No abstract

The aim of the present study was to compare thalidomide-dexamethasone (THAL-DEX) and vincristine-doxorubicin-dexamethasone (VAD) as primary therapy for newly diagnosed multiple myeloma (MM). For this purpose, we performed a retrospective matched case-control analysis of 200 patients who were treated with THAL-DEX (n=100) or VAD (n=100) on two consecutive studies from 1996 to 2003. Thalidomide was given orally at the daily dose of 200 mg, while VAD was administered by continuous infusion. Pulsed dexamethasone combined with thalidomide or vincristine-doxorubicin was given at the monthly dose of 40 mg/d for 4 days (1 to 4), with courses repeated on days 9 to 12 and 17 to 20 on odd cycles. By design of both studies, THAL-DEX and VAD were planned to be given for 4 months in an attempt to reduce tumor cell mass before collection of peripheral blood stem cells (PBSC) and subsequent autologous transplantation. Matching criteria were age (within 2 years), clinical stage and serum β2-microglobulin (within 1 mg/l). In addition to the above mentioned criteria, all other relevant baseline patient characteristics were comparable between the two groups. Response to therapy was evaluated using an intent-to-treat approach and stringently defined criteria (EBMT). In comparison with VAD, THAL-DEX resulted in a significantly higher ≥ partial response rate (76% versus 52%, respectively; P=0.0004) and effected more profound reduction in serum IgG (P=0.002) and IgA (P=0.01) M protein levels. Nine patients treated with THAL-DEX and 9 patients who received VAD did not proceeded to PBSC mobilization, mainly because of death while on study treatment (THAL-DEX=5 patients; VAD=6 patients) or nonfatal toxicity (THAL-DEX=3 patients; VAD=2 patients). The median number of CD34+ cells collected following high dose cyclophosphamide 7 g/m2 was 7.85 x 106/kg in the THAL-DEX group and 10.5 x 106/kg in the VAD group. Considering 4 x 106 CD 34+/kg as the minimum number of stem cells required to safely perform double autologous transplantation, adequate cell yields were obtained in 83% of patients with prior exposure to THAL-DEX and in 88% of patients treated with VAD (P=0.3). In conclusion, results of the present study (to the best of our knowledge, the first comparing THAL-DEX with VAD as initial cytoreductive therapy in preparation for autologous transplantation) extend and confirm prior observations by our group and others showing that THAL-DEX is an effective and relatively well tolerated induction regimen for previously untreated patients with MM. In comparison with VAD, THAL-DEX significantly augmented tumor cytoreduction without increasing the toxicity or interfering with subsequent collection of PBSC. Based on these data, thalidomide-dexamethasone may be considered an oral, and easy to administer, alternative to the more complex, and cumbersome to administer, combination of vincristine-doxorubicin-dexamethasone as front-line therapy for MM patients who are candidates to subsequent autologous transplantation.

The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.

3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi: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. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.