ALBERT

Description: Journal of the American Chemical Society DOI: 10.1021/ja305031y

Description: Abstract 4915 Multiple studies have highlighted the critical role of mutation and loss of p53 function in multiple myeloma (MM) when acquiring a more aggressive phenotype and refractoriness to treatment. Therefore, agents capable of overcoming p53 mutational status are important in the context of MM therapeutics. We have previously reported the in vitro and in vivo anti-MM activity of the multi-targeted small molecule inhibitor RGB-286638. Using a human MM cell xenograft model in SCID mice we demonstrated that RGB-286638 inhibited tumor growth and prolonged survival. Our data confirmed suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes in MM.1S MM cells containing wt-p53, which was correlated with rapid downregulation of Rb phosphorylation, resulting in effective G2/M cell cycle blockage and increased sub-G1phase. RGB-286638 induced dose and time-dependent inhibition of RNA pol II phosphorylation as an early event promptly followed by p53 induction. Moreover, RGB-286638 treatment was associated with p53 phosphorylation at ser 15, indicative of DNA damage followed by apoptosis, evidenced by caspases 8, 9 and 3 cleavage and confirmed by Annexin V/PI staining. All together these data suggested that RGB-286638-induced RNA pol II inhibition triggers cytotoxicity in MM cells via p53-dependent apoptosis. Interestingly, RGB-286638 demonstrated cytotoxic activity even in p53-deficient conventional drug-resistant RPMI 8226/Dox 40 MM cells. RGB-286638 treatment of RPMI 8226/Dox40 MM cells showed increased PARP response associated with enhanced NAD depletion followed by increased ATP consumption. Furthermore, concomitant assessment of RGB-286638-induced ATP depletion versus cytotoxicity demonstrated more than 60% ATP loss preceded cell death in RPMI 8226/Dox40 but not in MM.1S. This data suggests the role of either p53-mediated apoptosis (when active) or PARP-induced NAD/ATP depletion and bioenergetic crisis (when absent). Interestingly, the knockdown of p53 did not rescue MM.1S cells from RGB 286638-induced death, suggesting the existence of alternative p53-independent pathways through which RGB-286638 exerts its cytotoxic activity. Ongoing studies are addressing the molecular effects of p53 silencing in MM cells. In addition, dissecting the mechanism of RGB-286638 p53-independent cytotoxicity in MM cells will provide insights for future therapeutic strategies in patients with aggressive MM and associated mutated/deleted-p53. Disclosures Loferer: GPC Biotech AG: Employment. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Raje:Celgene: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding.

Description: Vitamin C (ascorbic acid, AA) has antioxydative effects and is widely taken as a food supplement. Previous studies have shown that AA inhibits bortezomib-induced cytotoxicity in human cancer cell lines in vitro thought a direct interaction of both agents. Since bortezomib is a standard treatment option in multiple myeloma (MM), we have determined whether daily oral intake of AA could alter anti-MM activities of bortezomib treatment in MM patients. We first examined plasma levels of vitamin C after oral intake of AA 1000 mg (about 15mg/kg). The mean peak plasma value was 135 μmol/l, and the median concentration at steady state in healthy volunteers ranged from 60 to 100 μmol/l. We next determined direct cytoxicity of AA in several MM cell lines, assessed by [3H]-thymidine uptake assay at 24h culture. The growth inhibition of AA at physiological concentration of 125 μM was 20% in MM1S, 15% in OPM1, 0% in RPMI8226, 17% in U266, 13% in OPM2 and 1% in H9299 cells, indicating marginal growth inhibitory effect. Higher concentration of AA (1mM), induced remarkable cytotoxicity (20–99%) in the majority of MM cell lines. To confirm inhibitory effect of AA on bortezomib-induced cell death, we treated RPMI 8226 cells with therapeutic doses of bortezomib (5nM), in the absence or presence of AA. Importantly, AA (125 μM and 250 μM) significantly inhibited bortezomib (5 ηM)-triggered growth inhibition in a dose-dependent fashion in RPMI8226 cells. We also examined the inhibition of proteasome activity by bortezomib in the presence of AA (125 μM), assessed by poly-ubiquitinated protein level in RPMI cells treated with bortezomib (20 ηM, 8h). Consistent with growth inhibition assays, anti-ubiquitin immunoblotting showed significant reduction of ubiquinated protein level in the presence of AA, confirming that AA blocked inhibition of proteasomal degradation of ubiquitineted proteins by bortezomib. Similar results were observed with other antioxidant agents (ie, LNAC). We further examined whether AA blocks other classes of proteasome inhibitors. Although AA strongly inhibited peptide boronate (MG262 and bortezomib), it did not block lactacystin or MG132-triggered proteasomal inhibition. Importantly, AA could not overcome proteasome inhibition by NPI0052, currently under evaluation in clinical trials in MM. These results confirmed that AA only blocked inhibition of proteasome activity and growth of MM cells triggered by peptide boronate proteasome bortezomib. To investigate whether a vitamin C-rich diet affects bortezomib-induced cytotoxicity we used plasma collected from healthy volunteers (n=4) taking 1000 mg AA for 4 consequtive days The mean baseline AA level was 35μmol/l, with peak concentration (120 μmol/l) at 4h after taking AA. Bortezomib treatment (10 ηM, 24h) triggered 67% growth inhibition in RPMI8226 cells, which was significantly abrogated (30%) by vitamin-C enriched plasma. We confirmed that inhibition of 20S proteasome activity by bortezomib was also markedly blocked (52% vs 7%) by those plasma samples used in growth inhibition assay. Specifically accumulation of polyubiquitinated proteins triggered by bortezomib was also reduced by the plasma. Ongoing studies in a human MM cell xenograft mouse model will delineate the inhibitory effect of AA against anti-tumor activities of bortezomib in vivo.

Description: Cyclin dependent kinases (CDKs) and their cyclin complexes play a crucial role in cell cycle control and transcriptional regulation. In multiple myeloma (MM), the abnormal activation of different CDKs and their cyclin partners, especially CDK4/cyclin D1 and CDK6/Cyclin D2, mediate uncontrolled cell cycle progression. Therefore CDKs represent promising novel therapeutic targets for MM. Additionally the cytokine dependent PI3K/Akt signaling pathway mediates growth, survival, drug resistance, migration and cell cycle regulation in MM. Activated Akt in turn phosphorylates downstream target molecules like glycogen synthase kinase (GSK)-3 β impacting growth and survival. Here we investigated the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519 in MM. In vitro kinase assays demonstrated more potent inhibition of CDK 1, 2, 4, 5 and 9 compared to CDK 3, 6, and 7. AT7519 also demonstrated potent inhibitory activity against GSK-3 β. No significant inhibitory effects against other kinases were observed. We next investigated the growth inhibitory effect of AT7519 on MM cell lines. Maximal cytotoxicity was observed in 48 hour culture with IC50 values ranging from 0.5μM (MM.1S, U266) to 4 μM (MM1R). AT7519 was also effective against primary tumor cells from MM patients with no significant cytotoxicity noted in peripheral blood mononuclear cells from healthy volunteers. To delineate the underlying mechanism of cytotoxicity induced by AT7519, cell cycle analysis using PI staining in MM.1S cell line was performed. No significant accumulation of cells in a particular phase of cell cycle was noted; however, AT7519 showed an increased sub-G1 population, indicative of apoptosis, which was confirmed by Annexin V+PI+ staining and associated with caspase-8-9 and -3 cleavage. Importantly, we found that AT7519 markedly inhibited phosphorylation (serine 2 and serine 5 sites) of the carboxyl terminal domain of RNA polymerase II (RNA pol II) within 6 hours of treatment. Non-cell cycle CDKs including CDK9 are responsible for phosphorylation and activation of RNA pol II. Similarly, AT7519 also inhibited phosphorylation of GSK-3β while no significant effects on CDK expression levels were evident at early time points. To investigate whether there was a correlation between inhibition of phosphorylation of GSK-3β and RNA pol II, MM.1S cells were cultured with α-amanitin, a specific inhibitor of RNA pol II. Although phosphorylation of RNA pol II was significantly inhibited, phosphorylation of GSK-3β was not altered by amanitin (10 μM for up to 24 hours). These results suggest that GSK-3β and RNA pol II dephosphorylation at serine 2 and serine 5 may be two independent mechanisms by which AT7519 induces apoptosis in MM cells. Ongoing studies are confirming the role of GSK-3 β in AT7519 induced cytotoxicity of MM cells. Finally, the in vivo efficacy of AT7519 was examined using a xenograft mouse model of human MM. Mice treated with AT7519 demonstrated slower tumor growth compared to the control group without adverse effects. Moreover, AT7519 resulted in a significant prolongation in median overall survival in treated mice (40 days in the treatment group versus 27.5 days in the control cohort, p = 0.0324). In conclusion, these results show significant anti-MM activity of AT7519, and provide the rationale for its clinical evaluation in MM.

Description: Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. We sought to identify the effects of targeting cyclin D1 for the treatment of MM. We first studied gene expression of cyclin D1 in MM cells from 64 patients and normal plasma cells from healthy volunteers as controls (n=24). Cyclin D1 was over-expressed in 42/64 MM patients (65%). To assess the importance of targeting cyclin D1, we next used shRNA lentiviral constructs to silence cyclin D1 in INA6 and MM.1S MM cells. Our data demonstrated 〉 50% of apoptotic cell death in cyclin D1 shRNA versus control transfected MM cells, indicating that small molecule cyclin dependent kinase inhibitors may provide a valuable therapeutic tool to specifically target cyclin D1. We next evaluated a clinical grade small molecule cyclin D1 specific inhibitor P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally, in vitro kinase assays against a broad range of other kinases have also confirmed its specificity at nanomolar concentrations for D1 and B cyclins. P276-00 treatment of MM cell lines and patient derived cells induced both time and dose dependent in vitro growth inhibition in a broad range of MM cells sensitive and resistant to conventional agents including dexamethasone, doxorubicin, and melphalan, with IC50 ranging from 400–800nM. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells. Apoptosis was, at least in part, caspase-dependent, since cytotoxicity was partially reversed by Z-VAD Fmk. P276-00, in a dose and time dependent fashion inhibited Rb-1 phosphorylation as early as 6 hours associated with down-regulation of cdk4, suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceded growth arrest and apoptosis in MM cells. Proliferative stimuli such as IL-6, IGF-1, and adherence to bone marrow stromal cells induced cyclin D1 and B cyclins, contributing to the development of drug resistance; P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Mice were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirmed in vivo anti-tumor activity of P276-00, evidenced by a significant decrease in biluminesence of GFP+ MM cells (p

Description: 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.

Description: Multiple studies have shown the importance of PI3K/Akt/mTOR pathway in the pathogenesis of multiple myeloma (MM). Specifically, it mediates cell proliferation and development of drug resistance. Identifying mTOR as a key kinase acting downstream of Akt led to the prediction that rapamycin, a universal inhibitor of mTORC1-dependent S6K1 phosphorylation, may be useful in the treatment of MM. Cumulative evidence support the hypothesis that rapamycin-induced cytotoxicity is predominantly triggered as a consequence of autophagy (programmed cell death type II) via excessive cell digestion. Therefore activated Akt can be a key upstream inhibitor of two cell death-inducing events: autophagy via mTOR activation and apoptosis via phosphorylation of BAD and inhibition of the catalytic subunit of caspase-9. Importantly, recent studies have demonstrated the existence of strong positive feedback loops from mTOR/S6K1 to Akt, resulting in Akt activation in some cancer types. We confirmed that suppression of mTOR signaling by rapamycin was associated with upregulation of Akt phosphorylation, which may reduce sensitivity to rapamycin. We therefore hypothesized that inhibition of this positive feedback by a potent Akt inhibitor perifosine could augment rapamycin-induced cytotoxicity in MM cells. As expected, perifosine inhibited rapamycin-induced p-Akt, associated with synergistic cytotoxicity, even in the presence of IL-6, IGF-1 or bone marrow stromal cells. Synergism was confirmed by the Chou-Talalay model, demonstrating that a combination index was 〈 1 over a range of doses evaluated. We further examined the molecular mechanisms whereby this combination treatment induced synergistic cytotoxicity. We first studied its effect on ultrastructure in MM cells assessed by electron microscopy, demonstrating that rapamycin-induced autophagy was augmented by perifosine in MM.1S cells. We also confirmed autophagy by immunofluorescent staining of LC3, an autophagosome membrane-associated protein. Interestingly, both autophagy and apoptosis were observed when cells were cultured for longer periods with rapamycin and perifosine co-treatment. The apoptotic effect of the combination was further assessed by Annexin/PI flow cytometric analysis and caspase/PARP cleavage. Neither inhibition of autophagy by 3-MA, nor the blockade of apoptosis by Z-VAD-FMK rescued MM cell death from the combination treatment. Finally, we demonstrate that the combination of nanoparticle albumin-bound (nab) -rapamycin and perifosine had significant antitumor activity in an in vivo human MM cell xenograft murine model, associated with prolonged survival. We observed pAkt upregulation induced by rapamycin, which was inhibited by perifosine evidenced by immunohistochemical analyses on tumor tissue from the xenograft model. Importantly, autophagy and apoptosis were also confirmed by LC3 and caspase 3 staining on tumor tissue. Collectively our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine co-treatment induces autophagy and apoptosis, resulting in synergistic cytotoxicity in MM, providing the rationale for use of this combination in future clinical trial in patients with MM.

Description: Constitutive B-cell lymphoma 6 (Bcl-6) expression was undetectable in multiple myeloma (MM) cell lines, except U266 cells. However, it was up-regulated by coculture with bone marrow (BM) stromal cell-culture supernatant (SCCS). Bcl-6 expression in patient MM cells in the BM was positive. Anti–interleukin-6 (IL-6)–neutralizing antibody significantly blocked SCCS-induced Bcl-6 in MM cells. Indeed, IL-6 strongly triggered Bcl-6 expression in MM cells, whereas Janus kinase inhibitor and STAT3 siRNA down-regulated Bcl-6. Tumor necrosis factor-α (TNF-α) also triggered Bcl-6, but independently of STAT3, whereas IκB kinaseβ inhibitor down-regulated TNF-α–induced Bcl-6, indicating that the canonical nuclear factor-κB pathway mediates TNF-α–induced Bcl-6 expression. Importantly, down-regulation of Bcl-6 by shRNA significantly inhibited MM cell growth in the presence of SCCS. Our results therefore suggest that Bcl-6 expression in MM cells is modulated, at least in part, via Janus kinase/STAT3 and canonical nuclear factor-κB pathways and that targeting Bcl-6, either directly or via these cascades, inhibits MM cell growth in the BM milieu.

Description: Nuclear factor-κB (NF-κB) has an important role in multiple myeloma (MM) cell pathogenesis in the context of the bone marrow (BM) microenvironment. In NF-κB signaling cascades, IκB kinase α (IKKα) and IKKβ are key molecules that predominantly mediate noncanonical and canonical pathways, respectively. In this study, we examined the biologic sequelae of the inhibition of IKKα versus IKKβ in MM cell lines. All MM cell lines have constitutive canonical NF-κB activity, and a subset of MM cell lines shows noncanonical NF-κB activity. Adhesion to BM stromal cells further activates both canonical and noncanonical NF-κB activity. IKKβ inhibitor MLN120B blocks canonical pathway and growth of MM cell lines but does not inhibit the noncanonical NF-κB pathway. Although IKKα knockdown induces significant growth inhibition in the cell lines with both canonical and noncanonical pathways, it does not inhibit NF-κB activation. Importantly, IKKα down-regulation decreases expression of β-catenin and aurora-A, which are known to mediate MM cell growth and survival. Finally, IKKβ inhibitor enhances the growth inhibition triggered by IKKα down-regulation in MM cells with both canonical and noncanonical NF-κB activity. Combination therapy targeting these kinases therefore represents a promising treatment strategy in MM.

Description: Heat-shock protein 90 (Hsp90) acts as a molecular chaperone required for maintaining the conformational stability of client proteins regulating cell proliferation, survival, and apoptosis. Here we investigate the biologic significance of Hsp90 inhibition in multiple myeloma (MM) and other hematologic tumors using an orally available novel small molecule inhibitor SNX-2112, which exhibits unique activities relative to 17-allyamino-17-demethoxy-geldanamycin (17-AAG). SNX-2112 triggers growth inhibition and is more potent than 17-AAG against MM and other malignancies. It induces apoptosis via caspase-8, -9, -3, and poly (ADP-ribose) polymerase cleavage. SNX-2112 inhibits cytokine-induced Akt and extracellular signal-related kinase (ERK) activation and also overcomes the growth advantages conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cells. Importantly, SNX-2112 inhibits tube formation by human umbilical vein endothelial cells via abrogation of eNOS/Akt pathway and markedly inhibits osteoclast formation via down-regulation of ERK/c-fos and PU.1. Finally, SNX-2112, delivered by its prodrug SNX-5422, inhibits MM cell growth and prolongs survival in a xenograft murine model. Our results indicate that blockade of Hsp90 by SNX-2112 not only inhibits MM cell growth but also acts in the bone marrow microenvironment to block angiogenesis and osteoclastogenesis. Taken together, our data provide the framework for clinical studies of SNX-2112 to improve patient outcome in MM and other hematologic malignancies.