Comparative pre-clinical evaluation of receptor tyrosine kinase inhibitors for the treatment of multiple myeloma
Introduction
Multiple myeloma (MM) is a disease of terminally differentiated plasma cells, characterised by the infiltration and accumulation of malignant cells at several sites within the bone marrow compartment [1], [2]. MM constitutes approximately 1% of all cancers, 10% of all haematological malignancies, and is responsible for 20% of all haematological malignancy deaths [3], [4]. In the past two decades, advances in autologous stem cell transplantation and in chemotherapeutic treatments have improved the long-term survival of MM patients; however, despite such advances, MM remains an incurable disease and is associated with severe symptoms such as pain and pathological fractures.
Recent progress in the understanding of the molecular pathology of MM has led to the identification of a number of non-random chromosomal translocations which are found in up to 60% of primary MM samples [5]. Four of these recurrent translocations involve the immunoglobulin heavy chain locus (IGH) at 14q32 and result in the deregulation of genes encoding cyclins D1 (11q13), D3 (6p21), c-MAF (16q23), fibroblast growth factor receptor 3 (FGFR3) and Wolf–Hirschhorn syndrome candidate 1 (FGFR3/WHSC1; (4p16.3)) [1]. These translocation events are implicated in myelomagenesis, and the resultant deregulated proteins are therefore candidates for chemotherapeutic intervention.
The t(4;14)(p16;q32) translocation is present in 10–20% of MM patients, is associated with a poor prognosis and results in the over-expression of functional FGFR3 and WHSC1 genes, compared to that of normal plasma cells [6], [7], [8]. FGFR3 is a tyrosine kinase receptor of the FGFR family which also comprises FGFR1, 2 and 4, and is activated by the binding of fibroblast growth factors (FGFs) [9]. The FGFR3 protein consists of an extracellular domain bearing three immunoglobulin-like sub-domains, and a cytoplasmic domain containing a split tyrosine kinase sub-domain. The extracellular and cytoplasmic domains are separated by a trans-membrane domain. Upon FGF stimulation, FGFRs activate several signal transduction pathways, including the mitogen activated protein kinase (MAPK), phosphotidylinositol 3 kinase and phospholipase C-γ pathways, which play an important role in a number of cellular processes including proliferation, migration and cell survival [9]. In some t(4;14) MM cases and cell lines, FGFR3 is constitutively activated by somatic mutations, predominantly found in the ligand, transmembrane and kinase domains, rendering the receptor ligand independent [10], or by amplification [11].
Several studies have shown that overexpression of both constitutively activated and wild-type FGFR3 is oncogenic in MM [12], [13], [14], which can promote proliferation, survival, and induce resistance to dexamethasone [14]. In addition, overexpression of mutant FGFR3 in the interleukin 6 (IL-6) dependent murine B9 plasmacytoma cell line resulted in IL-6 independence, decreased apoptosis and an enhanced proliferative response to IL-6 [13]. In the same system, wild-type FGFR3 in the presence of ligand also produced enhanced proliferation and survival of the B9 cell line. Furthermore, expression of mutant FGFR3 (K650E), and to a lesser extent wild-type FGFR3, was demonstrated to have in vivo transforming properties in a mouse model [12].
The oncogenic role of constitutively activated FGFR3 and its validation as a therapeutic target in t(4;14) MM has been demonstrated by the ability of anti-FGFR3 antibodies [15], [16] and several small molecule receptor tyrosine kinase (RTK) inhibitors, i.e. SU5406, PD173074, CHIR258 and PKC412 [17], [18], [19], [20], [21], [22], to inhibit the kinase activity and MM cell growth in vitro and/or in vivo. However, the most pronounced effect has often been seen in MM cells with very high levels of FGFR3, e.g. KMS-11 cells. Furthermore, these previous studies did not directly compare the activities of the RTK inhibitors in relation to potency and selectivity of FGFR3 tyrosine kinase inhibition. In this study, we further validate wild-type FGFR3 as a target for chemotherapeutic intervention in t(4;14) MM using a panel of cell lines and small molecule RTK inhibitors that vary in potency and selectivity. These studies led to the evaluation of one compound, sunitinib, in human tumour xenograft models of MM.
Section snippets
Materials
PD173074, sunitinib (SU-11248), vandetanib (ZD6474) and vatalanib (PTK-787) were synthesised by Syngene (Bangalore, India) with compound purity and structure being verified using both NMR and LCMS. Compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM and stored at −20 °C. Acidic fibroblast growth factor (aFGF/FGF1) and heparin were purchased from R & D Systems (Abingdon, UK) and Sigma–Aldrich (Dorset, UK), respectively. The anti-FGFR3 antibodies (C-15 and B9)
PD173074 and sunitinib potently inhibit FGFR3 kinase activity
The activity of PD173074, sunitinib, vandetanib and vatalanib were evaluated against FGFR3 in an isolated enzyme kinase assay (Table 1). PD173074 and sunitinib were the most potent FGFR3 inhibitors, sunitinib being 30-fold less potent than PD173074. The potency of PD173074 and sunitinib against FGFR3 is similar to that previously reported [23], [24], [25]. In contrast, vandetanib was a weaker inhibitor of FGFR3 receptor kinase activity and vatalanib did not inhibit the kinase at concentrations
Discussion
In the past two decades, understanding of the molecular pathology of haematological malignancies has increased significantly and has led to the discovery of several non-random chromosomal translocations whose products are targets for chemotherapeutic intervention. One such example is the inhibition of Bcr-Abl kinase activity by imatinib mesylate in chronic myelogenous leukaemia (CML). Imatinib mesylate has revolutionised the treatment of CML, producing high response rates with minimal toxic
Conflict of interest
All authors declare no conflict of interest.
Acknowledgements
We would like to thank Prof. Nick Cross and Prof. Takemi Otsuki for kindly providing the Ba/f3 cells transfected with the ZNF198-FGFR1 construct and the KMS-11 cell line, respectively. This project was supported by grants from Cancer Research UK, Leukaemia and Lymphoma Research and Astex Therapeutics.
Contributions. JI, HN, AH, GJ, and HL initially conceived and gained funding for the study. All authors were involved in the acquisition, analysis and interpretation of data. LB and JI wrote a
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