Abstract
Background & Aims Fas receptor is expressed on the surface of many malignant cells and its activation represents a potentially relevant anticancer target. APO010 is a recombinant form of Fas Ligand with hexameric structure, which is currently being evaluated in Phase 1 clinical trials. In order to identify possible targeted indications, we tested the in vitro and in vivo anti-tumor efficacy of APO010 on multiple myeloma (MM) cells.
Material & methods In vitro cytotoxicity was tested by MTT and Annexin V staining in 8 MM cell lines and PBMCs from 3 healthy donors. Other techniques used for mechanistic studies were propidium iodide uptake by flow cytometry, Western-blotting, BrdU uptake and gene expression profile analysis. The in vivo antimyeloma effect of APO010 was tested in a xenograft of human plasmocytoma in CB17-SCID mice. When tumors became palpable mice were randomized to receive APO010 15 μg/Kg ip × 5d/sem (n=7), APO010 5 μg/Kg ip × 5d/sem (n=8) or vehicle alone (n=8). Tumor volumes, clinical features and weight were monitored three times a week.
Results Six of the 8 MM cell lines studied by MTT were highly sensitive to APO010 with IC-50 at 24h of 0.5–20 ng/ml (2.5–100 pM), whereas two were resistant (RPMI-8226 and OPM-1). This sensitivity was correlated with the expression of Fas receptor by flow cytometry. Activation of apoptosis was rapid (within two hours of incubation) with maximum effect at 10 hours, as determined by Annexin V staining. Interestingly, APO010 was not toxic against PBMCs (both resting and activated) from 3 healthy donors at doses effective against MM cell lines. The presence of the microenvironment, as simulated by the coculture of MM1S cells with IL-6, IGF-1 and BMSCs, was not able to abrogate the APO010 antimyeloma effect. The combination of APO010 with Doxorubicin and Bortezomib, and, to a less extent, with Melphalan and Lenalidomide, potentiated the efficacy of the drugs alone. Regarding the mechanism of action, APO010 antiproliferative activity is mediated through caspase dependent apoptosis (Annexin-V staining, and PARP, caspase-3, caspase-7, caspase-8 and caspase-9 cleavage) and is independent of variations on the cell cycle profile. In this sense, the presence of the pan-caspase or caspase-8 inhibitors (Z-VAD-FMK and Z-IETD-FMK respectively) were able to completely abrogate APO010-induced cell death. Treatment of MM1S cells with APO010 for just one hour induced changes in the expression of 52 genes, many of them implicated in regulation of transduction (n=16). Three of the 4 most upregulated genes were the 3 members of the nuclear receptor subfamily 4, group A (Nurr1, Nor1 and Nur77). Other upregulated transcription factors were members of the Fos/Jun family such as Jun, JunB or FosL2. Western-Blot studies revealed that APO010 also provoked cleavage of MCL-1 and BIM, a decrease of BID and an important downregulation of pAKT. In the in vivo studies, APO010 treatment inhibited tumor growth as compared with the control group (p=0.02) without differences among the two doses of APO010. No significant toxicity was observed regarding body weight loss or increase in liver enzymes.
Conclusions These data show that Fas activation with APO010 induces in vitro and in vivo cytotoxicity in MM cell lines, mainly through transcriptional regulation. This study provides an initial rationale for the use of this compound for treatment of MM patients.
In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis
