Systemic analysis of tyrosine kinase signaling reveals a common adaptive response program in a HER2-positive breast cancer.

Drug-induced compensatory signaling and subsequent rewiring of the signaling pathways that support cell proliferation and survival promote the development of acquired drug resistance in tumors. Here, we sought to analyze the adaptive kinase response in cancer cells after distinct treatment with agents targeting human epidermal growth factor receptor 2 (HER2), specifically those that induce either only temporary cell cycle arrest or, alternatively, apoptosis in HER2-overexpressing cancers. We compared trastuzumab, ARRY380, the combination thereof, and a biparatopic, HER2-targeted designed ankyrin repeat protein (DARPin; specifically, 6L1G) and quantified the phosphoproteome by isobaric tagging using tandem mass tag liquid chromatography/tandem mass spectrometry (TMT LC-MS/MS). We found a specific signature of persistently phosphorylated tyrosine peptides after the nonapoptotic treatments, which we used to distinguish between different treatment-induced cancer cell fates. Next, we analyzed the activation of serine/threonine and tyrosine kinases after treatment using a bait peptide chip array and predicted the corresponding active kinases. Through a combined system-wide analysis, we identified a common adaptive kinase response program that involved the activation of focal adhesion kinase 1 (FAK1), protein kinase C-δ (PRKCD), and Ephrin (EPH) family receptors. These findings reveal potential targets to prevent adaptive resistance to HER2-targeted therapies.

Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy.

Structural basis for eliciting a cytotoxic effect in HER2-overexpressing cancer cells via binding to the extracellular domain of HER2.

Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.

Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.

Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation.

The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3'-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser(166) and Ser(188). Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser(166) and Ser(188) in a phosphatidylinositide 3'-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3'-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser(166) and Ser(188) and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBalpha displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21(Cip1), resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2.p53 signaling pathway by regulating Mdm2 stability.

NDR family of AGC kinases--essential regulators of the cell cycle and morphogenesis.

The nuclear Dbf2-related (NDR) family of protein serine/threonine kinases comprises mammalian NDR and large tumor suppressor (LATS) kinases, their orthologs from Drosophila melanogaster and Caenorhabditis elegans, and a number of related kinases from yeast and plants. The members of this family were independently implicated in various aspects of the control of cell division and morphogenesis. They are crucial regulators of the actin and tubulin cytoskeletal organization during polarized growth and cytokinesis in yeast. Furthermore, they are key players in control of proliferation and morphology of many cell types in D. melanogaster and C. elegans. In mammalians, the LATS kinase is a tumor suppressor, negatively regulating the cyclin-dependent kinase CDK1, cell proliferation rate, and modulating cell survival.