Potential Treatment For Multiple Myeloma Research Articles

Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells

We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.

Targeting the oncogenic transcription factor c-Maf for the treatment of multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy derived from clonal expansion of plasma cells within the bone marrow and it may progress to the extramedullary region in late stage of the disease course. c-Maf, an oncogenic zipper leucine transcription factor, is overexpressed in more than 50% MM cell lines and primary species in association with chromosomal translocation, aberrant signaling transduction and modulation of stability. By triggering the transcription of critical genes including CCND2, ITGB7, CCR1, ARK5, c-Maf promotes MM progress, proliferation, survival and chemoresistance. Notably, c-Maf is usually expressed at the embryonic stage to promote cell differentiation but less expressed in healthy adult cells. c-Maf has long been proposed as a promising therapeutic target of MM and a panel of small molecule compounds have been identified to downregulate c-Maf and display potent anti-myeloma activities. In the current article, we take a concise summary on the advances in c-Maf biology, pathophysiology, and targeted drug discovery in the potential treatment of MM.

Potential Treatment for Multiple Myeloma: Immune Checkpoint Inhibitors

Multiple myeloma (MM) is a malignant disease in which monoclonal plasma cells proliferate abnormally. The poor prognosis of MM is always closely related to the immunosuppression of T cells. Restore the immune function of suppressed T cells may reverse the immunodeficiency in the MM microenvironment and improve prognosis. In recent years, immunosuppressive receptors such as programmed cell death receptor-1 (PD-1), T cells immunoglobulin and ITIM domain (TIGIT), Lymphocyte-Activation Gene-3 (LAG-3), T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3), Leukocyte immunoglobulin-like receptor B1 (LILRB1) and Cytotoxic T- lymphocyte antigen 4 (CTLA-4) have been discovered playing a key role in the tumor immunodeficiency microenvironment. For patients with solid tumors and some leukemia, immunotherapy targeting such receptors can significantly improve the T cells immunodeficiency. However, similar positive results were not found in MM patients, which is related to the complex immunosuppressive mechanism. At present, the understanding of immunosuppressive receptors in MM is insufficient. In this review, this paper summarized part of the studies on PD-1, TCLA-4, Tim-3, TIGIT and other popular immunosuppressive receptors in MM, in order to attract more attention, and in-depth research on the immunotherapy also to promote the immunotherapy of MM from basic research to clinical transformation as soon as possible.

Abstract 2377: Design, synthesis, and characterization of 4-aminopyrazole quinazolines as potent inhibitors of G protein-coupled receptor kinase GRK6 for the potential treatment of multiple myeloma

Abstract Multiple myeloma (MM) is one of the most common hematological malignancies, but current therapeutic options are limited to high-dose chemotherapy or high-risk stem-cell transplantation. In a kinome-wide RNAi study by Tiedemann and colleagues (2010), the G-protein coupled receptor kinase GRK6 was identified as a critical kinase required for survival of MM cells. This study also suggests that MM cells, but not other cell types, are dependent on GRK6; and that gene silencing by shRNA or siRNA of GRK6, but not other members of the GRK family, results in decreased survival. Through gene silencing techniques, we determined that a functional GRK6 kinase domain is required for survival of MM cells. These findings helped validate that the kinase domain of GRK6 is a promising target for MM, and therefore encouraged us to embark on an effort to identify potent small molecule kinase inhibitors of GRK6. Toward that end, by screening a focused kinase-directed library of small molecule inhibitors, compounds with moderate potency against GRK6 in biochemical assays were identified. Through this exercise, we discovered that two structurally distinct aminopyrazoles AZ-960 and ASC-082 had modest inhibition of GRK6. By combining structural features of these hits and further optimization we identified the quinazoline analogue OICR9945, a potent GRK6 inhibitor with good selectivity against a panel of diverse kinases and anti-proliferative activity against MM cell lines. Herein, we describe the design, synthesis and early pharmacological characterization of OICR9945 along with structure activity relationships (SAR) of this series against GRK6 and other kinases. Citation Format: David Uehling, Babu Joseph, Carly Griffin, Ratheesh Subramaniam, Ayome Abibi, Richard Marcellus, Michael Prakesch, Gennadiy Poda, Methvin Isaac, Chungyee Leung-Hagesteijn, Rodger Tiedemann, Rima Al-awar. Design, synthesis, and characterization of 4-aminopyrazole quinazolines as potent inhibitors of G protein-coupled receptor kinase GRK6 for the potential treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2377.

Ibrutinib targets microRNA-21 in multiple myeloma cells by inhibiting NF-κB and STAT3.

The oncogenic microRNA-21 contributes to the pathogenesis of multiple myeloma. Ibrutinib (also referred to as PCI-32765), an inhibitor of Bruton's tyrosine kinase, while its effects on multiple myeloma have not been well described. Here, we show that microRNA-21 is an oncogenic marker closely linked with progression of multiple myeloma. Moreover, ibrutinib attenuates microRNA-21 expression in multiple myeloma cells by inhibiting nuclear factor-κB and signal transducer and activator of transcription 3 signaling pathways. Taken together, our results suggest that ibrutinib is a promising potential treatment for multiple myeloma. Further investigation of mechanisms of ibrutinib function in multiple myeloma will be necessary to evaluate its use as a novel multiple myeloma treatment.

CYP1A1-Mediated Intramolecular Rearrangement of Aminoazepane in GDC-0339.

GDC-0339 is a novel small molecule pan-Pim kinase inhibitor that was discovered as a potential treatment of multiple myeloma. During the in vitro and in vivo metabolite profiling of GDC-0339, a metabolite was detected that had the same elemental composition as the parent but was distinct with respect to its chromatographic separation and mass spectrometric fragmentation pattern. High resolution tandem mass spectrometry data indicated the metabolite was modified at the aminoazepane moiety. The structure was solved by nuclear magnetic resonance analysis of the isolated metabolite and further confirmed by comparing it to a synthetic standard. These results indicated that the metabolite was formed by an intramolecular amine replacement reaction with the primary amine forming a new attachment to pyrazole without any change in stereochemistry. In vitro experiments showed cytochrome P450s catalyzed the reaction and demonstrated high isoform selectivity by CYP1A1. Results from kinetic experiments showed that the CYP1A1-mediated rearrangement of GDC-0339 was an efficient reaction with apparent turnover number (kcat) and Michaelis constant (Km) of 8.4 minutes-1 and 0.6 μM, respectively. The binding of GDC-0339 to the cytochrome P450 active site was examined by characterizing the direct inhibition of CYP1A1-mediated phenacetin O-deethylation, and GDC-0339 was a potent competitive inhibitor with Ki of 0.9 μM. This high affinity binding was unexpected given a narrow active site for CYP1A1 and GDC-0339 does not conform structurally to known CYP1A1 substrates, which are mostly polyaromatic planar molecules. Further, we explored some of the structural requirements for the rearrangement reaction and identified several analogs to GDC-0339 that undergo this biotransformation.

Abstract 4720: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma

Abstract Introduction: Selinexor is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. XPO1 also mediates the export of key signaling molecules in multiple myeloma (MM) including c-MYC mRNA and NF-κB. Selinexor is currently being investigated in phase 2 clinical trials in MM in combination with dexamethasone, pomalidamide, lenalidomide, bortezomib and carfilzomib. In preclinical studies selinexor has been shown to synergize broadly with these anti-MM drugs making it an excellent candidate partner for combination therapies in MM. To identify additional synergistic pairings, we investigated the use of selinexor in combination with panobinostat, a pan-histone deacetylase inhibitor (HDAC) recently approved by the FDA in combination with bortezomib for 3rd line treatment of MM, as a potential treatment for MM both in-vitro and in-vivo. Methods: The effects of selinexor and panobinostat alone or in combination on cell viability were tested on MM1.S and NCI-H929 cell lines using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, MM.1S cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of selinexor and panobinostat alone or in combination and with the therapeutic dose of selinexor. Tumor growth was monitored for 17 days and% Tumor Growth Inhibition (%TGI) was determined. Xenografts were harvested and analyzed microscopically and by immunohistochemestry (IHC). Results: Selinexor-panobinostat combination was highly effective both in-vitro and in-vivo. In MTT assays, both selinexor and panobinostat demonstrated low IC50 values (nanomolar) and when combined, they synergistically/ additively inhibit proliferation. Gene expression and western blot analyses showed that the combination treatment leads to a synergistic reduction in c-MYC mRNA and protein levels. In addition, an overall reduction in expression of anti-apoptotic signaling molecules including NF-κB and p21 and an increase of expression of pro-apoptotic molecules including cleaved caspase 3 and BAX were observed. Importantly, in-vivo, while%TGI of sub-therapeutic doses of selinexor and panobinostat measured 58% and 52% respectively, the combination showed a synergistic effect, measuring 93%. Importantly this high%TGI exceeded the effect of the therapeutic dose of selinexor alone 79%. Conclusion: Selinexor-panobinostat combination synergizes to induce apoptosis in MM cells and amplifies anti-tumor effect in a MM xenograft model. These data provide rational support for study of selinexor/ panobinostat combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4720.

Cell adhesion induces overexpression of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) and contributes to cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma cells

Previous studies have shown that chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) exerts its anti-apoptotic function in many solid cancers. However, its role in human multiple myeloma (MM) has not been thoroughly elucidated. In this study, we investigate the role of CHD1L in MM. Preliminarily, up-regulation and down-regulation assay verified that CHD1L exerts its anti-apoptotic role through the apoptotic pathway involving caspase-9–caspase-3 apoptotic pathway in MM cells. In addition, we determined that CHD1L expression is increased when MM cells were adhered to fibronectin (FN) or bone marrow stromal cell line HS-5 cells and cell adhesion assay indicated that CHD1L siRNA reversed the high cell adhesion rate. Consistent with the reduced adhesion rate, the cells translated to a compromised cell adhesion-mediated drug resistance (CAM-DR) phenotype in MM. In summary, we will propose strategies for developing a CHD1L inhibitor for potential treatment of MM.

Abstract 2066: Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors.

Abstract Purpose. Despite improved treatments using proteosome inhibitor- and immunomodulator-based (thalidomide) therapies human multiple myeloma (MM) still remains an incurable disease. New therapeutic targets or unique drug-combinations are needed to further improve treatment outcomes. Our purpose was to investigate the use of small-molecule inhibitors of the nuclear export (SINE) in combination with proteosome inhibitors (PI), bortezomib (BTZ) and carfilzomib (CFZ) as a potential treatment for MM both in vitro and ex vivo. Methods. Previously, we reported that blocking nuclear export of topo IIα with SINE sensitized MM cells to topo II poisons. In this study, we used SINE inhibitors developed by Karyopharm Therapeutics, KPT-185, -249 and a new clinical compound KPT-330. In addition we investigated the use of KOS-2464 (BMS), an effective and relatively non-toxic analog of the known CRM1 inhibitor LMB. To test the efficacy of these inhibitors, we treated human MM cell lines, RPMI-8226, U266B1 and NCI-H929 with SINE +/- PI. MM cell lines or MM patient bone marrow aspirates were placed at high-density conditions and treated for 20 hours with KPT-drugs (300 nM) or KOS-2464 (10 nM), either concurrently or sequentially with 10-40 nM BTZ or CFZ. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. Additional experiments were performed to investigate time-course of drug action and dose-dependence. In addition, treated cell lines or CD138+/light chain+ patient MM cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the SINE blocked CRM1-mediated export, treated cells were separated into nuclear and cytoplasmic fractions and assayed for export of p53 and topo IIα. Immunofluorescent microscopy was used to confirm fractionation results. In addition, CRM1 expression levels were measured in all treated cell lines by both Western blot and quantitative PCR. Results. SINE used in combination with PI were highly effective against both MM cell lines and patient MM cells. We found that SINE blocked nuclear export of both p53 and topo IIα as shown by nuclear/cytoplasmic fractionation and also by immunocytochemical staining. Using the CellTiter-Blue assay, we found that all SINE tested had low IC50 values (nanomolar) as single agents and functioned synergistically with BTZ and CFZ (CI <0.5). SINE/PI combination therapy also induced apoptosis in MM cell cultures and patient MM cells (ex vivo), as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions. We found that specific, small-molecule CRM1 inhibitors, sensitized MM cells to BTZ and CFZ as shown by apoptosis and cell viability assays. Sensitization was found to be synergistic. In addition, CRM1 inhibiting activity was verified by nuclear/cytoplasmic fractionation and immunocytochemical staining in MM cells. This combination therapy may be an effective treatment for multiple myeloma. Citation Format: Joel G. Turner, Jana L. Dawson, Christopher L. Cubitt, Michael Kauffman, Sharon Shacham, Daniel M. Sullivan. Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2066. doi:10.1158/1538-7445.AM2013-2066

Abstract 2852: Combination therapy: Galectin-3C and bortezomib potent anticancer activity and inhibition of angiogenesis, chemotaxis, and NF-kB activation in human multiple myeloma

Abstract Introduction Galectin-3 is a human lectin involved in cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Methods We analyzed 9 human MM cell lines tested in vitro for cell proliferation/viability; chemotaxis. The human MM cell lines used in this study were RPMI-8226, U266 (American Type Culture Collection, VA, USA), ARP-1, ARK-B, (gifts from J. Epstein, University of Arkansas for Medical Sciences, Little Rock, AR, USA). The 8226/Dox and 8226/LR-5 cell lines were kindly provided by Dr. William S. Dalton of the H. Lee Moffitt Cancer Center and Research Institute in Tampa, Florida. All MM cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. The 8226/Dox cells were cultured with 40 nM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). The 8226/LR-5 cells were cultured in media containing 5 µM mephalan (Sigma-Aldrich). 8226/Dox and 8226/LR-5 cells were maintained in drug-free medium for 1 week prior to drug sensitivity assays. Human umbilical cord vascular endothelial cells (HUVEC, American Type Culture Collection, VA, USA) were maintained in EGM-2 medium (Lonza, Houston, TX, USA) and were used within 10 passages. Furthermore we used U266 MM model in the NOD/SCID mice to evaluate the efficacy of galectin-3C in single or in combination with BOR. Results Galectin-3C exhibited modest anti-proliferative effects on MM cells in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. Bortezomib, a proteasome inhibitor approved by the F.D.A. for MM treatment, also inhibited the growth of the U266 tumors. Treatment with both agents was more effective than either alone, such that the average tumor volume of the combination group was just 14.0% of controls at day 35. We show that mechanisms by which galectin-3C facilitates the anticancer activity of BOR include inhibition of angiogenesis, chemotaxis, and NF-kB activation. Conclusion In conclusion, our study shows that in a mouse model of human MM, treatment with galectin-3C alone is efficacious, and that it enhances the anti-tumor activity of bortezomib. These findings provide the proof-of-principle to test galectin-3C clinically for human MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2852. doi:1538-7445.AM2012-2852

Galectin-3C Inhibits Tumor Growth and Increases the Anticancer Activity of Bortezomib in a Murine Model of Human Multiple Myeloma

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.

Combining the Ras Inhibitor Salirasib and Proteasome Inhibitors: A Potential Treatment for Multiple Myeloma

Multiple myeloma (MM) is an incurable disease that develops resistance to chemotherapy. New treatments with thalidomide or bortezomib are partially successful. Drug resistance, differentiation block, and increased survival in MM result from genomic alterations, including high overexpression of cyclin D and fibroblast growth factor receptor 3 (FGFR3) and mutations in NRas. Oncogenic Ras isoforms can be inhibited by the Ras inhibitor farnesylthiosalicylic acid (FTS, salirasib), which also inhibits fibroblast growth factor (FGF)-stimulated Ras activation. Here we compared the effects of FTS on the proliferation of NCIH929 (harboring oncogenic NRas) and of two other MM cell lines, MM.1S and U266, which do not harbor oncogenic NRas. NCIH929 responded significantly better than the other cell lines to FTS treatment. FTS also inhibited FGF-stimulated GTP loading of wild-type NRas, and hence ERK activation, in MM-NCIH929. Gene-expression analysis of FTS-treated NCIH929 cells demonstrated downregulation of FGFR3, and the FGFR3 protein in these cells declined after FTS treatment. Combined treatment with FTS and the proteasome inhibitor MG132 or bortezomib yielded synergistic inhibition of NCIH929 MM cell growth. These data strongly suggest that FGFR3 acts together with NRas to activate the MAPK pathway, and that inhibition of Ras by FTS affects both early Ras-dependent signaling and long-term Ras-dependent control of gene expression and protein translation. We suggest that salirasib be considered, both alone and in combination with proteasome inhibitors, as a potential treatment for MM.

Combining the Ras Inhibitor Salirasib and Proteasome Inhibitors: A Potential Treatment for Multiple Myeloma

AbstractAbstract 1810Multiple Myeloma (MM) is characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomide, Bortezomib (Velcade) and Lenalidomide improve response rates and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations, including high cyclin D and fibroblast growth factor receptor 3 (FGFR3) over-expression as well as mutations in NRas. Interactions between myeloma cells and stromal cells in the tumor microenvironment play a major role in MM resistance. Particularly, activation of NF-κB-mediated upregulation of IL-6 secretion by stromal cells is in connection with signal transduction of the Ras oncogene pathway. Activating mutations of Ras have been reported in 30–50% of MM patients. KRas and NRas are the most frequent mutated, suggesting that active Ras is an appropriate target in MM. Development of oncogenic Ras isoforms can be inhibited by Ras inhibitor, farnesylthiosalicylic acid (FTS, salirasib) which also inhibits fibroblast growth factor (FGF)-stimulated Ras activation. We, therefore, compared the effects of FTS on proliferation of NCIH929 (harboring oncogenic NRas) and of two other MM cell lines, MM1.S and U266, which do not harbor oncogenic NRas. Inhibition of cell proliferation was evident by the reduction in BrdU incorporation into the DNA of cells treated for 24 h with FTS (50, 75, or 100 μM) and by counting cells stained with alamarBlue. NCIH929 responded better than the others cell lines to FTS-induced growth inhibition (P<0.05). The IC50 values were 64μM, 82μM and 82–100μM for NCIH929, MM.1S and U266 MM tumor cell lines, respectively (n=3). Treatment with FTS also significantly reduced total Ras and NRas-GTP in NCIH929 but not in the two other cell types, which was accompanied by a significant decrease in the amount of c-Myc (62±2%), p-ERK (38±5%) and p-Akt (52±1.6%) (n=3). All of these proteins are essential for the proliferation, growth, and survival of myelomas. Gene-expression patterns of control and of FTS-treated NCIH929 cells demonstrated down-regulation of FGFR3 (by 2.44 fold) and FGFR3 protein expression declined significantly (36±5%) in these cells after FTS treatment. FTS also inhibited FGF-stimulated GTP loading of wild-type NRas, and hence ERK activation, in MM-NCIH929. These findings suggested that FGFR3 acts together with NRas to activate the MAPK pathway, and also pointing to the possibility that treatment with FTS affected both early Ras-dependent signaling and long-term Ras-dependent control of gene expression and protein translation. Proteasome inhibitors have emerged as powerful tools for inhibiting NFκB activity in myelomas. We therefore examined the combined effect of the proteasome inhibitor MG132 (0.5-2.5 μM) or bortezomib (2.5μM) and the Ras inhibitor FTS (50 or 75 μM) on the growth of NCIH929 cells. Combination of FTS with the proteasome inhibitor MG132 or bortezomib yielded synergistic inhibitory effect (up to 86±6.4%) of NCIH929 MM cell growth (P<0.001; P<0.05, respectively) (n=3). Lastly, we tested the potential inhibitory capabilities of new FTS derivatives including FTS-esters and amides. The FTS-amides exhibited substantially higher activity (50% higher) than FTS itself, while the FTS-esters were completely inactive. In conclusion, the dependence of MM on FGF3R and Ras pathways make them sensitive to Ras inhibitors such as FTS. The synergistic effects of bortezomib and FTS in NCIH929 cells and presumably in MM might be explained by the two distinct pathways that they affect. Based on these results, we suggest that salirasib (FTS) may be considered, both alone and moreover in combination with proteasome inhibitors, as a potential treatment for MM. Disclosures:No relevant conflicts of interest to declare.

Abstract 725: Protein kinase-CK2 inhibition as a potential treatment for multiple myeloma

Abstract Hypothesis Nuclear export of topo IIα to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. Background We have previously demonstrated that topo IIα is exported from the nucleus of human multiple myeloma cells by a CRM1-dependent mechanism at densities similar to those in myeloma patient bone marrow (Engel et al, Exp. Cell Res. 295:421-31, 2004). We have also identified the nuclear export signals for topo IIα at amino acids 1017-1028 and 1054-1066 using mutated full-length FLAG-topo IIα protein and fluorescence microscopy, (Turner et al, J. Cell Sci. 117:3061-71, 2004). In addition, blocking nuclear export with a CRM1 inhibitor or by using CRM1-siRNA sensitizes myeloma cells to topo II poisons (Turner et al. Cancer Res. 69:6899-905, 2009). Therefore, preventing nuclear export may sensitize myeloma cells to topo II inhibitors. Methods To determine what signals CRM1-mediated nuclear export of topo IIα we investigated the phosphorylation status of topo IIα, both in the nucleus and in the cytoplasm, using proteomic technologies. Topo IIα was isolated from the nuclei and cytoplasm of human myeloma cells by immunoprecipitation. Purified topo IIα fractions were analyzed by Orbitrap mass spectroscopy. We found that serine 1524 was highly phosphorylated in the cytoplasmic fraction. Using site-directed mutagenesis we converted serine 1524 to an alanine to determine if mutated FLAG-tagged topo IIα export was reduced as compared to wild-type FLAG-tagged topo IIα. Serine 1524 is a CK2 phosphorylation site, therefore, we tested three recently published and highly specific inhibitors of CK2; quinilizarin, resorufin and hematein. These drugs were used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry. Results Using mass spectroscopy we found that cytoplasmic but not nuclear topo IIα was phosphorylated at serine 1524, a published CK2 motif. Using site-directed mutagenesis we converted serine 1524 to an alanine and found that mutated FLAG-topo IIα export was reduced, as compared to wild-type FLAG- topo IIα. Of the tested CK2 inhibitors, hematein, quinalizarin and resorufin, we found that the CK2 inhibitor quinalizarin (1,2,5,8-tetrahydroxyanthracene-9,10-dione) produced significantly more apoptosis in the nanomolar range than both resorufin and hematein. In addition, we found that quinalizarin prevented nuclear export of topo IIα in high-density cells. These data were duplicated using a CK2 specific siRNA to knockdown CK2 expression (90% knockdown). Conclusion We found that blocking nuclear export of topo IIα with the CK2 inhibitor quinalizarin or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poisons doxorubicin and VP-16. These data may have potential clinical implications in the treatment of multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 725.

CYT997 Causes apoptosis in human multiple myeloma

Multiple Myeloma (MM) is an incurable malignancy of mature plasma cells. Microtubule targeting agents (MTAs) are an established class of drug that include many conventional and some novel compounds. MTAs function by inhibiting the polymerisation or depolymerisation of microtubules (MTs) within the cell, disrupting various important cellular functions. We have investigated pre-clinically the novel tubulin polymerisation inhibitor CYT997 for the potential treatment of MM. Here we demonstrate the promising anti-myeloma activity of CYT997 as evidenced by tubulin disruption, inhibition of growth and proliferation, cell cycle arrest and most importantly apoptosis of both human myeloma cell lines (HMCLs) and primary MM cells using nanomolar drug concentrations. CYT997 also synergises with bortezomib to produce more potent anti-MM activity. These in vitro observations were validated in vivo by the ability of CYT997 to significantly prolong survival in a murine model of aggressive systemic myelomatosis. These findings provide a basis for continuing pre-clinical and clinical investigations into the anti-MM effects of CYT997.