Genetic alterations that result in changes within signaling pathways have been implicated in tumor initiation, propagation and resistance to cancer therapy. In particular, amplification and/or over-expression of receptor tyrosine kinases (RTK) of which EGFR and Her-2 are prototypic examples, has been implicated as being initiating events in a variety of human malignancies1
. These kinases as well as their downstream signaling kinases therefore make a very attractive target for therapeutic interventions. Indeed, recent clinical success of Trastuzumab in Her-2 over-expressing breast cancer2,3
and Erlotinib in EGFR over-expressing cancers4
as well as Gleevec for chronic myeloid leukemia5
provides impetus for targeted inhibition of kinases as a viable therapeutic paradigm. The serine/threonine kinase PKB/Akt is a key mediator of survival and resistance to therapy. Akt functions as a signaling hub wherein many upstream signaling pathways such as those activated in response to RTK activation, eventually converge. The integration of these intracellular signals at the level of Akt and its kinase activity, regulates the phosphorylation of several downstream effectors, such as NF-kappa B, mTOR, Forkhead, Bad, GSK-3 and MDM-26-12
. These phosphorylation events in turn mediate the effects of Akt on cell growth, proliferation, protection from pro-apoptotic stimuli, and stimulation of neo-angiogenesis13
. The activation of Akt is regulated by both translocation to the plasma membrane and phosphorylation at Thr308 and Ser473. Constitutive phosphorylation of Akt at these sites is frequently observed in a wide range of solid tumors and hematologic malignancies. Ongoing efforts have focused on therapeutically targeting Akt and its biologic sequelae, either at the level of Akt itself or at the levels of its upstream regulators and downstream effectors14
. Because Akt is also important for proliferative and anti-apoptotic signaling pathways critical for normal cells, particular emphasis is placed on the fine-tuning the targeting of individual components of this pathway to maximize the therapeutic index of anti-cancer strategies based on the Akt pathway. Although, there have been major developments in our understanding of the biology of Akt and its role in human malignancies, the development of molecular imaging technologies to monitor and quantify Akt activity is still in its infancy.
Non-invasive technologies like MRI, MRS, PET and Optical Imaging will contribute significantly to drug discovery research with emphasis on drug efficacy, on the mechanism of action and on target validation studies in animal disease models in vivo15-24
. Molecular imaging techniques bridge the gap between preclinical and clinical research for the development of successful drug candidates that have target specificity, optimal safety, pharmacokinetics/pharmacodynamics and efficacy25-28
. The use of molecular imaging endpoints instead of time-consuming dissection and histology significantly decreases the workload involved in tissue analysis and thereby speeds up the evaluation of drug candidates. As imaging methods are non-invasive, they allow for longitudinal studies in a single animal. This increases the statistical relevance of a study, allows for more clinically relevant study designs and decreases the number of animals required. These tools also provide much earlier surrogate markers of therapeutic success and thus speed up the process of drug discovery and clinical evaluation.
In previous studies we described a reporter molecule whose expression provides the ability to non-invasively image Akt activity using bioluminescence imaging (BLI)29
. This bioluminescent Akt reporter (BAR) was designed to be expressed within the cytosolic compartment. Based on the reasoning that Akt, when activated is recruited to the plasma membrane, here we describe the development of a membrane targeted bioluminescent Akt reporter (MyrPalm-BAR). Fusion of 10 amino-terminal residues of Lyn, which enables recruitment of Lyn to the plasma membrane by virtue of myristoylation and palmitoylation30
, to the amino-terminus BAR resulted in membrane association of the reporter. Using various inhibitors of the Akt pathway we demonstrate that membrane targeted BAR is a more sensitive reporter of Akt activity. MyrPalm-BAR would therefore be better suited for studies wherein Akt activity was being evaluated in a biological context as well as studies for the identification of novel compounds targeting the Akt pathway.