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Mapping Cytokine Signalling Networks using Engineered Surrogate Ligands

Periodic Reporting for period 3 - Cytokine Signalosome (Mapping Cytokine Signalling Networks using Engineered Surrogate Ligands)

Reporting period: 2020-04-01 to 2021-09-30

Cells use an intricate network of intracellular signaling molecules to translate environmental changes, sensed via surface receptors, into cellular responses. Despite the prominent role that these networks play in life itself, we still lack a comprehensive understanding of how extracellular information is conveyed through them into specific bioactivities and fate decisions. In order to rationally manipulate cell fate, which could fundamentally change the way that we currently treat human diseases, first we need a systematic understanding of how signaling is initiated and propagated inside the cell. My primary research interest is to understand the cellular and molecular determinants of signaling specificity by cell surface receptors.

To address this important question, we use cytokines as a model system. Cytokines are a large family of secreted ligands that control every aspect of the mammalian physiology. They act as the major source of mid- and long-distance communication between cells, ensuring the coordinated response between different cell subsets. A clear example of this can be found in their regulation of the immune response. Cytokines critically contribute to initiate, maintain and define the type of immune response generated upon an external harm, thus making them highly relevant to human health. Indeed, cytokines are often found dysregulated in many human disorders including inflammation, cancer and auto-immunity. Despite their high clinical value, very few cytokines are used in the clinic, due to the high toxicity associated with their use. A better understanding of the molecular bases for cytokine-induced signaling and activities, could help us to design more efficient and less toxic therapies.

This program aims to tackle two important questions/objectives. The first one focuses on understanding the molecular bases defining signal activation by cytokines. The second one aims at use the information generated from objective 1 to engineer cytokine variants with a more defined set of biological responses. The primary research interest of the project is IL-6, a well-studied cytokine that plays a crucial role in immune regulation by controlling the intensity and duration of the inflammatory response. The information generated from these objectives will provide us with new insight into how cytokines elicit such complex biological responses, which in turn will allow us to design more targeted therapies with reduce toxicities.
Since the project started we have advanced significantly in the development of the objectives described in this program. The program is divided in two aims focused on understanding IL-6 functional pleiotropy in human T cells.

The first aim focuses on characterizing the signalling network engaged by IL-6 in T cells and how these networks contribute to fine-tune IL-6 responses. This project is the bases of a manuscript that we have recently deposited in BioRXiv and it is currently submitted. We have shown that in response to IL-6, STAT3 recruits CDK8 to responsive promoters, where CDK8 phosphorylate STAT3 on Ser, regulating its DNA binding dwell time and therefore its transcriptional potential. Overall, our study provides new molecular evidences that establish CDK8 as a master regulator of STAT3 transcriptional activities and present a new strategy to harness the therapeutic potential of cytokines by fine-tuning CDK8 expression levels and activities in different immune cells.

The second aim focuses on identifying the molecular determinants that define signal activation by the IL-6 receptor complex. On the one hand, we have engineered variants of IL-6 with altered IL-6 receptor binding properties and show that the kinetics of receptor internalization critically contribute to bias signalling responses by IL-6. This study was recently published in eLife. On the other hand, we have engineered additional IL-6 surrogate ligands using antibodies that bind gp130 in different epitopes. These antibodies produce unique signaling signatures that differ from those engaged by IL-6. Currently, we are defining the molecular basis for these differences using crystallography studies as well as single-particle TIRF microscopy.

Overall, the program in reaching the expected outcomes and it is well on its way to deliver the objective proposed in the application. The results generated from this program will provide us with mechanistic bases for cytokine signal activation, which in turn with set the guide-lines to design more defined and less toxic cytokine-based therapies.
To date we lack a clear understanding on how cytokines trigger unique signaling signatures and biological responses. This has hindered our ability to translate this important family of ligands to the clinic. The outcome of this program will move significantly forward our understanding of cytokine biology and provide us with a set of biophysical and structural boundaries determining signal activation potency and nature by cytokines. In principle, this could be exploited to engineer synthetic agonists with tailored functional properties to interrogate and manipulate cellular responses promoted by cytokines for potential pharmaceutical applications.