Small-molecule exploitation of ZBP1-driven nuclear necroptosis for cancer immunotherapy

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PROJECT SUMMARY/ABSTRACT. Immune checkpoint blockade (ICB) and other immunotherapies have revolutionized cancer treatment, but the non-responsiveness of most cancers to ICB-based monotherapy remains a significant problem. A major reason for the non-responsiveness of these so-called ‘cold’ tumors is that they lack an immunogenic tumor microenvironment (TME) and thus escape T-cell killing despite expressing ICB targets. How to selectively intensify the immunogenicity of the TME has been an unmet challenge. Here we propose a new small-molecule approach that activates necroptosis and triggers robust innate immune responses in the TME. This new avenue derives from our work on influenza A virus (IAV). Our early findings showed that IAV activates necroptosis in infected cells. Necroptosis kills infected cells and is highly immunogenic. It is initiated when viral RNAs activating the host protein ZBP1. Recently, and highly relevant for cancer immunotherapy, we found that ZBP1 activates necroptosis from the nucleus. Such ‘nuclear necroptosis’ is significantly more immunogenic than conventional (cytoplasm-initiated) necroptosis because it ruptures the nucleus and releases hyper-inflammatory nuclear DAMPs into the extracellular space. We also found that the viral RNAs that activate ZBP1 are Z-RNAs. Although these unique ZBP1 activators should be superb adjuvants for ICB, Z-RNA is unstable and hard to produce absent virus infection. Z-DNA, however, is structurally almost identical to Z-RNA, binds ZBP1 with the same affinity, and can be stably produced in eukaryotic cells by distorting DNA into the Z-conformation. This suggested that a compound that can generate Z-DNA in cells would activate ZBP1 and trigger on-demand nuclear necroptosis without need for virus infection. Such a compound would fill the long-unmet need for a necroptosis agonist for use in cancer immunotherapy. We have now identified a small molecule, curaxin, which induces Z-DNA formation in live cells and directly activates ZBP1 to trigger ‘on-demand’ nuclear necroptosis in cells of the TME. These and other findings allow us to propose the hypotheses that curaxin alters chromatin structure and induces the formation of Z-DNA; that such Z-DNA recruits ZBP1 to the nucleus and triggers nuclear necroptosis; and that curaxin-induced nuclear necroptosis will greatly improve ICB treatment outcomes. In this proposal, we will ask how curaxin triggers Z-DNA formation (Aim 1), how Z-DNA activates ZBP1 and nuclear necroptosis (Aim 2), and whether induction of nuclear necroptosis by curaxin has combinatorial benefit with ICB in clinically-relevant mouse models of melanoma (Aim 3). The successful completion of these Aims will outline an entirely new small- molecule based strategy to activate a highly inflammatory form of necroptosis and potentiate ICB-based immunotherapies, with important clinical ramifications.