Establishing a rationale for selectively blocking mTORC1 fibrogenic signalling in fibrosis and cancer using CRISPR-Cas9 genome editing

Fibrosis is the concluding pathological outcome and major cause of morbidity and mortality in a number of common chronic inflammatory, immune-mediated and metabolic diseases [1]. The progressive and relentless deposition of a collagen-rich matrix is the cornerstone of the fibrotic response and culminates in organ failure and premature death. Epithelial dysfunction is recognised as a key mechanism underlying the development of fibrosis with type II alveolar epithelial cells (AEC2s), the major stem cells of the distal adult lung that maintain lung homeostasis and contribute to epithelial repair, showing an impaired renewal capacity. Despite the rising incidence of fibrotic disease and intense research efforts, there remains a pressing need to develop novel anti-fibrotic therapeutic strategies.

Previous studies from our laboratory underpinned the scientific rationale for progressing the potent PI3K/mTOR inhibitor, Omipalisib, to a first-in-human proof of mechanism trial in patients with idiopathic pulmonary fibrosis (IPF), based on the potential of this compound to interfere with fibroblast function, including TGFβ1-induced collagen deposition [1, 2]. Mechanistic target of rapamycin (mTOR) is a nodal serine/threonine protein kinase which regulates key cellular processes. mTOR forms the catalytic subunit of two complexes, mTORC1 and mTORC2, which are activated by different upstream inputs, elicit different downstream responses, and exhibit differential sensitivity to the allosteric partial mTORC1 inhibitor, rapamycin. We recently reported a key role for mTORC1 in mediating the potent fibrogenic effects of TGFβ1 in primary human lung fibroblasts (pHLFs) and in cancer associated fibroblasts (CAFs) [3, 4]. ATP competitive dual mTOR inhibitors (targeting mTORC1 and mTORC2) are currently in development but the tolerability of long-term dual mTORC1 and mTORC2 inhibition remains unknown. Our recent observation that the TGFβ1-induced collagen response is mediated via the rapamycin-insensitive mTORC1/4E-BP1 axis raises the prospect of developing strategies to selectively inhibit this axis in the context of multiple fibrotic conditions. However, to date, there are no specific mTORC1 inhibitors available so that novel strategies aimed at targeting the mTORC1/4EBP1 axis need to be explored. Unpublished microarray data of laser-captured myofibroblasts from human IPF lung tissue from our laboratory further revealed that the TSC2/Rheb axis, a major upstream regulator of mTORC1 activation formed one of the major cell signalling clusters that correlated with collagen gene expression. Taken together these observations provide human tissue-based evidence for a potential role for Rheb in influencing mTORC1 activation and fibrogenesis in the context of a fibrotic tissue microenvironment.

The overall aim of this project is therefore to perform target validation using gene-editing, genomics and informatics approaches to support the scientific rationale of targeting Rheb as a novel therapeutic approach in the context of fibrosis and the stromal reaction in cancer.

This project will address three objectives:
1. Determine whether Rheb is involved in mediating TGFβ1-induced mTORC1 activation and collagen production in Rheb-deficient IPF fibroblasts and CAFs from multiple donors following CRISPR-Cas9 gene editing
2. Define the involvement of Rheb in regulating the TGFβ1-induced transcriptome and translatome in Rheb-deficient IPF fibroblasts and CAFs using RNA-Seq and Frac-Seq in order to further our understanding of the mechanism by which Rheb influences the fibrogenic response
3. Determine the impact of Rheb on lung epithelial regenerative potential using CRISPR-Cas9 gene editing of alveolar organoids

Applications
Applications must be complete, including both references, by 24th Jan 2020