PROJECT SUMMARY / ABSTRACT Despite the impressive clinical success of Immune checkpoint blockade (ICB) therapy in cancer, therapy resistance is common and only a small subset of patients mounts lasting antitumor responses, highlighting the urgent need to identify strategies to overcome resistance and improve responsiveness to immunotherapy. Such resistance is related with the fact that many tumors are immunologically ‘cold’. Activating innate immunity in tumor cells is a potent means by which ‘cold’ tumors can be turned ‘hot’. One promising strategy to trigger an innate immune response within tumors is by inducing the intracellular accumulation of endogenous ‘virus-mimetic’ nucleic acids in cancer cells. Our preliminary data demonstrate that targeting the RNA/DNA helicase DHX9 is a promising and unexplored strategy by which ‘viral mimicry’ can be triggered in tumors. 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. Here, we provide data that ablating DHX9 induces the formation of double- stranded RNAs (dsRNAs) derived from endogenous retroviral elements (EREs), provoking an antitumor immune response in ‘cold’ tumors. Intriguingly, eliminating DHX9 also induced the accumulation of RNA-DNA hybrids (R- loops), leading not only to an antiviral immune response but also to DNA replication stress and DNA damage in Small Cell Lung Cancer (SCLC) cells. In vivo, eliminating DHX9 causes a significant decrease in tumor growth while inducing a more immunogenic Tumor Microenvironment (TME) in SCLC mouse models, dramatically enhancing responsiveness to ICB. To our knowledge, this is the first description of the role of DHX9 on tumor immunity and genomic instability and the identification of a novel viral mimicry-inducing strategy to enhance antitumor immunity and boost cancer immunotherapy. Excitingly, we now present a first-in-class, orally bioavailable small molecule inhibitor of DHX9, which will allow us to evaluate the translational relevance of targeting DHX9 in SCLC and in immunotherapy settings. Our overarching hypothesis is that targeting DHX9 induces dsRNA accumulation, causing antiviral signaling, while concurrently triggering the formation of R-loops, compromising genome stability, and triggering these tumor-intrinsic events greatly improves ICB. In Aim 1, we will determine which dsRNAs are substrates of DHX9 and whether they are immunogenic, as well as how DHX9 senses and suppresses R-loops. We will also delineate the mechanism by which DHX9 loss triggers cell death in SCLCs. In Aim 2, we will dissect the impact of DHX9 loss or inhibition on the TME at a single cell resolution, and we will evaluate whether DHX9 depletion or inhibition induces a robust antitumor immunity able to boost cancer immunotherapy using murine immunocompetent SCLC models and a SCLC GEMM. The successful completion of these Aims will outline an entirely new viral mimicry-inducing strategy with widespread application in cancer treatment, as stand-alone anti-cancer therapy to improve responsiveness to immunotherapy.

PROJECT NARRATIVE/RELEVANCE TO PUBLIC HEALTH Immunotherapy has revolutionized cancer treatment, but therapy resistance remains a significant problem. One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing the accumulation of endogenous ‘virus-mimetic’ nucleic acids in cancer cells. We have uncovered a novel viral mimicry-inducing factor, DHX9, involved in the suppression of double-stranded RNAs and RNA/DNA hybrids in cancer cells and aim to study how DHX9 controls these endogenous nucleic acids, as well as targeting DHX9 to enhance antitumor immunity and boost immunotherapy in ‘cold’ tumors, such as Small Cell Lung Cancer.