The addition of rituximab to the therapeutic regimens used to treat B-cell non-hodgkin lymphoma (B-NHL) has improved patient response rates, but it has also alter the biology of the disease in the relapse/refractory setting. The patients that relapse after receiving induction therapy containing rituximab respond less well to salvage chemotherapy regimens, and have a decreased overall survival rate. To study the molecular mechanisms controlling rituximab resistance in B-cell malignancies our group developed rituximab resistant cell lines (RRCL). These RRCLs are resistant to rituximab, as well as to chemotherapy agents. This resistance to chemotherapy agents closely mirrors what is observed clinically when lymphoma patients develop rituximab resistant disease.
The resistance to rituximab in RRCLs in mediated through down-regulation of CD20 (the target of rituximab), along with an upregulation of the complement inhibitory proteins CD55 and CD59. The molecular changes responsible for RRCL chemotherapy resistance involve alterations to the apoptotic response pathway, specifically loss of expression of the pro-apoptotic Bcl-2 proteins Bak and Bax. Without these two proteins RRCLs are highly resistant to any stimuli that activates the intrinsic apoptotic pathway, which is the cell death pathway triggered by many cytotoxic chemotherapy agents, including those used for the clinical management of lymphoma. These RRCLs also exhibit upregulated expression of survivin and livin, which are two members of the inhibitor of apoptosis protein (IAP) family. The IAPs act by directly inhibiting active caspases 3 and 7, which are the primary effectors of apoptosis. Our group previously determined that restoring expression of Bax in RRCLs can trigger spontaneous apoptosis, which indicated to us that restoring the balance of pro- and anti-apoptotic factors in RRCLs could improve anti-tumor activity of chemotherapy agents in these cells. Although forced expression of Bax was an effective strategy in RRCLs, it has little translational potential. The potential for IAPs to inhibit the effector caspases led us to hypothesize that targeting IAPs in RRCLs would magnify the low level of natural apoptotic activity in these cells, and potential increase chemotherapy sensitivity.
To investigate if survivin or livin were regulating chemotherapy resistance in RRCLs we used a transient siRNA knockdown of each. Cells were then incubated with chemotherapy agents and the induction of apoptosis was measure through annexin-V/Sytox Blue staining. We observed no difference in the rates of apoptosis between Raji 4RH cells transfected with a scramble control, and those transfected with either the survivin or livin knockdown. We also observed no increase in apoptosis when we knocked down two additional IAPs, specifically cIAP1/2, either separately or together. When we used the same transient siRNA knockdown of XIAP we did observe a change in the rates of apoptosis. Knockdown of XIAP in the RRCL Raji 4RH increased the cytotoxic effect of both 50 µM gemcitabine, as well as 50 µM etoposide. Knockdown of XIAP in RL 4RH (another RRCL) was cytotoxic even without the addition of chemotherapy, and triggered spontaneous apoptosis measured by annexinV/Sytox Blue staining, as well as through western blot for PARP cleavage.
To determine if knockdown of XIAP could improve the anti-tumor effect of chemotherapy in vivo we generated a version of the Raji 4RH cell line with a stable knockdown of XIAP, and we implanted these cells into SCID mice at 10 × 106 cells per animal. Animals were treated with a combination of rituximab, ifosfamide, etoposide, and carboplatin (R-ICE). Animals implanted with Raji 4RH_XIAP KD responded to R-ICE chemotherapy better than animals implanted with the Raji 4RH_scramble control line; however, the results of this experiment were not repeatable. When we investigated the Raji 4RH_XIAP KD cell line we observed that over approximately 2 months in culture it had become chemotherapy resistant once again. We observed several protein changes in the newly resistant Raji 4RH_XIAP KD cell line, including overexpression of the IAP livin; however, XIAP remained knocked down, indicating that chemotherapy resistance in these new cells involves a novel mechanism.
The data indicating that XIAP knockdown can increase chemotherapy activity in RRCLs prompted us to investigate if small molecule IAP inhibitors could produce similar results. We selected two IAP inhibitors, LCL-161 and BMT-062789, each of which has reported inhibitory activity with XIAP. We exposed a panel of lymphoma cell lines, including RRCLs, to LCL-161 and BMT-062789 at escalating concentrations. LCL-161 single agent IC50 concentrations were in the 20 µM to 50 µM range for most cell lines. Single agent IC 50 values for BMT-062789 were in the low micromolar range for most cell lines tested. BMT-062789 is a more selective XIAP inhibitor, and it appears to be active in lymphoma cell lines at lower concentration that LCL-161. (Abstract shortened by ProQuest.)
|Advisor:||Hernandez-Ilizaliturri, Francisco J.|
|Commitee:||Li, Fengzhi, Nemeth, Michael J., Seon, Ben|
|School:||State University of New York at Buffalo|
|Department:||Microbiology and Immunology|
|School Location:||United States -- New York|
|Source:||DAI-B 80/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Immunology|
|Keywords:||Apoptosis, Lymphoma, Resistance|
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