Elsevier

Leukemia Research

Volume 35, Issue 9, September 2011, Pages 1233-1240
Leukemia Research

Comparative pre-clinical evaluation of receptor tyrosine kinase inhibitors for the treatment of multiple myeloma

https://doi.org/10.1016/j.leukres.2011.01.011Get rights and content

Abstract

Background

Fibroblast growth factor receptor 3 (FGFR3) is up-regulated as a result of the t(4;14)(p16;q32) translocation that occurs in up to 20% of multiple myeloma (MM) patients. Recent studies have demonstrated that up-regulation of FGFR3 promotes cell survival, growth and drug resistance in malignant plasma cells, both in vitro and in vivo. Therefore, inhibition of FGFR3 signalling is potential target for the chemotherapeutic intervention in t(4;14) MM.

Methods

Small molecule receptor tyrosine kinase inhibitors (PD173074, sunitinib (SU-11248), vandetanib (ZD6474) and vatalanib (PTK-787)) with varying degrees of inhibitory activity and selectivity against FGFR, were assessed in Ba/f3 cells expressing ZNF198-FGFR1 and MM cell lines. Cell viability, FGFR3 and ZNF198-FGFR1 phosphorylation and apoptosis were evaluated by growth inhibition assays, immunoblotting and fluorescence-activated cell sorting analysis, respectively. An in vivo study was performed with sunitinib in t(4;14)-positive and t(4;14)-negative human MM tumour xenograft models.

Results

PD173074 and sunitinib differentially inhibited the growth of Ba/f3 cells expressing ZNF198-FGFR1 (GI50 = 10 nM and 730 nM, versus GI50 >1 μM and 2.7 μM for parental cells; p < 0.0001) and t(4;14) positive MM cell lines (GI50 = 4–10 μM and 1–3 μM, versus GI50 = 14–15 μM and 4–5 μM for t(4;14) negative MM cells; p  0.002). In addition, both PD173074 and sunitinib inhibited the activation of FGFR3 in t(4;14)-positive MM cells. PD173074 and sunitinib induced an apoptotic response in a concentration and time-dependent manner in a t(4;14)-positive (PD174073 and sunitinib) but not a t(4;14)-negative MM cell line (sunitinib only); however, in in vivo tumours derived from the same cell lines, sunitinib was only active in the t(4;14)-negative model.

Conclusions

These data demonstrate that PD173074 and sunitinib are inhibitors of FGFR3 in MM cell lines, and that sunitinib has in vivo activity in a human MM tumour xenograft model. However, caution should be exercised in using the t(4;14) translocation as a predictive biomarker for patient selection in clinical trials with sunitinib.

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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