Inhibition of Hsp90 Targets Multiple Myeloma Cell Growth, Angiogenesis, and Osteoclastogenesis in the BM Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2522-2522
Author(s):  
Yutaka Okawa ◽  
Teru Hideshima ◽  
Hiroshi Ikeda ◽  
Sonia Vallet ◽  
Tanyel Kiziltepe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Small Molecule ◽  
Cell Apoptosis ◽  
Osteoclast Formation ◽  
Erk Signaling ◽  
Client Proteins

Abstract Heat-shock protein 90 (hsp90) is a important molecular chaperone required for protein folding, assembly, and maintenance of conformational integrity of several client proteins regulating cell survival, proliferation, and apoptosis. In this study, we investigate whether targeting hsp90 induces cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) microenvironment using the small molecule hsp90 inhibitor SNX-2112. SNX-2112 induces growth inhibition in both MM cell lines and patient MM cells resistant to conventional therapeutic agents, with IC50 of 0.019-7.339 uM. Interestingly, SNX-2112 is more potent against all MM cell lines than 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and orally bioavailable. These data suggest its potential utility to overcome conventional drug resistance in MM. MM cell apoptosis triggered by SNX-2112 is mediated via caspase-8,-9,-3 and PARP cleavage. In addition, SNX-2112 significantly inhibits Akt and extracellular signal-related kinase (ERK) signaling pathways induced by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), which mediate cell proliferation and survival in MM cells, both known client proteins of hsp90. Moreover, SNX-2112 overcomes the survival and growth advantages conferred by IL-6, IGF-1 and bone marrow stromal cells (BMSC). Importantly, SNX-2112 inhibits in vitro capillary-like tube formation by human umbilical vein endothelial cells (HUVEC) via abrogation of endothelial nitric oxide (eNOS)/Akt pathway which is essential cascade in angiogenesis. It also markedly inhibits osteoclast formation associated with downregulation of ERK/c-fos, p38MAPK, and PU.1 pathways, which mediate osteoclastogenesis. Furthermore SNX-5422, a prodrug of SNX-2112, significantly inhibits MM tumor growth and prolongs survival in vivo in a xenograft murine model. We also confirm that SNX-5422 triggers MM cell apoptosis in vivo by TUNEL assay, associated with an inhibitory effect on microvessel density (MVD), evidenced by immunohistochemical analysis for CD34 expression. Therefore, targeting hsp90 by novel small molecule inhibitor SNX-2112 not only inhibits MM cell growth, but also acts in the BM microenvironment to block angiogenesis and osteoclast formation associated with downregulation of Akt and ERK signaling. Taken together, our data provide the preclinical rationale for clinical evaluation of SNX-2112 to improve patient outcome in MM.

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2056-2056 ◽  
Author(s):  
Chantiya Chanswangphuwana ◽  
Michael P. Rettig ◽  
Walter Akers ◽  
Deep Hathi ◽  
Matthew Holt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Molecular Diagnostic ◽  
Fluorescent Intensity ◽  
Variable Expression ◽  
Rpmi 8226

Abstract Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM. Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study. Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection. Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. Disclosures No relevant conflicts of interest to declare.

Centrosomal Clustering – a Novel Therapeutic Target for Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 300-300 ◽  
Author(s):  
Marc S Raab ◽  
Iris Breitkreutz ◽  
Blanka Rebacz ◽  
Thomas O Larsen ◽  
Ludmila Wagner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Malignant Cells ◽  
Multipolar Spindles

Abstract Abstract 300 The lack of tumor-specific targets that would allow for the selective eradication of malignant cells without affecting healthy tissues is a major challenge in the development of novel therapies for multiple myeloma (MM). In contrast to normal cells, malignant plasma cells frequently contain multiple centrosomes, associated with the transient formation of multipolar mitotic spindles that lead to segregation defects and chromosomal instability. As in most tumor types, mitotic stability in these cells is maintained by coalescence of multiple centrosomes into two functional spindle poles, termed “centrosomal clustering”. As we have recently shown, this mechanism is an attractive therapeutic target with specificity for tumor cells. To identify potent and selective inhibitors of centrosomal clustering, we performed a phenotype-based small molecule screen in order to force tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. We here describe the characterization of a novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis in MM cells. We tested a wide array of MM cell lines, including those resistant to conventional chemotherapeutic agents, and primary patient cells. We found mean inhibitory concentrations (IC50) of proliferation and survival in the range of 1-5 mM, associated with annexin V conversion and activation of caspases 8, 9, and 3. Importantly, GF-15 also overcomes the tumor cell growth advantage conferred by both bone marrow stromal cell-MM, and endothelial cell-MM, co-culture systems. Moreover, non-malignant cells without supernumerary centrosomes like activated PBMCs, immortalized hepatocytes, and bone marrow stromal cells did not reach their IC50 at doses of up to 50 mM. To further demonstrate the specificity of GF-15, we generated resistant MM cell lines by long-term culture with sub-IC50 doses of GF-15. In resistant cell lines, therapeutic doses of GF-15 no longer induce multipolar spindles, consistent with a significant loss of centrosomal aberrations in these cells, as observed by immunoflourescence microscopy. Mechanistically, cell cycle analysis of synchronized MM cells showed marked G2/M arrest within 12-16h followed by a dramatic increase of the sub-G1 fraction after treatment with GF-15. In addition, short term treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered MM cell migration. Co-treatment assays to assess potential partners for therapeutic combinations revealed at least additive effects for GF-15 together with bortezomib and marked synergism with paclitaxel at very low doses (1-5 nM), while the combination with melphalan resulted in antagonistic effects due to the S-phase arrest of tumor cells induced by melphalan. Finally and most importantly, i.p. as well as oral treatment of murine xenograft models of human MM resulted in tumor growth inhibition and significantly prolonged survival in vivo. Growth inhibition of xenograft tumor samples was associated with a dramatic increase of mitotic aberrations and multipolarity, as assessed by immunohistochemistry. In vivo biodistribution studies are ongoing. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a prototype small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and therefore strongly support its further evaluation and development as a lead compound of a new class of therapeutics for human malignancies. Disclosures: No relevant conflicts of interest to declare.

Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4080-4080
Author(s):  
Yuhuan Zheng ◽  
Jing Yang ◽  
Liang Zhang ◽  
Jianfei Qian ◽  
Jairo Matthews ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Compound A

Abstract Abstract 4080 Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM. Disclosures: No relevant conflicts of interest to declare.

ARRY-797, a Potent and Selective Inhibitor of p38 Map Kinase, Inhibits LPS-Induced IL-6 and In Vivo Growth of RPMI-8226 Human Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3478-3478
Author(s):  
Dale Wright ◽  
Shannon L. Winski ◽  
Deborah Anderson ◽  
Patrice Lee ◽  
Mark Munson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Small Molecule ◽  
Single Agent ◽  
Tnf Alpha ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

KRN5500, a Spicamycin Derivative, Exerts Anti-Myeloma Effects through Impairing Both Myeloma Cells and Osteoclasts.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1669-1669
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shingen Nakamura ◽  
Ayako Nakano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Osteolytic Bone Disease ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by devastating bone destruction due to enhanced bone resorption and suppressed bone formation. Although high-dose chemotherapy and new agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains incurable due to drug resistance mediated by interactions with osteoclasts or stroma cells. Moreover, osteolytic bone disease continues to be a major problem for many patients. Therefore, alternative approaches are necessary to overcome drug resistance and inhibit osteoclasts activity in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan), which potently inhibits protein synthesis and induces cell death in human tumor cell lines. Phase I studies of KRN5500 in patients with solid tumors such as colon cancer and gastric cancer showed acceptable toxicity with Cmax values of 1000––3000 nM. In this study, we investigated the effects of KRN5500 against MM cells and osteoclasts in vitro and in vivo. MM cell lines such as RPMI 8226, MM.1S, INA-6, KMS12-BM, UTMC-2, TSPC-1, and OPC were incubated with various concentrations of KRN5500 for 3 days. Cell proliferation assay showed marked inhibition of cell growth with G1 arrest in these MM cells (IC50: 4–100 nM). KRN5500 (100 nM) also induced 30–90% of cell death in primary MM cells (n=7). Annexin V/propidium iodide staining showed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Western blot analysis confirmed activation of caspase-8, -9, and −3, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of Mcl-1. We next examined the effect of KRN5500 against MM cell lines and primary MM cells in the presence of bone marrow stroma cells and osteoclasts. Co-culture of these cells enhanced viability of MM cells; however, KRN5500 still induced strong cytotoxicity to MM cells. Of interest, KRN5500 specifically mediated apoptosis in osteoclasts but not stroma cells as assessed by TUNEL staining. More than 90% of osteoclasts were killed even at a low concentration of KRN5500 (20 nM). Finally, we evaluated the effect of KRN5500 against MM cells and osteoclasts in vivo. Two xenograft models were established in SCID mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells into subcutaneously implanted rabbit bones (SCID-rab model). These mice were treated with intraperitoneal injection of KRN5500 (5 mg/kg/dose) or saline thrice a week for 3 weeks after tumor development. In a subcutaneous tumor model, KRN5500 inhibited the tumor growth compared with control mice (increased tumor size, 232 ± 54% vs 950 ± 422%, p&lt;0.001, n=6 per group). In a SCID-rab model, KRN5500 also inhibited MM cell growth in the bone marrow (increase of serum human sIL6-R derived from INA-6, 134 ± 19% vs 1112 ± 101%, p&lt;0.001, n=5 per group). Notably, the destruction of the rabbit bones was also prevented in the KRN5500-treated mice as evaluated by radiography. Therefore, these results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts and that this unique mechanism of action provides a valuable therapeutic option to improve the prognosis in patients with MM.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

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