Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer
Authors: Cañadas Israel 
Published: April 2024

Abstract
Immune checkpoint blockade (ICB) therapy has permanently altered cancer medicine and can result in impressive clinical responses of multiple cancer types. However, therapy resistance is common and only a small subset of patients mounts lasting responses to ICB monotherapies, highlighting the urgent need to identify mechanisms of resistance as well as new strategies to improve responsiveness. In large part, such resistance is related with the fact that many tumors are immunologically silent, or ‘cold’. Emerging data suggest that activating innate immunity in tumor cells is a potent means by which cold tumors can be turned ‘hot’, invigorating responsiveness to ICB modalities. One promising strategy to trigger an innate immune response within tumors, and boost cancer immunotherapy is by inducing the intracellular accumulation of endogenous ‘virus-mimetic’ nucleic acids in cancer cells and triggering an active antiviral response in the tumor, phenomenon known as ‘viral mimicry’. These nucleic acids are sensed by the host innate immune system as evidence of viral replication, and provoke an immediate and robust Interferon (IFN)-driven antiviral response. Such an antiviral response is potently immunogenic, and ‘viral mimicry’ inducing therapies have shown very promising results in pre-clinical models and in clinical trials. Reawakening Endogenous Retroviral Elements (EREs), including Endogenous Retroviruses (ERVs) has emerged as an effective strategy to induce ‘viral mimicry’ and boost ICB responsiveness in cold tumors. In fact, we previously reported a novel subclass of ERVs that engages innate immune signaling in mesenchymal subpopulations of Small Cell Lung Cancer (SCLC) (Canadas I, et al. Nat Med 2018). The unique feature of these ERVs is that they are located in the 3’ untranslated regions (3’UTRs) of IFN-induced genes, and oriented in an antisense fashion, which results in double-stranded RNA (dsRNA) production and IFN response. More recently, and to discover novel dsRNA regulators, we carried out a curated CRISPR screen of RNA helicases and identified DHX9 as a potent inducer of dsRNA. DHX9 is an abundant RNA/DNA helicase capable of unwinding both RNA and DNA duplexes, as well as more complex nucleic acid structures. Its functions include regulation of DNA replication and transcription, RNA processing and transport, and maintenance of genomic stability.
We initially focused on SCLC, since it is the tumor type with higher DHX9 gene expression levels in the Cancer Cell Line Encyclopedia (CCLE) database. SCLC is the most aggressive and lethal form of lung cancer, with a 5-year overall survival of less than 5%. Importantly, while having one of the highest mutational burdens because of its strong association with tobacco smoking, SCLC is often characterized by a reduced antigen presentation and an immunologically cold Tumor Microenvironment (TME). Despite promising advances in the use of immunotherapy, SCLC remains a devastating disease and only a very small fraction of SCLC patients respond to these therapies. We now present a novel and effective way to restore the immunogenicity of the TME through ‘viral mimicry’, and also to selectively kill SCLC cells while doing so.
Our study demonstrates that targeting DHX9 is a promising and unexplored strategy by which ‘viral mimicry’ can be triggered in tumors. Ablating DHX9 induces the formation of dsRNA derived from EREs, provoking a strong antitumor immune response in ‘cold’ tumors. Intriguingly, eliminating DHX9 also induced the accumulation of RNA-DNA hybrids (R-loops), which resulted in DNA replication stress and DNA damage in SCLC cells. In vivo, eliminating DHX9 causes a significant decrease in tumor growth while inducing a more immunogenic TME in mouse models of SCLC, dramatically enhancing responsiveness to ICB therapy. Notably, DHX9 expression is negatively correlated with immune signatures and associated with poor clinical outcomes across cancer patient datasets. These discoveries have potentially profound implications for inducing ‘viral mimicry’ in ‘cold’ tumors, because if we can induce a more immunogenic TME while selectively killing cancer cells, we could more efficiently convert these ‘cold’ tumors to ‘hot’, and therefore targeting DHX9 could be an attractive therapeutic strategy as an adjuvant for ICB-based cancer immunotherapy. To our knowledge, this is the first description of the role of DHX9 on tumor immunity and genomic instability and the identification of a completely novel viral mimicry-inducing strategy to enhance antitumor immunity and boost immunotherapy in immunologically ‘cold’ tumors, such as SCLC, one of the least responsive and deadliest tumors. All this work, which I am the senior corresponding author of, has been recently published in Cancer Discovery (Murayama T, et al. Cancer Discov. 2024).
In summary, our study demonstrates that targeting DHX9 (1) induces immunogenic dsRNAs, causing tumor cell-intrinsic antiviral signaling; that (2) alters transcription and DNA replication in cancer cells to induce formation of R-loops, compromising genome stability and cell viability; and that (3) triggering these tumor-intrinsic events greatly improves ICB treatment outcomes. Thus, our work outlines an entirely new viral mimicry-inducing strategy with widespread application in cancer treatment.
Citation Format: Israel Cañadas. Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG04.