Mutations in RAS oncogenes are responsible for driving some 20% of all human malignancies, occurring in many major killers, such as lung, pancreatic, and colon cancers, but attempts to develop therapeutic interventions for RAS mutant cancers have yet to provide clinical benefit. By inhibiting pathways downstream of RAS along with other key signaling nodes, we have developed combination therapies that cause major regression of KRAS mutant lung cancer in mouse models. However, a major limitation is that the tumours are not eradicated and rapidly recur once treatment is withdrawn. Lung cancer is partly responsive to immunotherapies in the clinic, suggesting dependence on immune evasive signaling. We would like to understand whether RAS driven oncogenic signaling pathways act to protect tumours from the immune system. If so, what mechanisms does RAS use to evade tumour immune destruction and can these be specifically targeted to unleash the immune system on the tumour? Could we develop effective therapies rationally combining these with our existing RAS pathway therapies to achieve complete tumour eradication? We will use clinical samples to establish whether activation of RAS signaling pathways correlates with the ability of lung tumours to evade the immune system and by what mechanisms. We will develop appropriate preclinical models to test the impact of targeting immune evasion in RAS driven lung cancer, recognising the major limitations of existing mouse models for this purpose. We will also utilize these immunogenic preclinical models to seek novel mechanisms of tumour immune evasion, including through the use of in vivo functional genomic screens. Finally, we will establish how our existing optimal strategies for achieving RAS signaling pathway inhibition in lung cancer impact on the tumour immune microenvironment and establish strategies for combining these with interventions to subvert immune evasion, thus enabling optimal immune-assisted tumour destruction.

Maintenance of genome stability is challenged by obstacles that interfere with normal progression of essential DNA-associated transactions, such as DNA replication and transcription. One such obstacle is the G-quadruplex (G4) DNA secondary structure, which can form in G-rich repetitive DNA sequences. Transcribed G4-DNA loci often co-exist with stable RNA-DNA hybrids (R-loops), potentially causing deleterious transcription-replication conflicts (TRCs). Therefore, the ability of cells to mitigate TRCs formation is critical for cell and organismal fitness. While the pathways involved in repairing other DNA lesions are relatively well understood, the mechanisms by which cells respond to G4/R-loop-induced TRCs remain uncharted, largely due to a lack of efficient approaches for inducing site-specific G4s/R-loops into the genome. Several studies reported dedicated helicases capable of unwinding G4s/R-loops, yet whether additional factors exist and how different types of TRCs are resolved remains unknown. In this project, I aim to dissect the fundamental mechanisms that protect cells from TRCs at DNA loci harbouring G4s/R-loops. To do so, I will establish a novel system with site-specific G4/R-loop substrates in living cells and I will systematically investigate their protein composition to identify novel factors in G4/R-loop metabolism. I will further examine the genetic vulnerabilities of candidate G4/R-loop helicase-deficient cells and the regulatory mechanisms underlying G4/R-loop unwinding by these helicases. As mutations in several G4/R-loop helicases predispose to various cancers and genetic disorders, a detailed understanding of the basic mechanisms controlling G4/R-loop removal is of utmost importance, and will shed light on disease aetiology and the rational development of more targeted therapeutic strategies.

MicroRNAs (miRNAs) can be transferred between cells, representing an exciting new dimension to intercellular communication, referred to as non-cell-autonomous gene regulation. We recently identified that distinct miRNAs are packaged and exported from TREG cells and delivered directly to TH1 cells, suppressing T cell-mediated disease. Different T cell populations express different miRNAs and release a distinctive set of extracellular miRNAs. In this proposal we will identify whether the transfer of miRNAs between cells contributes to T cell development, T cell differentiation and TH2-mediated allergy and anti-helminth immunity. miRNA-mediated gene silencing requires one of four catalytically active Argonaut (Ago) proteins to regulate gene expression. To investigate miRNA transport between cells, we have generated novel mice with miRNA-deficient T cells that can (Dicer–/–) or cannot (Dicer–/–Ago-1,-3,-4–/– Ago-2fl/fl) respond to exogenous miRNAs. Using these novel mice we will identify which Ago protein(s) specific miRNAs associate with and which Ago proteins are required for miRNA-mediated gene regulation in T cells. TH2 cells express unique miRNAs, which can be found within TH2 cells and in extracellular vesicles released from TH2 cells. We have generated several new TH2-associated miRNA-deficient mice to investigate the cell intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) role of these miRNAs in TH2-mediated allergy and anti-helminth immunity. Studies in plants and worms have identified various mechanisms of RNA transfer between cells, involving cell-contact dependent and independent mechanisms. We will translate these observations into mammalian systems and identify the mechanisms of miRNA transfer. Results from this work will identify novel miRNA-mediated pathways and incentivise state-of-the-art approaches for novel therapeutic intervention to treat inflammatory diseases.

Cells have strict control over their size to ensure proper cell physiology. They also have ways to correct for deviations, referred to as cell-size homeostasis, leading to a very narrow distribution in cell size at division. Although some of the genes involved in this process have been identified, the underlying molecular mechanism has remained elusive. One possibility is that the cell can “sense” its size through a protein whose concentrations are related to cell-size ‒ known as the sizer model. However, this would not completely explain the fact that diploid cells are approximately twice the size of haploids, suggesting that ploidy also plays a role ‒ something that has not garnered a lot of attention in the field. Live-cell fluorescence microscopy is well-suited to address these issues because we can directly visualize the dynamics and localization patterns of sizers, along with cell-size, and quantify these characteristics. I propose to develop a live single-cell fluorescence microscopy approach using a microfluidics device that allows mother cell lineages to be tracked across multiple generations, for use with haploid and diploid strains of the unicellular eukaryotic organism, Schizosaccharomyces pombe. This will allow me to measure protein levels of sizer candidates and their localization, along with cell length, across single lineages. This information could reveal long-term dynamics and correlations between sizer candidates and cell-size, and provide insight into the molecular mechanisms governing cell-size homeostasis and the role that ploidy plays in governing cell-size control within eukaryotes.

The engulfment of dead cells by type 1 conventional dendritic cells (cDC1s) is essential to maintain tolerance to self-antigens and to induce immunity to infected or malignant cells. cDC1s have the unique ability to engulf dead cells, in contrast to the closely related cDC2s. Moreover, uptake of apoptotic cells induces cDC1 maturation and serves as their key source of antigens. However, we still do not understand which receptors on cDC1s are involved in the recognition and internalisation of dead cell corpses and whether these receptors differ depending on the type of cell death (e.g. apoptosis vs necrosis) that led to corpse formation. Remarkably, our preliminary data indicates that cDC1s, unlike macrophages, 1) do not express many of the classical or canonical engulfment receptors, 2) they tend to nibble pieces from dead cells and 3) are able to ingest parts of live cells. Hence, in the proposed work, I aim to characterise live, apoptotic and necrotic cell engulfment by cDC1s and its phosphatidylserine-dependency using novel reagents and state-of-the-art instrumentation such as high-resolution live cell microscopy. Furthermore, I will use a validated CRISPR-editing protocol and establish a CRISPR screen with both primary and immortalized cDC1 cell lines to discover which receptors on cDC1s are necessary for the engulfment of live, apoptotic and/or necrotic cells. Lastly, I will interrogate in vivo the role of key cDC1-dependent phagocytic receptors in a tumour and tolerance model using novel markers for DC maturation that I identified during my PhD. Altogether, the work in this proposal will elucidate a long-standing question in the DC and phagocytosis field, contribute to our knowledge on how cDC1s mediate anti-tumour immunity and tolerance, and may open new strategies to improve anti-tumour immunity and tolerance to self.