Inhibition of IGF-1 Signalling Enhances the Apoptotic Effect of AS602868, an IKK2 Inhibitor, in Multiple Myeloma Cell Lines

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-κB 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-κB inhibitors

The chemosensitivity of plasma cells to conventional treatments and the modulation of this sensivity by IGF-1 pathway

Le myélome multiple (MM) est une hémopathie dont la croissance et la prolifération sont liés à une variété de facteurs de croissance, y compris « insulin-like growth factor type 1 » (IGF-1). Bortézomib est le premier inhibiteur protéasome ayant une activité anti-tumorale significative dans le myélome multiple. Nous avons analysé l'impact de l'IGF-1 recombinant associé à l'inhibiteur du protéasome bortezomib sur des lignées humaines de MM, in vivo et sur des cellules de myélome frais humaines ex vivo. Nous avons montré que l'IGF-1 améliore l'activité cytotoxique du bortezomib in vitro, in vivo et ex vivo. Nous avons montré que l'accroissement de la toxicité peut être inhibé par la présence d'un anticorps monoclonal dirigé contre le récepteur de l'IGF-1 (IGF1-R). IGF-1 renforce l'activité cytotoxique des autres inhibiteurs de protéasome, y compris MG115, MG132, PSI et epoxomicin. Nos résultats confirment le fait que l'IGF-1sensibilise des cellules de myélome à l'activité cytotoxique des inhibiteurs du protéasome tels que le bortezomib, en raison du niveau accru du stress de réticulum endoplasmique et l'induction de la une réponse protéine dépliée (UPR)Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib in MM cell lines, in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro, in vivo and ex vivo. We showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 enhances the cytotoxic activity of other proteasome inhibitors, including MG115, MG132, PSI and epoxomicin. Our results support the fact that IGF-1sensitize myeloma cells to the cytotoxic activity of proteasome inhibitors such as bortezomib, as a consequence of enhanced level of endoplasmic reticulum stress and the induction of an unfolded protein response (UPR

Abstract LB-2: Insulin-like growth factor 1 potentiates the cytotoxic activity of bortezomib against myeloma cells

Abstract Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. The co-administration of IGF-1 bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content of Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-2. doi:10.1158/1538-7445.AM2011-LB-2</jats:p

Effect of Imids on Gene Expression Profiles of Fresh Human Myeloma Cells

Abstract Abstract 5013 Immunomodulatory drugs represent a major therapeutic advance in the treatment of patients with multiple myeloma. While these agents appear to exert various effects on the microenvironment, including effect on immune cells and angiogenesis, a direct effect on the tumor cells themselves is also likely. To describe and compare the effect of the three clinically available agents (thalidomide, lenalidomide, pomalidomide) we analyzed the gene expression profiles of fresh human myeloma cells exposed to thalidomide, lenalidomide or pomalidomide, using high density DNA arrays. Fresh human myeloma samples were obtained from bone marrow aspirates of patients with myeloma, and myeloma cells were immunopurified using anti CD138 magnetic beads. Purified myeloma cells (1.106 cells/ml) were incubated for 24 hours in RPMI 1640 medium supplemented with 10% fetal calf serum under each of the four following conditions: 1) DMSO; 2) thalidomide 40 microM; 3) lenalidomide 1 microM; 4) pomalidomide 100 nM. These levels are achievable in the plasma of MM pts. Pangenomic array experiments were performed usingWhole Human Genome 4 × 44K Agilent one-color microarrays. Data were normalized using the quantile normalization method. Samples were analysed for differentially expressed genes, taking into account both the level of significance and the fold-change. Ten evaluable samples were processed. Exposure to thalidomide, lenalidomide and pomalidomide induced differential expression of 36, 50 and 75 genes, respectively, in comparison to DMSO-exposed controls, the total list including 101 genes. Twelve of these were found to be differentially expressed after exposure to all of the three agents, including trophoblast glycoprotein, WAS protein family member 1, dickkopf homolog 1, pentraxin-related gene, CD28, interleukin 12B, tissue factor pathway inhibitor 2, phospholipase A2, dehydrogenase/reductase (SDR family) member 9, hypothetical LOC145788 and betacellulin. These commonly altered genes could be mechanistically involved in themultiple activities of these agents in multiple myeloma or may represent epiphenoma mechanistically unrelated to drug-induced cell death. Genes differentially expressed between the treatment with each of these agents could be indicative of the different and non-overlapping actions these agents have in multiple myeloma. An example of this is the recent demonstration that pomalidomide is clinically active in lenalidomide refractory patients. These results suggest that exposure to IMIDs induce various intracellular signalization pathways in myeloma cells which might be involved in the cytotoxic activity of these compounds. Disclosures: Dumontet: Celgene: Research Funding. </jats:sec

RNA-Sequencing Of Paired Pre-Treatment and Relapse Samples Reveals Differentially Expressed Genes Associated With Lenalidomide Resistance In Multiple Myeloma

Abstract Background Immunomodulatory (IMiDs) drugs’ cytotoxicity in MM cells is mediated through their binding to cereblon (CRBN) an adaptor protein of the Cul4a-DDB1-ROC1 ubiquitin E3 ligase complex. Loss of CRBN is clearly associated with resistance to IMiDs (Zhu et al. Blood. 2011) however this does not appear to be the sole mechanism through which MM cells may acquire resistance to this class of drugs. In addition, the acquisition of CRBN mutations within the Thalidomide binding domain (exons 10-11) have not been well studied. Similarly and while splice variants of CRBN were recently reported (Ghandi et al. Blood. 2012), no correlations has been made between the presence of these isoforms and IMiDs resistance in primary samples. Methods and Results In this study, we first validated CRBN as biomarker of clinical response to lenalidomide. At the mRNA level, using qRT-PCR (n=26, amplicons with 2 sets of primers overlapping exons 8-9 and exons 10-11) low CRBN in CD138 sorted bone plasma cells was significantly associated with shorter PFS (p=0.008) and lack of response to lenalidomide. We next compared CRBN mRNA (qRT-PCR) expression in paired samples (n=17 patients - 34 pairs) collected immediately pre-treatment and at the time of disease progression post-lenalidomide. In 10/17 (58.8%), a significant reduction (2-ΔΔCT &lt; 0.8) in the CRBN amplicon levels was observed between the paired pre- and post-treatment samples. These data suggest that in nearly half of the patients, CRBN-independent mechanisms may be mediating resistance to IMiDs. In order to elucidate these CRBN-independent mechanisms of resistance to lenalidomide and identify potential targets that may overcome it, RNA-seq analysis was performed in 12 paired patients samples obtained sequentially from the same patient prior to lenalidomide treatment initiation and after development of resistance. Transcriptome sequence data was generated by RNA-seq performed for each sample on Ion Torrent Proton sequencer with a minimum of 70 million reads per sample. Poly(A) is captured with poly(T) magnetic beads, fragmented and copied to cDNA libraries with reverse transcriptase and primers. Filtered Fastq files are processed with TopHat-Fusion alignment against hg19 as reference genome. Cufflinks and Cuffdiff algorithms were used to detect differentially expressed transcripts and spliced isoforms. A total of 830 genes were identified as differentially expressed (p value and FDR &lt;0.05) between the pre- and post-lenalidomide paired samples. Selected differentially expressed genes were also measured by RT-PCR analysis to confirm the validity of the RNA-seq analysis. In order to gain insight into the cellular and molecular functions of this identified gene set, we compared it to the expression of 395 gene sets curated in the Molecular Signatures Database (MSigDB), using the Gene set enrichment analysis (GSEA) algorithm. Among the most enriched gene sets in this analysis were KRAS.KIDNEY_UP.V1_UP; KRAS.LUNG_UP.V1_UP; KRAS.600.LUNG.BREAST_UP.V1_UP; RAF_UP.V1_DN; MEK_UP.V1_DN; and STK33_DN suggesting a role for the RAS-RAF-MAPK pathway in the acquired resistance to IMiDs. Other gene sets of interest included AKT_UP_MTOR_DN.V1_DN, E2F3_UP.V1_UP; EIF4E_UP and RPS14_DN.V1_DN; consistent with a role for the translational machinery activity (mTOR-4EBP1-eIF4E pathway) in IMiDs resistance. Of interest variant splice isoforms of CRBN were visualized using the Integrative Genomics Viewer (IGV) tool including isoforms lacking exon 10, which contains a portion of the IMiD-binding domain. However these CRBN splice variants are unlikely to be implicated in resistance to IMiDs since they were not enriched in the relapsed paired samples. Conclusions Study of the transcriptome of paired pre- and post-IMIDs in myeloma primary cells confirms that acquired resistance to this class of drugs is associated with the direct loss of CRBN as well as through the modulation of other CRBN-independent pathways. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Insulin-Like Growth Factor 1 Potentiates the Cytotoxic Activity of Bortezomib Against Myeloma Cells

Abstract Abstract 3987 Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis by reducing NF-κB signalling. Other cytotoxic mechanisms have been suggested, including increased reticulum stress leading to an unfolded protein response. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. Using an MTT assay, we found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines, at a concentration of IGF-1 of 100 ng/mL. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. Mice received weekly administrations of bortezomib (0.5 mg/kg, i.p.) with or without recombinant IGF-1 (0.03 mg/kg, i.p.). The co-administration of IGF-1 with bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content o Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Exploration the NFkB pathway showed that exposure to IGF-1 and bortezomib induced a reduction of IkBalpha, an increase in phosphor-IKBalpha as well as a decrease in NFkB p65. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Cell cycle distributions of cells exposed to bortezomib alone or the IGF-1/bortezomib combination were similar. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells. Disclosures: No relevant conflicts of interest to declare. </jats:sec