The Role of the Antiapoptotic Protein c-FLIP in Multiple Myeloma.

Multiple myeloma (MM) is an incurable blood cancer. Treatments which target key proteins that play a role in drug resistance should improve treatment outcomes. The cellular FLICE-inhibitory protein (c-FLIP) is an antiapoptotic protein which confers resistance to death receptor-mediated apoptosis. Further, c-FLIP overexpression has been identified in various cancers, and in MM specifically, gene expression profiling has demonstrated that c-FLIP is overexpressed in a patient's MM cells compared to the normal plasma cells of an identical twin. However, the precise clinical significance of c-FLIP overexpression and its potential as a therapeutic target in MM have not been established.

To determine the potential of c-FLIP as a therapeutic target by exploring the in vitro and in vivo effects of c-FLIP knockdown in MM cells, using a doxycycline-inducible lentiviral vector containing c-FLIP shRNA.

Doxycycline treatment in vitro induced c-FLIP shRNA transcription and reduced c-FLIP levels more than 80% in H929 cells. Correspondingly, we observed a greater than 95 % reduction in the growth of H929 cells, compared to the cell line containing an empty vector control. In addition, immunoblots of MM cell lysates showed an accumulation of the cleaved products of caspases 8 and 3, suggesting that c-FLIP knockdown sufficiently induced caspase activation and apoptosis in MM cells.

To explore the cytotoxicity of c-FLIP knockdown in MM cells in vivo, we used two different mouse models. In the first model, MM cells containing the doxycycline inducible, c-FLIP shRNA containing, lentiviral vector, were transplanted subcutaneously into NOD/Scid mice. Two weeks post transplantation, when accurate tumor measurements could be made, the mice were divided into two groups, of approximately equal tumor volumes, and one group was given doxycycline treated water. A significant reduction in tumor size was observed in doxycycline treated mice compared to the controls (Figure 1).

Figure 1

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Tumor measurement from mice transplanted with MM cells containing a doxycycline inducible c-FLIP shRNA vector (+/− doxycycline).

Figure 1

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Tumor measurement from mice transplanted with MM cells containing a doxycycline inducible c-FLIP shRNA vector (+/− doxycycline).

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In a second mouse model, NOD/Scid mice were transplanted with MM cells by tail vein injection and divided into two groups. The first group (“early dox”; n=10) was given doxycycline treated water 4 hours post transplantation, while the second group (n=20) was given regular water until they developed tumors which could be determined by measuring human IgA kappa levels in the mouse serum. Within this group, mice were randomized base on the IgA levels to receive doxycycline treatment (“late dox”; n=6) or regular water (n=6). ELISA measurements of human IgA kappa light chain from treated mice are shown in Figure 2. The “early dox” group had barely detectable IgA kappa levels while the “late dox” group had significantly lower levels than the mice that never received any doxycycline.

Figure 2

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Serum ELISA for human IgA kappa light chain from mice IV transplanted with MM cells containing a doxycycline inducible c-FLIP shRNA vector (+/− doxycycline).

Figure 2

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Serum ELISA for human IgA kappa light chain from mice IV transplanted with MM cells containing a doxycycline inducible c-FLIP shRNA vector (+/− doxycycline).

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An inducible c-FLIP shRNA system was created in order to observe the effects of c-FLIP knockdown on the viability of MM cells both in vitro and in vivo. A nearly complete c-FLIP knockdown could be generated in vitro which had a dramatic effect on the viability of the MM cells, and this translated to a significant reduction in tumor volumes when these cells were later transplanted into mice, both subcutaneously and intravenously, and then treated with doxycycline. This work provides evidence that targeting c-FLIP in MM, either directly or via gene regulation, holds significant promise.

Abonour: Celgene: Honoraria; Millennium: Honoraria.