inhibitors of EGFR or PDGFRa to determine their impact on flue and rluc respectively. We will use these chimeric EGFR-fluc and PDGFRa-rluc alleles to generate xenograft models of glioma, to test the impact of treating tumors with inhibitors directed against either receptor tyrosine kinase alone, or against both tyrosine kinases together. Our long-term goal is to use these models to develop treatment paradigms that diminish the emergence of therapeutic resistance.
The Specific Aims are: ? ? Aim 1: To develop chimeric activated alleles of EGFR fused to split firefly luciferase, and to separately generate activated alleles of PDGFRa fused to split Renilla luciferase (rluc). We propose to fuse the N- and C-terminal halves of each respective luciferase separately to two individual RTK monomers. Since docking proteins bind to the cytoplasmic domains of EGFR and PDGFRa in response to activation, we will also test each RTK-N-terminal luc chimera in conjunction with a chimera consisting of a docking protein fused to the C-terminal half of the appropriate split luc. ? ? Aim 2. To create cell and animal models for glioma in which PDGFR and EGFR pathways are activated and can be inhibited independently. We will generate cell culture and xenograft models for glioma driven by chimeras of EGFR with split flue, PDGFRa with split rluc, or both. The reconstitution of luc activity upon receptor activation will then be imaged non-invasively. Upon treatment with an appropriate inhibitor, we hypothesize that the bioluminescence of flue, rluc, or both will decrease, allowing us to image separately the blockade of EGFR or PDGFRa even when both RTKs are active in an individual tumor. ? ?
