Monday November 23 2009 | Biotechnology feed | All feeds

Despite the therapeutic success of Avastin as a strategy for limiting tumor neovascularization and growth (see Innovative Cancer Therapies: Targeted therapy, a clinical and commercial revolution), as a class, the clinical efficacy of angiogenesis inhibitors has been rather limited.

Avastin (bevacizumab), developed by Genentech/Roche's, gained US approval in 2004 as a first-line treatment for metastatic colorectal cancer in combination with intravenous 5-FU-based chemotherapy. This was followed closely by European approval. Within the past few months the addition of Avastin to a paclitaxel regimen has been shown to produce a dramatic survival advantage in women with metastatic breast cancer.

In contrast to Avastin which acts systemically to neutralize VEGF, compounds developed to inhibit the tyrosine kinase activity of the VEGF receptors have met with more modest success. For example, LeadDiscovery's DailyUpdates service has recently highlighted disappointing data from studies of PTK/ZK, a VEGR receptor tyrosine kinase inhibitor under development by Schering and Novartis. Following demonstrations of safety in phase I/II trials the CONFIRM 1 and CONFIRM 2 phase III trials were initiated. Data from CONFIRM 1, a phase III study designed to investigate the efficacy of PTK/ZK failed to meet its end point. More recently data from CONFIRM 2 demonstrated that the primary endpoint of overall survival in this study is unlikely to achieve statistical significance (for the press release and our editorial click here).

One of the potential problems of Avastin is that it neutralizes VEGF both in the vicinity of the tumor and away from the tumor mileau. VEGF is known to play a role in wound healing and also has a number of effects on cardiovascular homeostasis. The inhibition of such effects is likely to contribute to the gastrointestinal perforation and wound dehiscence (wound rupture) and hemoptysis (blood in the sputum) observed in some treated patients. The limited success to date of VEGF receptor inhibition is unfortunate therefore since this strategy promised to offer a more targeted approach than that achievable with Avastin. Unfortunate as they may be, given the complexities of the VEGF pathway, the problems encountered by therapies such as PTK/ZK are however perhaps not surprising.

VEGF exists in multiple isoforms, VEGF-A, -B, -C, -D and PlGF. The original VEGF, now denoted VEGF-A, is regulated under the hypoxic conditions found within tumors, and binds to VEGFR-1 as well as –2. Whereas the function of VEGFR-1 is still enigmatic, VEGFR-2 is expressed in endothelial cells and, in concert with VEGF-A, appears to play a pivotal role in endothelial cell differentiation during embryogenesis as well as in formation of new vessels in adulthood. The complexity of the situation is further extended by the existence of multiple VEGF splice variants. For example VEGF-A is expressed as VEGF189, VEGF165, and VEGF121, which have distinct biological effects. The binding of VEGF isoforms to their receptors causes  dimerization of receptors thereby activating the receptor tyrosine kinase and consequently intracellular signaling pathways. VEGFR-2 has been investigated in some detail and residues have been identified which regulate the effector function of the receptor. The intracellular domain of VEGFR-2 contains 19 tyrosine residues, 11 of which are located in the noncatalytic part of the receptor, potentially serving as phosphorylation sites. In their recent EMBO J. paper Matsumoto et al create a pan phosphorylation site map of VEGFR-2 thus allowing this group to investigate the functional role of the various residues.

Tyrosine residues Y1054 and Y1059 in the kinase domain have previously been shown to serve as positive regulatory sites while phosphorylation at Y1175 in the C-terminal tail allows binding, phosphorylation, and activation of phospholipase Cg1 and eventually activation of protein kinase C. Phosphorylated Y1175 also binds the adapter molecule Shb, which mediates activation of PI3-kinase and assembly of focal adhesions. A further residue, Y951 in the kinase insert has been linked to migration of endothelial cells and binds another adapter molecule, TSAd (also know as VRAP).

The complexity conferred to the system by the roles of the various residues suggests that the development of VEGFR-2 kinase inhibitors per se may be a supoptimal approach to the development of angiogenesis inhibitors and a more effective strategy may be to target key sites within the receptor. Such an approach may not only increase efficacy but also reduce potential side effects, for example VEGFR-2 activation plays a role in neurogenesis while neurological adverse effect are dose-limiting for a number of existing angiogenesis inhibitors.

To allow more selective targeting of VEGFR-2, Dr Lena Claesson-Welsh and colleagues describe in the featured EMBO paper a pan-phosphorylation map of VEGFR-2 and the roles of the various phosphorylation sites in endothelial cell function. One of the key findings of this study was that Y951 phosphorylation was restricted to endothelial cells expressing features characteristic of vessels undergoing active angiogenesis. Further investigation revealed that although Y951 did not apparently contribute to the regulation of VEGFR-2 downstream targets such as ERK, p38 MAPK, PLCg1, and PI3-kinase/Akt, phosphorylation of this residue was required for actin stress fiber formation in endothelial cells and consequently for endothelial cell migration, which is a prerequisite for angiogenesis. Phosphorylation of Y951 allowed coupling of VEGFR-2 to TSAd and interrupting this complex by specific siRNA targeting of TSAd prevented endothelial cell chemotaxis. Consistent with its key down-stream effector function, inhibiting TSAd expression in a mouse model was shown to be critical for tumor vascularization and growth.

The present study maps the various phosphorylation sites within VEGFR-2 forming a basis for further studies aimed at developing therapeutic candidates that more selectively inhibit the response to receptor activation. This approach will hopefully lead to the identification of molecules with improved therapeutic margins and less likely to suffer the dose-limiting adverse effects of currently available molecules. In particular, the present study suggests that screening for candidates that target Y951 may yield molecules that effectively prevent tumor angiogenesis.