Targeted therapies have been widely used in tumors driven by RAS oncogenes. Unfortunately, no effective RAS inhibitors have been translated to the clinic, and attempts to block other signaling nodes usually fail due to the emergence of drug resistance. Chromatin dependent signal transduction and transcription are a point of confluence of multiple signaling networks elicited by hyperactive RAS. Hence, pharmacologic disruption of gene-regulatory dependencies imposed by mutant RAS represents an attractive therapeutic interface less prone to the emergence of resistances. The urgent clinical need of RAS-related therapies is well exemplified by pancreatic cancer, one of the most aggressive and deadly cancers, which will be the disease background of my studies. Using the innovative approach of targeted protein degradation, I want to characterize and understand the consequences of acute mutant KRAS degradation on chromatin remodeling and transcription. Further engineering the models of acute KRAS degradation will enable to devise cellular reporters of KRAS-dependent chromatin regulation amenable to high-throughput phenotypic drug and genetic screens. Coupled to a facile readout via high-throughput microscopy, these screens will allow me to identify molecules and genetic perturbations that interfere with KRAS-dependent, transcriptionally active chromatin. Lead molecules will be characterized for the underpinning mechanism of action and assessed for therapeutic potential. Building on already existing experimental and computational pipelines in the Winter laboratory at CeMM-Research Center for Molecular Medicine of the Austrian Academy of Sciences, this project will increase the understanding of transcriptional control elicited by oncogenic KRAS and could open new avenues for the treatment of RAS-driven tumors based on chemical modulation of critical chromatin and transcription regulators.

Two major issues hamper successful treatment of solid cancers with CAR T cells. First, immunosuppression imposed by the tumor microenvironment (TME) can compromise CAR T cell function. Second, CAR T cells require specific antigens for precise tumor recognition to limit/avoid toxicities associated with on-target/off-tumor activity. Initial attempts to overcome these challenges included immune checkpoint blockade and tunable CAR T cells. Here, we propose to develop a conceptually new framework for solid tumor therapy that introduces genetically modified, blood-derived TME cells as Trojan horses to deliver inflammation and broadcast “intratumor” signals that activate programmable CAR T cells. Overall, we will design a hematopoietic stem cell (HSC)-directed strategy for Tumor microenvironment-Regulated Activation of Programmable CAR T (TRAP-CART). This project builds on the host lab’s expertise in high-throughput genomics, bioinformatics, and CAR T cells, and on my own background in HSC engineering. Through epigenome/transcriptome profiling, we will systematically identify gene-regulatory elements that are specific to blood-derived cells in the TME. We will use such regulatory sequences for HSC-directed gene therapy, such that their progeny will express a programmed transgene only in the immediate vicinity of tumor cells. This TME-regulated transgene will induce inflammation (to help overcome the hostile immune environment of many solid tumors) and/or release localized activatory signals for CAR T-cells (making it possible to use weakly specific CARs while avoiding on-target/off-tumor activity elsewhere in the body). We will focus on melanoma as our “model cancer”, which has a high mutational burden, good mouse models and is widely used for testing cutting-edge immunotherapies. Specific Objectives: 1)Develop a promoter/enhancer toolbox for HSC gene therapy targeting the TME 2)Induce a CAR T-supportive TME that activates programmable CAR T-cells in situ

Intestinal microbial communities expand the functional capabilities of the host via their metabolic attributes. From energy harvest to the production of vitamins, the gut microbiota shapes mammalian physiology and is often considered a postnatally developed “organ”. Yet, the microbiome poses a formidable challenge to the immune system: How can we host trillions of bacteria without mounting an inflammatory response? Gut immune homeostasis relies on the balanced action of suppressive and inflammatory T cell subsets. I discovered that bacterial metabolism of bile acids and dietary fibers promotes the differentiation of suppressive T cells. Given the complexity of the microbiome, finding other immunoregulatory cues deployed by gut bacteria and their mechanisms of action remains a major challenge, and the logic behind these tolerance mechanisms is not understood. I will use a novel conceptual framework to bridge this gap: based on my previous findings, I postulate that immunoregulatory bacterial molecules are produced in response to food intake. Within this emerging paradigm, I selected two new groups of bacterial molecules for immediate investigation and developed a strategy to identify novel putative immunoregulatory candidates based on a careful examination of microbial metabolism after food intake. I will find the molecular targets of active molecules using chemical screening and chemoproteomic methods and test metabolites in vivo by colonizing germ-free mice with genetically manipulated bacterial strains. The proposed work is grounded on my strong expertise in host-microbe interactions and takes advantage of the state-of-the-art biochemistry facilities at my hosting institution and of the complementary skillsets of my collaboration network. This synergistic combination will allow for a comprehensive interrogation of immunological tolerance to gut commensals: from metabolites and their molecular targets to their functional relevance for intestinal health.