Inhibition of IGF-1 Signalling Enhances the Apoptotic Effect of AS602868, an IKK2 Inhibitor, in Multiple Myeloma Cell Lines Ines Tagoug1,2,3,4, Ame´ lie Sauty De Chalon1,2,3,4, Charles Dumontet1,2,3,4* 1 Universite´ de Lyon, Lyon, France, 2 INSERM U1052, Centre de Recherche de Cance´rologie de Lyon, Lyon, France, 3 CNRS UMR 5286, Centre de Recherche de Cance´rologie de Lyon, Lyon, France, 4 HCL, Lyon, France Abstract Multiple myeloma (MM) is a B cell neoplasm characterized by bone marrow infiltration with malignant plasma cells. IGF-1 signalling has been explored as a therapeutic target in this disease. We analyzed the effect of the IKK2 inhibitor AS602868, in combination with a monoclonal antibody targeting IGF-1 receptor (anti-IGF-1R) in human MM cell lines. We found that anti- IGF-1R potentiated the apoptotic effect of AS602868 in LP1 and RPMI8226 MM cell lines which express high levels of IGF-1R. Anti-IGF-1R enhanced the inhibitory effect of AS602868 on NF-kB pathway signalling and potentiated the disruption of mitochondrial membrane potential caused by AS602868. These results support the role of IGF-1 signalling in MM and suggest that inhibition of this pathway could sensitize MM cells to NF-kB inhibitors. Citation: Tagoug I, Sauty De Chalon A, Dumontet C (2011) Inhibition of IGF-1 Signalling Enhances the Apoptotic Effect of AS602868, an IKK2 Inhibitor, in Multiple Myeloma Cell Lines. PLoS ONE 6(7): e22641. doi:10.1371/journal.pone.0022641 Editor: Marianne Koritzinsky, University Health Network, Canada Received January 7, 2011; Accepted July 1, 2011; Published July 25, 2011 Copyright: 2011 Tagoug et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the Ligue contre le Cancer du Rhoˆ ne and the Association pour la Recherche contre le Cancer and the Ministry of Higher Education and Scientific Research of Tunisia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: charles.dumontet@chu-lyon.fr Introduction Multiple myeloma is characterized by unrestrained accumula- tion of antibody-secreting plasma cells in the bone marrow, attributed to loss of apoptotic control and cell cycle deregulation [1,2]. Its incidence is approximately 4/100,000 persons per year, but is predicted to increase in the future due to the expected increase in longevity. The proliferation and the survival of MM cell lines and fresh human cells has been shown to be related to the activation of several pathways such as phosphatidylinositol-3 kinase (PI-3K)/Akt, Janus kinase (JAK)/signal transducer and activator of transduction 3 (STAT3), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-kB) [3,4,5,6,7]. Several growth factors produced by the microenvironment induce the activation of these pathways such as interleukin 6 (IL-6), and insulin-like growth factor 1(IGF-1) [8,9]. In vivo and in vitro, IGF-1 decreases drug sensitivity of MM cells, and upregulates a series of anti-apoptotic proteins such as A1/Bfl-1, XIAP and Bcl-2 [10,11,12]. Several strategies seek to target and inhibit the IGF-1 signalisation pathway, either by blocking the IGF-1 receptor or by inhibiting the signals downstream of this receptor [13,14,15]. Monoclonal antibodies targeting the IGF-1R constitute a very attractive approach to block the IGF-1/IGF-1R interaction, subsequently blocking the stimulation of several signalisation pathways, especially PI-3K/Akt and NF-kB [16]. Inactive NF-kB is located in the cytosol associated to different NF-kB inhibitory proteins (IkB) [17]. Many stimuli such as growth factors, stress and inflammatory response induce the phosphorylation of IkB proteins, thereby activating their ubiquitination and degradation by the 26S proteasome [18]. The IkB kinase complex (IKK) is responsible for the phosphorylation of IkB. IKK include three subunits, the two catalytic subunits IKK1 (IKKa) and IKK2 (IKKb), and the regulatory subunit Nemo (IKKc) [19,20,21]. The IKK kinase complex indirectly regulates the translocation of NF- kB to the nucleus and the transcription of growth and survival genes. While inhibition of the NF-kB pathway has been suggested to be a significant mechanism of proteasome inhibitors such as bortezomib [22], another approach to inhibit this pathway consists in the inhibition of IKK [23]. IKK inhibitors could have anticancer effects per se or could potentiate agents interfering on signalisation pathways upstream of NF-kB, as seems to be the case for IGF-1R inhibitors. AS602868 is an anilinopyrimide derivative and adenosine triphosphatase competitor selected for its inhibitory effect on IKK2 in vitro. AS602868 induces the apoptosis of MM, acute myeloid leukemia and sarcoma cell lines in vitro [24,25,26]. It has also been shown that AS602868 blocks the canonical NF-kB pathway and the proliferation of MM cell lines [27]. Recent work from our laboratory recently identified gene expression profiles in fresh human acute myeloid leukemia cells exposed to AS602868 and suggests that multiple mechanisms, including pathways other than NF-kB may be induced by this agent [28]. In this study, we analysed the effect of the combination of the IKK2 inhibitor AS602868 and a monoclonal antibody directed against IGF-1R on MM cell lines. AS602868 was found to block the activation of NF-kB, induced a dissipation of mitochondrial PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22641