The inhibitor of apoptosis (IAP) proteins contribute to acquired chemotherapy resistance in B-cell neoplasms
MetadataShow full item record
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 50uM gemcitabine, as well as 50uM 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 10x106 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 20uM to 50uM range for most cell lines. Single agent IC50 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.We also used western blot to investigate the how LCL-161 exposure impacted the expression levels of IAPs in RRCLs. The strongest effect was seen against cIAP1 in both the Raji 4RH and RL 4RH cell lines, which was totally depleted at 1uM concentration of LCL-161. The expression levels of survivin and livin were also decreased at higher concentrations of LCL-161. To determine if LCL-161 could enhance chemotherapy activity in RRCLs we cultured Raji 4RH cells with LCL-161 and various chemotherapy agents for 48 hours. We observed that LCL-161 synergistically enhanced the anti-tumor activity of etoposide, gemcitabine, and vincristine in the Raji 4RH cell line, with less synergistic responses seen in the Raji cell line. To determine if the combination of BMT-062789 and chemotherapy was more active than either alone we incubated RRCLs with BMT-062789 and etoposide. The rates of apoptosis (measured by annexin-V positivity) increased when BMT-062789 and etoposide were combined in both the Raji 4RH and RL 4RH cell lines. To quantify the in vivo anti-tumor effect of LCL-161 we used a SCID mouse model implanted with a disseminated Raji 4RH tumor model. LCL-161 combined with chemotherapy produced a statistically significant increase long term survival compared to chemotherapy alone. We also investigated the anti-tumor effect of LCL-161 and BMT-062789 in ex vivo lymphoma patient samples, and we observed that LCL-161 at a 10uM concentration enhanced the anti-tumor effect of carfilzomib in tissue samples from patients with a variety of lymphomas, including therapy relapse lymphomas. BMT-062789 at a 3uM concentration was active as a single agent in all ex vivo samples tested, and resulted in almost total tumor cell death, as measure by luminescent ATP assays. These results support our hypothesis that targeted XIAP inhibition with small molecule compounds is an effective anti-lymphoma strategy. In addition, selective XIAP inhibition appear to be a more effective anti-tumor approach in RRCL models.