Targeting the X-Linked Inhibitor of Apoptosis Protein (XIAP) Can Promote Tumor Cell Death, and Increase the Cytotoxic Effects of Chemotherapy Agents in in Vitro and In Vivo Models of Rituximab-Resistant Lymphoma

In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease.  To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents.  These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax.  We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models.  To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents.  Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis.  On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells).  An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH.  Results were confirmed by western blot (PARP cleavage).  In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine).  To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging.  SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x10⁶ cells per animal given i.v.).  Tumors were allowed to engraft for 7 days prior to treatment administration.  Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression.  At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE.  InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease.  In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models.  Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death.  Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma.