Development and characterization of protein kinase B/AKT isoform-specific nanobodies

The serine/threonine protein kinase AKT is frequently over-activated in cancer and is associated with poor prognosis. As a central node in the PI3K/AKT/mTOR pathway, which regulates various processes considered to be hallmarks of cancer, this kinase has become a prime target for cancer therapy. However, AKT has proven to be a highly complex target as it comes in three isoforms (AKT1, AKT2 and AKT3) which are highly homologous, yet non-redundant. The isoform-specific functions of the AKT kinases can be dependent on context (i.e. different types of cancer) and even opposed to one another. To date, there is no isoform-specific inhibitor available and no alternative to genetic approaches to study the function of a single AKT isoform. We have developed and characterized nanobodies that specifically interact with the AKT1 or AKT2 isoforms. These new tools should enable future studies of AKT1 and AKT2 isoform-specific functions. Furthermore, for both isoforms we obtained a nanobody that interferes with the AKT-PIP3-interaction, an essential step in the activation of the kinase. The nanobodies characterized in this study are a new stepping stone towards unravelling AKT isoform-specific signalling

An AKT2-specific nanobody that targets the hydrophobic motif induces cell cycle arrest, autophagy and loss of focal adhesions in MDA-MB-231 cells

The AKT kinase family is a high-profile target for cancer therapy. Despite their high degree of homology the three AKT isoforms (AKT1, AKT2 and AKT3) are non-redundant and can even have opposing functions. Small-molecule AKT inhibitors affect all three isoforms which severely limits their usefulness as research tool or therapeutic. Using AKT2-specific nanobodies we examined the function of endogenous AKT2 in breast cancer cells. Two AKT2 nanobodies (Nb8 and Nb9) modulate AKT2 and reduce MDA-MB-231 cell viability/proliferation. Nb8 binds the AKT2 hydrophobic motif and reduces IGF-1-induced phosphorylation of this site. This nanobody also affects the phosphorylation and/or expression levels of a wide range of proteins downstream of AKT, resulting in a G0/G1 cell cycle arrest, the induction of autophagy, a reduction in focal adhesion count and loss of stress fibers. While cell cycle progression is likely to be regulated by more than one isoform, our results indicate that both the effects on autophagy and the cytoskeleton are specific to AKT2. By using an isoform-specific nanobody we were able to map a part of the AKT2 pathway. Our results confirm AKT2 and the hydrophobic motif as targets for cancer therapy. Nb8 can be used as a research tool to study AKT2 signalling events and aid in the design of an AKT2-specific inhibitor