Abstract

Rituximab-chemotherapy relapse/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) patients have lower response rates to second-line chemotherapy, and have a poor overall prognosis. Pre-clinical models of rituximab resistance demonstrated that the acquirement of resistance to anti-CD20 antibodies leads to concomitant resistance to chemotherapy drugs, suggesting the existence of share pathways of resistance between rituximab and chemotherapy agents. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines (RRCL), which also display significant resistance to a wide range of chemotherapy agents. RRCLs exhibit a deregulation in the apoptotic response pathway at several levels. Previous work by our group demonstrated that the X linked inhibitor of apoptosis (XIAP) is a key regulator of chemotherapy resistance in RRCLs. Moreover, we have established that knocking down XIAP in RRCLs can enhance the anti-tumor cytotoxic effect of chemotherapy, but a clear mechanistic role for XIAP in RRCLs has yet to be established. To investigate how XIAP regulates chemotherapy response in resistant lymphoma models we first looked at expression levels of XIAP and its binding partners in RRCLs and rituximab sensitive cell lines (RSCL). Although we observed no change in XIAP expression between the RRCL Raji 4RH and RSCL Raji, we did observe changes in several of the XIAP interacting proteins. Protein levels for both of the primary apoptotic effector caspases (caspase 3 and caspase 7) were substantially decreased in the Raji 4RH cell line compared to Raji cells. In addition, when we exposed Raji and Raji 4RH cells to gemcitabine (50uM) for 28 hours we observed substantially less release of cytochrome C from the mitochondria in Raji 4RH compared to Raji. Cytochrome C is an essential part of the apoptosome, which functions as a primary activator of caspases 3 and 7. Both of these observations support the hypothesis that impaired effector caspase activation and signaling contributes to chemotherapy resistance in RRCLs. To determine if targeting XIAP could restore caspase activation in RRCLs we used a transient siRNA knockdown of XIAP in the Raji 4RH cell line. Cells were treated with either the XIAP targeting siRNA, or a scramble sequence control, and then exposed to chemotherapy for 36 hours. Cellular viability was measured with the Promega CellTiter-Glo ATP assay, while caspase 3 and 7 activity was determined with the Caspase 3/7-Glo assay, also purchased from Promega. XIAP knockdown increased caspase activity by more than 500% compared to controls when cells were exposed to etoposide (20uM). This correlated with a 60% decrease in cellular viability in cells with an XIAP knockdown. In addition, the addition of the caspase inhibitor QVD-OPh along with the RIP kinase inhibitor necrostatin-1 (both at 10uM) was enough to restore cellular viability to the same levels observed in scramble treated controls exposed to the same dose of etoposide. Additional experiments looking at activation of caspases 2 and 6 in Raji 4RH cells following XIAP knockdown did not show any difference compared to scramble controls. In summary, XIAP appears to directly inhibit the caspase dependent apoptotic response in RRCLs. In addition, targeting XIAP can restore the apoptotic response in cell line models of rituximab resistant disease. These results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against rituximab rel/ref B-cell lymphoma.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.