Birinapant (TL32711)

Therapeutically Increasing MHC-I Expression Potentiates Immune Checkpoint Blockade

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-na?ve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. SIGNIFICANCE: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell-based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials.This article is highlighted in the In This Issue feature, p. 1307.

Birinapant Enhances Gemcitabine's Antitumor Efficacy in Triple-Negative Breast Cancer by Inducing Intrinsic Pathway-Dependent Apoptosis

Triple-negative breast cancer (TNBC) is the most aggressive subgroup of breast cancer, and patients with TNBC have few therapeutic options. Apoptosis resistance is a hallmark of human cancer, and apoptosis regulators have been targeted for drug development for cancer treatment. One class of apoptosis regulators is the inhibitors of apoptosis proteins (IAPs). Dysregulated IAP expression has been reported in many cancers, including breast cancer, and has been shown to be responsible for resistance to chemotherapy. Therefore, IAPs have become attractive molecular targets for cancer treatment. Here, we first investigated the antitumor efficacy of birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspases (SMACs), in TNBC. We found that birinapant as a single agent has differential antiproliferation effects in TNBC cells. We next assessed whether birinapant has a synergistic effect with commonly used anticancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), erlotinib (EGFR inhibitor), and gemcitabine, in TNBC. Among these tested drugs, gemcitabine showed a strong synergistic effect with birinapant. Birinapant significantly enhanced the antitumor activity of gemcitabine in TNBC both in vitro and in xenograft mouse models through activation of the intrinsic apoptosis pathway via degradation of cIAP2 and XIAP, leading to apoptotic cell death. Our findings demonstrate the therapeutic potential of birinapant to enhance the antitumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.

Retraction Note: Birinapant sensitizes platinum-resistant carcinomas with high levels of cIAP to carboplatin therapy

The IAP antagonist birinapant potentiates bortezomib anti-myeloma activity in vitro and in vivo

Background: Mechanisms by which Smac mimetics (SMs) interact with proteasome inhibitors (e.g., bortezomib) are largely unknown, particularly in multiple myeloma (MM), a disease in which bortezomib represents a mainstay of therapy.
Methods: Interactions between the clinically relevant IAP (inhibitor of apoptosis protein) antagonist birinapant (TL32711) and the proteasome inhibitor bortezomib were investigated in multiple myeloma (MM) cell lines and primary cells, as well as in vivo models. Induction of apoptosis and changes in gene and protein expression were monitored using MM cell lines and confirmed in primary MM cell populations. Genetically modified cells (e.g., exhibiting shRNA knockdown or ectopic expression) were employed to evaluate the functional significance of birinapant/bortezomib-induced changes in protein levels. A MM xenograft model was used to evaluate the in vivo activity of the birinapant/bortezomib regimen.
Results: Birinapant and bortezomib synergistically induced apoptosis in diverse cell lines, including bortezomib-resistant cells (PS-R). The regimen robustly downregulated cIAP1/2 but not the canonical NF-κB pathway, reflected by p65 phosphorylation and nuclear accumulation. In contrast, the bortezomib/birinapant regimen upregulated TRAF3, downregulated TRAF2, and diminished p52 processing and BCL-X_L expression, consistent with disruption of the non-canonical NF-κB pathway. TRAF3 knockdown, ectopic TRAF2, or BCL-X_L expression significantly diminished birinapant/bortezomib toxicity. The regimen sharply increased extrinsic apoptotic pathway activation, and cells expressing dominant-negative FADD or caspase-8 displayed markedly reduced birinapant/bortezomib sensitivity. Primary CD138⁺ (n = 43) and primitive MM populations (CD138^-/19⁺/20⁺/27⁺; n = 31) but not normal CD34⁺ cells exhibited significantly enhanced toxicity with combined treatment (P < 0.0001). The regimen was also fully active in the presence of HS-5 stromal cells or growth factors (e.g., IL-6 and VEGF). Finally, the regimen was well tolerated and significantly increased survival (P < 0.05 and P < 0.001) compared to single agents in a MM xenograft model. Combined treatment also downregulated cIAP1/2 and p52 while increasing PARP cleavage in MM cells in vivo.
Conclusions: Our data suggest that birinapant and bortezomib interact synergistically in MM cells, including those resistant to bortezomib, through inactivation of the non-canonical NF-κB and activation of the extrinsic apoptotic pathway both in vitro and in vivo. They also argue that a strategy combining cIAP antagonists and proteasome inhibitors warrants attention in MM.

Birinapant (TL32711) Improves Responses to GEM/AZD7762 Combination Therapy in Triple-negative Breast Cancer Cell Lines

Background: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer currently lacking targeted therapies. Our previous work demonstrated a therapeutic synergism with gemcitabine (GEM) and the CHK1 inhibitor (AZD7762) combination treatment in a TNBC cell line. We hypothesized that the response to this combination therapy would differ among heterogeneous TNBC patients and that addition of a SMAC mimetic (TL32711) could improve efficacy.
Materials and methods: Therapeutic responses to GEM, GEM/AZD7762, and GEM/AZD7762/TL32711 combinations were investigated by XTT assays and western blotting of cell cycle and apoptosis-related proteins in ten TNBC cell lines.
Results: TNBC cell lines harboring low levels of endogenous CHK1, cIAP1 and cIAP2 were responsive to GEM alone, whereas cell lines demonstrating a minimal increase in phospho-S345 CHK1 after treatment were responsive to GEM/AZD7762 or GEM/AZD7762/TL32711 combination.
Conclusion: The response of TNBC cells to particular therapies varies and will require development of predictive biomarkers.