Human Multiple Myeloma Cell Growth Research Articles

Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma.

Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.

APRIL Is Significantly Elevated at All Stages of Multiple Myeloma (MM) and Interferes with Anti-Bcma Monoclonal Antibody-Mediated Cytolysis, Supporting the Clinical Evaluation of Bion-1301 As a Novel Therapeutic Approach in MM

A proliferation inducing ligand (APRIL) is a natural ligand with higher affinity than BAFF for both B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), which are overexpressed on multiple myeloma (MM) cells. APRIL, which is abundantly secreted by myeloma-supporting osteoclasts and macrophages, promotes MM cell progression in vivo and further induces regulatory T cells (Treg) via TACI, but not BCMA, to promote an immunosuppressive MM bone marrow (BM) microenvironment (Blood 2017;130:3066). In preclinical studies, an antagonistic APRIL monoclonal antibody (mAb) significantly inhibited human MM cell growth in SCID-hu mice and abrogated APRIL-induced immunosuppression mediated by Tregs. Here we characterized the ability of BION-1301, a neutralizing APRIL mAb, currently under clinical development in MM (NCT03340883) to overcome protection conferred by APRIL against MM cell lysis induced by the anti-BCMA J6M0 mAb (Blood 2017 130:499).Our studies show that APRIL protects against MM cell lysis induced by J6M0 in the presence of FcR-expressing effector cells (PBMC, monocytes or NK), an effect which is blocked by neutralizing anti-APRIL mAb. APRIL also downregulates J6M0-induced cell membrane CD107a expression on NK cells co-cultured with BCMA-expressing MM cells, which is similarly abrogated by anti-APRIL mAb. Importantly, anti-APRIL mAb also significantly decreases protection conferred by osteoclasts against MM cell lysis induced by J6M0. These data indicate that blocking APRIL with anti-APRIL mAb may enhance BCMA mAb targeted (J6M0)-induced MM cell lysis. Next, using anti-human IgG1 to detect J6M0 binding to MM cell surface BCMA, we found that APRIL in a dose-dependent manner directly competes with J6M0 for binding to BCMA, which was confirmed by ELISA. In addition, APRIL effectively inhibits J6M0 binding to BCMA at 4°C, which argues against APRIL-induced BCMA receptor shedding and/or internalization. In contrast, BAFF affects J6M0 binding to BCMA only at higher concentrations (>1 µg/mL), consistent with > 2-log higher affinity of APRIL vs. BAFF for BCMA.We further assessed APRIL, BAFF, BCMA, and TACI levels in the serum of patients with MM at various stages of disease. Specifically, we used ELISA to measure free APRIL and the ELLA automated immunoassay platform to determine the levels of soluble BAFF, soluble BCMA, and soluble TACI in serum samples from patients with MM (n=193) as well as serum samples from healthy volunteers (HV, n=100). Patient samples included monoclonal gammopathy of undetermined significance (MGUS, n=12), smoldering MM (SMM, n=20), newly diagnosed MM (ND, n=39), post induction pre-autologous stem cell transplant (ASCT, n=55), post-ASCT (n=6), and relapsed refractory MM (RR, n=61). We found that free APRIL levels are significantly increased in serum samples from patients with MM at all stages of disease, when change from baseline levels were compared to those from HVs (post-ASCT: p=0.0003; other groups (MGUS, SMM, ND, pre-ASCT, and RR): p<0.0001). In contrast, average soluble BAFF levels gradually increase with progression of disease from 818.47 pg/mL at MGUS to 1685.50 pg/mL at RR MM. Both soluble BCMA and soluble TACI concentrations are highest in serum from a subset of patients with ND (593550 and 5999.63 pg/mL, respectively) and RR (1088897 and 5050.97 pg/mL, respectively) MM. A detailed analysis of clinical characteristics, treatment and response, as well as measurement of additional cytokines and chemokines is ongoing and will be reported when applicable.Our studies therefore indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-induced immunosuppression, and that combination strategies targeting APRIL with BCMA directed therapy may augment anti-MM activity. Moreover, elevated free APRIL serum levels in MGUS and all stages of MM suggest a role for APRIL in mediating immunosuppression during the development and in the pathogenesis of MM. DisclosuresDulos:Aduro Biotech: Employment. Guelen:Aduro Biotech Europe: Employment. van Zandvoort:Aduro Biotech Europe: Employment. van de Wiel:Aduro Biotech Europe: Employment. van de Crommert:Aduro Biotech Europe: Employment. Lejeune:Aduro Biotech Europe: Employment. Namini:Aduro Biotech: Employment. Eenennaam:Aduro Biotech Europe: Employment, Equity Ownership. van Elsas:Aduro Biotech Europe: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director. Anderson:Takeda Millennium: Consultancy; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Oncopep: Equity Ownership.

Caffeic acid phenethyl ester exerts apoptotic and oxidative stress on human multiple myeloma cells.

Caffeic acid phenethyl ester (CAPE) is a phenolic compound initially identified in bee glue. CAPE is reported to exhibit antitumor activity in many cancer models. However, the effect of CAPE on multiple myeloma (MM) is not well studied. We investigated the anti-myeloma effect of CAPE, and the data showed that CAPE inhibited the growth of human MM cells in a dose (1 ~ 30μM) and time (24 ~72h) dependent manner without altering the viability of normal human peripheral blood B cells. Stress and toxicity pathway analysis demonstrated that CAPE, in a dose- and time-related fashion, induced the expression of apoptotic and oxidative stress-response genes including growth arrest and DNA-damage inducible, alpha and gamma (GADD45A and GADD45G) and heme oxygenase-1. Apoptosis of MM cells by CAPE was further confirmed through flow cytometric analysis with up to 50% apoptotic cells induced by 50μM CAPE within 24h. Western blot analysis revealed the CAPE-induced activation of apoptosis executioner enzyme caspase-3, and corresponding cleavage of its downstream target poly(ADP-ribose)polymerase (PARP). The oxidative stress caused by CAPE cytotoxicity in MM cells was evaluated through measurement of reactive oxygen species (ROS) level, antioxidant intervention and glutathione depletion. The intracellular ROS level was not elevated by CAPE, but the pretreatment of antioxidant (N-acetyl cysteine) and glutathione synthesis inhibitor (buthionine sulfoximine) suggested that CAPE may cause oxidative stress by decrease of intracellular antioxidant level rather than over production of ROS. These data suggest that CAPE promotes apoptosis through oxidative stress in human multiple myeloma cells.

A clinically relevant in vivo zebrafish model of human multiple myeloma to study preclinical therapeutic efficacy

AbstractPatient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138+ MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138+ plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples.

Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.

Molecular Chaperone gp96 Is a Novel Therapeutic Target of Multiple Myeloma

gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro. We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay. Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically. gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 19(22); 6242-51. ©2013 AACR.

Promotion of human multiple myeloma cell growth in vitro and bone marrow invasion in vivo by Notch receptors and the CXCR4/SDF1 axis.

8591 Background: Multiple myeloma (MM) originates from post-germinal center B cells, and is caused by malignant plasma cells accumulating in the bone marrow. Interactions of MM cells with the bone marrow stroma promote tumor growth, migration and drug resistance. The chemokine receptor CXCR4 and its ligand SDF1 are critical regulators of this process. MM cells frequently hyper-express CXCR4 and respond to SDF1,2 enhancing MM cell infiltration, proliferation and osteolysis. Notch receptors similarly promote MM cell growth, drug resistance and the associated osteolytic process. We hypothesized that the CXCR4/SDF1 axis mediates the effects of Notch signals in MM. Methods: We used real-time PCR, flow-cytometry, E.L.I.S.A. and chemotaxis assay to explore the effects of CXCR4 in cultured human MM cell lines after Notch inhibition or over-stimulation. Additionally, we validated our findings in a NOD/SCID murine model xenografted with human MM cells. Results: Our results show that Notch blocking reduced CXCR4 and SDF1 expression by MM cells. Further, Notch activation was required for MM cell chemotactic and proliferative response to SDF1 in vitro. We then investigated the outcome of anti-Notch treatment on human MM cells bone invasion in NOD/SCID mice. Interfering with Notch activity dramatically reduced xenografted MM cell ability to infiltrate the bone marrow, ultimately resulting in diminished tumor burden. Notably, such effect was associated with a decrease of CXCR4 expression. Conclusions: This was the first time that Notch receptors were reported to regulate the CXCR4/SDF1 axis and bone marrow invasion in human MM. These findings indicate that specific Notch-tailored therapies may effectively hamper CXCR4-mediated bone infiltration and associated lesions, and are expected to significantly improve treatment outcome and survival.

A Clinically Relevant Zebrafish in Vivo Model of Human Multiple Myeloma (MM) to Study Disease Biology and Preclinical Therapeutic Efficacy

Abstract 4078Although a number of murine models of human MM have been developed and the SCID-hu murine model allows for growth of primary MM cells, these models have significant limitations including need for insertion of human bones, generation of limited number of mice with human MM Growth and over 2 months lag period, highlighting the need for a model that allows growth of human MM cells rapidly and in larger number of animals. We here describe a zebrafish model which can sustain human patient MM cell growth and be able to test efficacy of therapeutic agents. In this model we have utilized Casper fish, which are transparent and hence ideal for in vivo observation of tumor, Moreover, their 48 hours post fertilization (hpf) stage embryos are immune deficient allowing growth of xenogeneic human MM cells. We have tested growth of both MM cell lines as well as primary patient cells. About 50–200 cells from MM cell lines or CD138+ plasma cells from MM patient bone marrow samples labeled by CM-Dil were injected into the abdominal perivitelline space of 48 hpf stage Casper fish larvae and observed tumor progression, including tumor size and cell invasion by fluorescence microscopy at 24, 48 and 72 hrs post injection. MM cells were confirmed by immunohistochemistry. We observe over 80% larvae demonstrating MM cell survival for up to 9 days. As we require very small number of cells we were able to inject and observe growth of primary cells from all the patients producing over 50 larvae, We next treated the MM cells in vivo by adding first common anti-MM agents into fish water through 24 hrs to 72 hrs post injection (hpi) and observed change in tumor cell survival by fluorescence microscopy again at 24, 48 and 72 hours post treatment (hpt). We have here evaluated response of MM cell lines and MM primary patient cells to single or combination drug treatment, and defined response. We have observed response of the traditional and novel agents as predicted. For example, we have confirmed the response of dexamethasone to MM1.S and MM1.R cell lines which are sensitive or resistant to dexemathasone in this model. We observed that 100 nM dexemathasone treatment led to observation of MM cell survival, and found response ratio of MM1.S and MM1.R xenograft tumors to were 37.5% vs 87.5% fish at 24 hpt and 26.7% vs 87.5% at 48 hpt respectively. We have confirmed efficacy of bortezomib as well as lenalidomide in this model using MM cell lines as well as observed efficacy against primary MM cells. Importantly, we have been able to confirm similar drug resistance profile as patients; for example cells from a patient with bortezomib resistance survived bortezomib treatment in this model. Any combination treatment produced higher response than single drug treatment indicating that MM xenograft zebrafish model can predict drug response accurately. The zebrafish larvaes tolerated all the treatment at the dose utilized well. In conclusion, we have characterized a highly-reproducible zebrafish model of human myeloma that supports primary myeloma cell growth using very small number of cells and can allow generation of large number of fish with evalution of drug activity in a very short time of 3–5 days. This model provides a powerful tool to evaluate preclinical efficacy of novel agents and combinations and may provide opportunity to evaluate personalized therapy. Disclosures:No relevant conflicts of interest to declare.

The effect of selective inhibition of HDAC6 with ACY1215 on bortezomib activity in multiple myeloma (MM).

e18569 Background: HDACs are being studied as novel therapeutic targets in MM. Here, we investigated the preclinical activity of an HDAC6 selective inhibitor ACY1215 in MM, alone and in combination with bortezomib (BZ). Methods: In vitro enzyme assays were used to evaluate the selectivity of ACY1215. The cytotoxicity of ACY1215 versus pan-HDAC inhibitor (e.g. SAHA) were compared in MM cell lines, MM patient cells, MM osteoblasts (OB) and osteoclasts (OC), as well as peripheral blood mononuclear cells (PBMCs) from healthy donors. Western blotting and RT-PCR were used to identify the mechanisms of cell death. The activity of ACY1215 and BZ was confirmed in vivo using two different murine xenograft models of human MM, plasmacytoma model and disseminated MM model. Results: ACY1215 showed more potent inhibitory activity against HDAC6 (EC50 0.0054 µM) than SAHA. Like SAHA, ACY1215 triggered potent MM cytotoxicity but showed less toxicity than SAHA against PBMCs and activated T-cells. Low doses of ACY1215 with BZ showed synergistic anti MM activity, resulting in apoptosis via caspase-8,-9,-3 and PARP activation. Moreover, ACY1215 increased BZ-induced ER stress in MM cells, evidenced by induction of UPR components including PERK, CHOP and p-IRE1α. ACY1215, alone and with BZ, enhanced OB differentiation (increased alkaline phosphatase enzyme activity) and increased mRNA expression of β-catenin, osteocalcin, and Runx2 (OB markers). Importantly, both treatments inhibited OC differentiation (reduced number of multinucleated cells staining for TRAP). In vivo studies confirmed decreased human MM cell growth and prolonged host survival after combination ACY1215 and BZ therapy versus controls (34 vs 22 days, n=7, p<0.0011, in plasmacytoma model and 40 vs 17 days, n=12, p<0.0001, in disseminated model), without significant adverse effects. PK/PD studies showed ACY1215 plasma levels (2133 ng/ml) at 1 hr following treatment. Immunohistochemistry on excised human MM demonstrated increased in acetylated-α tubulin, a biomarker of HDAC6 inhibition, stained cells 4 hrs after ACY1215 treatment. Conclusions: These results provide the rationale for clinical evaluation of ACY1215, alone and together with BZ, in the treatment of MM.

Targeting MEK induces myeloma-cell cytotoxicity and inhibits osteoclastogenesis

AbstractActivation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade mediates human multiple myeloma (MM) growth and survival triggered by cytokines and adhesion to bone marrow stromal cells (BMSCs). Here, we examined the effect of AZD6244 (ARRY-142886), a novel and specific MEK1/2 inhibitor, on human MM cell growth in the bone marrow (BM) milieu. AZD6244 blocks constitutive and cytokine-stimulated ERK1/2 phosphorylation and inhibits proliferation and survival of human MM cell lines and patient MM cells, regardless of sensitivity to conventional chemotherapy. Importantly, AZD6244 (200 nM) induces apoptosis in patient MM cells, even in the presence of exogenous interleukin-6 or BMSCs associated with triggering of caspase 3 activity. AZD6244 sensitizes MM cells to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. AZD6244 down-regulates the expression/secretion of osteoclast (OC)–activating factors from MM cells and inhibits in vitro differentiation of MM patient PBMCs to OCs induced by ligand for receptor activator of NF-κB (RANKL) and macrophage-colony stimulating factor (M-CSF). Finally, AZD6244 inhibits tumor growth and prolongs survival in vivo in a human plasmacytoma xenograft model. Taken together, these results show that AZD6244 targets both MM cells and OCs in the BM microenvironment, providing the preclinical framework for clinical trials to improve patient outcome in MM.

Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x107 pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V+ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG0/G1 compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.

Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) inhibits human multiple myeloma cell growth in the bone marrow milieu in vitro and in vivo

AbstractAzaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine; trade name, Atiprimod) is an orally bioavailable cationic amphiphilic compound that significantly inhibits production of interleukin 6 (IL-6) and inflammation in rat arthritis and autoimmune animal models. We here characterize the effect of atiprimod on human multiple myeloma (MM) cells. Azaspirane significantly inhibited growth and induced caspase-mediated apoptosis in drug-sensitive and drug-resistant MM cell lines, as well as patient MM cells. IL-6, insulin-like growth factor 1 (IGF-1), or adherence of MM cells to bone marrow stromal cells (BMSCs) did not protect against atiprimod-induced apoptosis. Both conventional (dexamethasone, doxorubicin, melphalan) and novel (arsenic trioxide) agents augment apoptosis induced by atiprimod. Azaspirane inhibits signal transducer activator of transcription 3 (STAT3) and a PI3-K (phosphatidylinositol 3-kinase) target (Akt), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2), inhibits phosphorylation triggered by IL-6, and also inhibits inhibitorκBα (IκBα) and nuclear factor κB (NFκB) p65 phosphorylation triggered by tumor necrosis factor α (TNF-α). Of importance, azaspirane inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BMSCs triggered by MM cell binding and also inhibits angiogenesis on human umbilical vein cells (HUVECs). Finally, azaspirane demonstrates in vivo antitumor activity against human MM cell growth in severe combined immunodeficient (SCID) mice. These results, therefore, show that azaspirane both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for clinical trials to improve patient outcome in MM.

Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells, inhibits binding of MM cells in the bone marrow microenvironment, and inhibits cytokines mediating MM cell growth, survival, drug resistance, and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly, PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH(2)-terminal kinase (JNK), caspase-8, and caspase-3; and cleaving the DNA protein kinase catalytic subunit, ATM, and MDM2. Inhibition of JNK activity abrogates PS-341-induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation, more targeted therapies.

Novel therapies for multiple myeloma.

Novel therapies for multiple myeloma.

Novel therapies targeting the myeloma cell and its bone marrow microenvironment

Novel therapies in multiple myeloma (MM) target not only the tumor cell but also the bone marrow (BM) microenvironment. Thalidomide (Thal), as well as derivative immunomodulatory drugs (IMiDs), directly induce apoptosis or GI growth arrest in MM cell lines and patient's MM cells which are resistant to melphalan (Mel), doxorubicin (Dox), and dexamethasone (Dex). Although Thal and IMiDs do not alter adhesion of MM cells to bone marrow stromal cells (BMSCs), they inhibit the upregulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion triggered by the binding of MM cells to BMSCs. Proteasome inhibitors represent another potential anticancer therapy targeting the MM cell and the BM microenvironment. The proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis in both human MM cell lines and freshly isolated patient's MM cells which are resistant to Mel, Dox, and Dex. PS-341 inhibits p44/42 mitogen-activated protein kinase (MAPK) growth signaling triggered by IL-6 and induces apoptosis, despite induction of p21 and p27, in p53 wild-type and p53 mutant MM cells. PS-341 adds to the anti-MM activity of dexamethasone and overcomes IL-6-mediated protection against dexamethasone-induced apoptosis. PS-341 blocks the paracrine growth of human MM cells by decreasing their adherence to BMSCs and related NF-κB-dependent induction of IL-6 secretion in BMSCs. Moreover, proliferation and MAPK growth signaling of those residual adherent MM cells is also inhibited. Tumor necrosis factor-α (TNF-α), which is produced by some MM cells, induces only low-level MM proliferation and MAPK activation in MM cells, but markedly upregulates IL-6 secretion from BMSCs and upregulates expression of adhesion molecules (VLA-4 and LFA-I) on MM cells and their receptors (VCAM-I and ICAM-I) on BMSCs, with resultant increased binding of MM cells to BMSCs. Inhibition of TNF-α-induced NF-κB activation with PS-341 inhibits both the upregulation of these molecules on MM cells and BMSCs and the resultant increased adhesion. Therefore, inhibiting TNF-α and its sequelae may be useful treatment strategies in MM. Our data show that VEGF causes proliferation and enhances migration of MM as well as plasma cell leukemia (PCL) cells. VEGF induced twofold activation of cell migration in MM cells and more than 100-fold activation of cell migration in PCL cells, suggesting an important role of VEGF in the progression of MM to PCL. These data indicate that VEGF plays a pivotal role not only in neoangiogenesis in MM BM but also in proliferation and migration of tumor cells.

CD40 Ligand-Induced Apoptosis is Fas-Independent in Human Multiple Myeloma Cells

We and others previously demonstrated that human multiple myeloma (MM) cells express CD40 and have an active CD40-growth regulatory pathway. This study characterizes the growth outcome of soluble (gp39) or membrane-bound recombinant human CD40-ligand (rCD4()L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-MAb G28.5, gp39 inhibited 3H-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and HS-Sultan in a dose-dependent fashion by up to 82%. By comparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA→TTA, serine→leucine) at the 3rd cysteine-rich extramembrane region of CD40. Gp39 similarly reduced myeloma clono-genic colony (MCC) formation in patient primary bone marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibited the growth of ARH-77, U266, and HS-Sultan cells (66%, 63%, and 32%. respectively), whereas untransfected L cells did not. Growth inhibition by gp39 or CD40L+ L cells was neutralized by coincubation with the CD40L antibodies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleo-somal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines expressed the Fas/Apo-1/CD95 antigen (65-92% CD95+). However, only ARH-77 cells responded to the growth inhibitory effect of the CD95-agonistic antibody CH-II. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease in H-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 reversed the Fas-dependent growth inhibitory process but did not significantly alter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, whereas detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our observations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect corresponds to lower levels of cytoplasmic TRAF signaling elements, and appears independent of the Fas-signaling pathway. CD40 receptor mutation may lead to unresponsiveness to CD40 growth modulation in multiple myeloma cells.