Abstract #5118: Requirement of both isoforms of TRAIL-R2 (DR5) in death receptor signaling.

Colon cancer remains resistant to treatment with only 25-30% of patients remaining disease-free long term. For the remaining patients, the median survival rate is 18-22 months. Using a human colon adenocarcinoma xenograft model, we had previously shown that the addition of interferon-gamma (IFN-gamma) to the standard 5-fluorouracil/leucovorin (FUra/LV) treatment regimen caused complete volume regression of established tumors. Addition of IFN-gamma to FUra/LV has also shown promise in Phase I clinical trials (ClinCancer Res 8:2488, 2002) and is currently undergoing Phase II trials. As promising as the complete regression of the xenograft tumors upon initial treatment, regrowth of the xenograftis is observed after 7 weeks. In order to enhance the therapeutic response of FUra/LV/IFN-gamma, we targeted the TNF receptor superfamily, specifically the TRAIL receptor DR5. Addition of TRAIL to the FUra/LV/IFN-gamma regimen resulted in complete tumor regression without any tumor regrowth. The same result was also obtained with lexatumumab, an agonistic monoclonal antibody that targets DR5. These data suggested that death receptor activation could be synergistic in the enhancement of therapeutic response. In order to further understand the mechanism of TRAIL- and lexatumumab- induced cell death in colon carcinoma cells, we explored DR5 expression in a variety of colon carcinoma cell lines. We found that the majority of cell lines expressed the short isoform of DR5 (DR5S) at a higher level than the long isoform (DR5L). The two DR5 isoforms are generated by alternative splicing and differ in a 29 amino acid extracellular domain that is immediately adjacent to the membrane. To determine the specific function of each isoform in regulating death signaling, we utilized a BJAB-derived cell line that was selected for loss of DR5 expression (DR5-/-). With the loss of DR5, the cells were no longer sensitive to TRAIL or to lexatumumab-induced apoptosis. When each DR5 isoform was expressed individually in these cells, they remained resistant to TRAIL- and to lexatumumab- induced apoptosis despite the fact that the individual isoforms could still bind ligand (TRAIL). Apoptotic signaling was only restored when both the DR5L and DR5S isoforms were re-expressed together in the DR5-/- cells. Subsequently we varied the expression levels of the two isoforms in the reconstituted cells and found that as long as both isoforms were expressed, responsiveness to TRAIL and to lexatumumab were comparable to the sensitivity of wt BJAB cells. As each isoform is capable of binding ligand independently, we hypothesize that there is an isoform-specific recruitment of secondary signaling factors. This would indicate that functional DISC assembly is only generated by both DR5 isoforms. This hypothesis is currently under investigation. This work is supported by NCI awards CA 32613 and CA 108929. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5118.