The deregulation of specific cell signaling pathways and their downstream transcription factor targets plays an important pathogenic role in numerous brain disorders. Thus, having the capability to image second messenger and transcription factor activity in vivo will advance our understanding of disease mechanisms and our ability to target pathogenic pathways. In vivo transcription factor imaging has the exciting potential to allow for real time analysis of location and magnitude of specific transcriptional events within target tissues and will greatly facilitate the classification of brain diseases at the molecular level in vivo, the quantification of baseline activities of specific transcription factors/pathways, and the noninvasive monitoring of transcription factor responses to therapies that target transcription factors and their upstream regulators.
A primary role for dysfunctional transcription regulation in brain pathogenesis is well defined in primary brain tumors that are now the most common cause of cancer-related death in children and increasingly responsible for neurologic disability and death in middle-aged adults. The molecular mediators of dysfunctional brain tumor transcription are numerous, and many are considered promising therapeutic targets under active pre-clinical and clinical investigation. The clinical translation of these discoveries will be aided substantially by non-invasive transcription factor imaging. The hypothesis of this proposal is that reporter transgenes can be used to image and quantify the activity of endogenous brain tumor-associated transcription factors within brain at baseline and in response to therapeutics that alter transcription factor activity.
In Aim #1 we will engineer chimeric tri-reporter transgenes (coding for luciferase, red fluorescence protein, and a truncated herpes simplex sr39 thymidine kinase) for localizing and quantifying the activity of specific transcription factors (i.e. Gli and E2F) linked to brain tumor cell malignancy and validate their reporter function in vitro.
In Aim #2 we will establish that reporter transgenes can be used to quantify and localize transcription factor activity in intracranial tumor xenografts derived from tumor cell lines engineered to stably express the tri-reporters constructed and validated in aim #1. We will also image the effects of anti-tumor agents expected to down-regulate Gli- and E2F-dependent reporter expression in tumor xenografts using the HSV sr39 thymidine kinase (ttk) substrates 125I-FIAU (SPECT imaging) and 124I-FIAU (PET imaging).
In Aim #3 we will determine if reporter constructs delivered to wild-type brain tumor xenografts in vivo can be used to quantify tumor transcription factor activity and transcriptional responses to anti-tumor therapeutics. The successful completion of these experiments will establish the feasibility of monitoring endogenous disease-related transcription factors within brain and brain tumors in vivo. Establishing this capability will substantially impact upon the development of novel therapeutics that target cell signaling and transcriptional pathways.