Abstract 1703: Sensitizing cancer cells to TNF induced cell death by the BET-inhibitor RG6146
Authors: Lisa C. Wellinger, Simon J. Hogg, Dane Newman, Thomas Friess, Daniela Geiss, Marina Bacac, Tanja Fauti, Astrid Ruefli-Brasse, Ricky W. Johnstone, Daniel Rohle
Journal: Cancer Research (2020)

Abstract Bromodomain and extra-terminal family (BET) proteins bind to acetylated lysine residues on histone tails to modulate transcription. While the focus of BET inhibitors (BETi) has been to attenuate the transcription of oncogenes, recent work has shown that BETi suppress PD-L1 expression thereby possibly increasing anti-tumor immunity. Upon activation through immune-oncology (IO) agents, cytotoxic T-cells release pro-inflammatory cytokines such as IFNγ, TNF and Granzyme B leading to direct cancer cell cytolysis at the immunological synapse and bystander cancer cell death in the surrounding tumor microenvironment. However, rapid genetic and epigenetic tumor evolution can lead to immune escape and clinical resistance against IO agents. Therefore, our work examined how BETi reprogram cancer cells to become more sensitive to T-cell derived tumor necrosis factor (TNF) leading to increased bystander killing in combination with IO agents. Using TNF as a surrogate for activated T-cells, we tested a large panel of cell lines for enhanced sensitivity to TNF in the presence of BETi, RG6146. In a subset of cancer cells, BETi treatment sensitized the cells to TNF induced cell death irrespective of their histology or genetic background. The combination of TNF and RG6146 led to complete proliferation arrest and induction of cell death. We identified that RG6146 in this context suppressed the expression of important signaling partners in the pro-survival NF-κB pathway leading to potent Caspase-8 activation and induction of the extrinsic apoptotic pathway. In order to further confirm the phenotype, we activated T-cells using a tumor antigen targeted approach. The CEATCB is a (2:1) T-cell bispecific (TCB) antibody connecting cancer cells expressing carcinoembryonic antigen (CEA) on their cell surface with CD3 on the surface of T cells. This interaction induces T-cell activation, release of cytokines and subsequent killing of cancer cells. Addition of RG6146 to the supernatant of the CEATCB assay containing TNF, could significantly decrease viability of cancer cells compared to control treatment indicating a synergistic effect of the CEATCB and RG6146. We could verify these results in a co-culture experiment with a mixture of cancer cells expressing high and low levels of CEA and PBMCs. Even though treatment of this co-culture with the CEATCB alone increased bystander killing of cancer cells expressing low CEA levels, addition of RG6146 significantly enhanced this effect. We used syngeneic recipient mice to validate our findings in vivo. While single agent treatment of CEATCB or BETi decreased tumor growth, the combination of both molecules caused tumor regression. Taken together this data establishes a paradigm where BETi can rewire NF-κB signaling, leading to enhanced sensitivity to cytotoxic lymphocyte-derived TNF and therapeutically augmenting the anti-tumor activity of IO agents. Citation Format: Lisa C. Wellinger, Simon J. Hogg, Dane Newman, Thomas Friess, Daniela Geiss, Marina Bacac, Tanja Fauti, Astrid Ruefli-Brasse, Ricky W. Johnstone, Daniel Rohle. Sensitizing cancer cells to TNF induced cell death by the BET-inhibitor RG6146 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1703.