Myelosuppression and kinase selectivity of multikinase angiogenesis inhibitors

Abstract

Background: Myelosuppression has been observed with several multikinase angiogenesis inhibitors in clinical studies, although the frequency and severity varies among the different agents. Inhibitors targeting vascular endothelial growth factor receptor (VEGFR) often inhibit other kinases, which may contribute to their adverse-event profiles.

Methods: Kinase selectivity of pazopanib, sorafenib, and sunitinib was evaluated in a panel of 242 kinases. Cellular potency was measured using autophosphorylation assays. Effect on human bone marrow progenitor growth in the presence of multiple growth factors was evaluated and correlated with the kinase selectivity.

Results: Sunitinib inhibited more kinases than pazopanib and sorafenib, at potencies within 10-fold of VEGFR-2. All three compounds potently inhibited VEGFR-2, platelet-derived growth factor receptor-beta and c-Kit, However, pazopanib was less active against Flt-3 in both kinase and cellular assays. The inhibitory properties of pazopanib, sorafenib, and sunitinib were dependent on the growth factor used to initiate bone marrow colony formation. Addition of stem cell factor and/or Flt-3 ligand with granulocyte-macrophage colony stimulating factor resulted in significant shifts in potency for sorafenib and sunitinib but less so for pazopanib.

Conclusion: Activity against c-kit and Flt-3 by multikinase angiogenesis inhibitors provide a potential explanation for the differences in myelosuppression observed with these agents in patients.

Figures

Figure 1

Inhibition of receptor autophosphorylation by various tyrosine kinase inhibitors. To compare the activities of pazopanib, sunitinib, and sorafenib, we evaluated their inhibitory effects against wild-type VEGFR-2, c-Kit, PDGFR-β, and Flt-3 receptors in HUVEC (for VEGFR-2), NCI-H526 (c-Kit), HFF (PDGFR-β), and RS4;11 (Flt-3). Cells were serum-starved overnight and then treated with DMSO or different concentrations of pazopanib, sunitinib, or sorafenib for 2 h. Cells were then stimulated with respective ligands as described in the cellular autophosphorylation section under Materials and Methods. Total receptor was immunoprecipitated using antireceptor antibodies and phosphorylation was detected using anti-pTyr antibody following western blot analysis.

Figure 2

Effect of tyrosine kinase inhibitors (TKIs) on ligand-stimulated progenitor cell growth. (A) Human bone marrow progenitor cells were stimulated with GM-CSF (90 ng ml−1), SCF (50 ng ml−1), Flt-3 ligand (200 ng ml−1), and their various combinations for colony stimulating unit (CFU) formation. Less than one CFU formed in cultures without any growth factor addition, thus no background correction was needed. (B and C) Bone marrow progenitor cells were treated with pazopanib, sunitinib, and sorafenib at concentrations ranging from 0.1 nM to 10 μM in the presence of various ligands. Inhibitory potentials, IC50 (B) and IC90 (C), of TKIs were calculated for ligand-induced CFU formation. Data represent mean+s.d. from three human bone marrow preparations. *Significantly different than GM-CSF alone; P<0.01 by Dunnett's Multiple Comparison test.