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Clinical observations suggest the existence of shared resistance pathways between rituximab and chemotherapy agents. To explore the mechanisms of rituximab resistance, our group created rituximab-resistant cell lines (RRCLs), which display altered expression of several inhibitor of apoptosis (IAP) family proteins. Here, we provide evidence to support pharmacologically targeting IAPs in lymphoma with LCL-161, a small molecule mimetic of the second mitochondria-derived activator of caspases (SMAC). The antitumor effect of LCL-161 was determined using luminescent adenosine triphosphate assays, flow cytometry, SCID mouse xenografts, and ex vivo patient biopsy sample studies. In vitro exposure to LCL-161 also resulted in a dose-dependent decrease in IAP levels, along with synergistic enhancement of the antitumor effect of cytotoxic chemotherapy, in rituximab-sensitive cell lines and RRCLs. In addition, LCL-161 increased the cytotoxic effect of the proteasome inhibitor carfilzomib in ex vivo lymphoma patient samples. The combination of LCL-161 with the chemotherapy regimen rituximab, gemcitabine, and vinorelbine (RGV) improved in vivo survival compared with RGV alone in severe combined immunodeficient mice implanted with RRCLs but not in animals implanted with rituximab-sensitive cell lines. In summary, LCL-161 exhibits synergistic antitumor activity in both in vitro and in vivo models of resistant lymphoma. Our data support further preclinical investigation of LCL-161 as a novel antilymphoma agent.

Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69. Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib. Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. Disclosures No relevant conflicts of interest to declare.

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. Disclosures Czuczman: Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.

The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets. Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days). In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

Abstract 61 The loss of response to apoptotic stimulus in lymphoma is a major obstacle in the treatment of primary and refractory B-cell malignancies. While the role of the anti-apoptotic Bcl-2 family proteins in the pathogenesis, maintenance, and progression of many sub-types of B-cell lymphoma is well characterized, the impact of the expression level(s) of other key regulatory proteins of cell death pathways (i.e. inhibitor of apoptosis [IAP] proteins) is less defined. The role of IAP proteins in the acquirement of resistance to rituximab or chemotherapy in B-cell lymphoma is unclear. Overexpression of IAP proteins and loss of expression of its antagonist, the second mitochondria-derived activator of caspases (SMAC) correlates with inferior clinical outcomes in a range of malignancies. Perhaps related to the acquisition of resistance, we found that rituximab-resistant cell lines (RRCL) have a deregulation of pro-apoptotic (Bak/Bax) and anti-apoptotic (Mcl-1, Bcl-XL) Bcl-2 family protein expression along with increased expression of the IAP protein survivin. Small molecule SMAC mimetics like LCL-161 are promising agents for lowering the threshold of tumor cell apoptosis, and represent a potential new avenue of therapy for de novo and refractory drug-resistant lymphoma. To this end, we evaluated the anti-tumor activity of LCL-161 in a range of rituximab-sensitive (RSCL), RRCL, and primary lymphoma cells. Cells were exposed to escalating doses of LCL-161 alone or in combination with various chemotherapy agents (i.e. etoposide, doxorubicin, vincristine, gemcitabine, carboplatin, oxaliplatin, bortezomib and cytarabine) for 48 and 72 hrs. Changes in cell viability and ATP content were determined by the CellTiter-Glo viability assay. Protein lysates were obtained from RSCL and RRCL to determine baseline levels of IAP protein family members. LCL-161 displayed significant anti-tumor activity against Burkitt's lymphoma (BL), diffuse large B-cell (DLBCL) and mantle cell lymphoma (MCL) cell lines. Activity was observed in both RSCL and RRCL cell lines. IC50 values for LCL-161 alone were between 35uM and 45uM for the DLBCL lines. Responses were slightly lower in BL and MCL compared to DLBCL cell lines. Synergistic activity between LCL-161 and several chemotherapy agents (e.g. gemcitabine, cytarabine, carboplatin, vincristine, etoposide and bortezomib) commonly used in the management of aggressive lymphoma was seen at physiologically-relevant doses. In vitro exposure of lymphoma cells to LCL-161 decreased the cytotoxic threshold of chemotherapy by 50%, while ex vivo studies with primary patient lymphoma samples showed a decrease of nearly 60%. In vivo studies using a xenograft SCID murine model are planned. In summary, LCL-161 has shown activity both as a single agent, and when combined with several chemotherapy agents in BL, MCL, and DLBCL cell lines as well as primary patient samples. Additionally, LCL-161 exhibits significant cytotoxic activity against RRCLs suggesting an ability to antagonize IAP proteins. This data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of de novo and refractory aggressive B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.