Investigating the role of galectins in oncogenic KRAS signalling

About the Project

Background

The RAS family of small GTPases serves as signalling hubs that regulate cell proliferation and differentiation. Notably, approximately 18% of all human cancers contain mutations in KRAS, one of the RAS isoforms (Catalogue Of Somatic Mutations In Cancer (COSMIC)). Although recent advancements in targeting oncogenic KRAS molecules with small-molecule inhibitors have shown promise for treating KRAS-driven cancers, their relatively low efficacy and the quick development of acquired resistance underscore the need for the continued development of additional treatment strategies (1).

KRAS activation is triggered by epidermal growth factor (EGF), which induces oligomerisation of both the EGF receptor (EGFR) and KRAS at the plasma membrane, with the help of scaffold and adaptor proteins. Activated KRAS then transduce the signal to multiple effector molecules via direct protein-protein interactions. Galectins 1 and 3 (GAL1/3) play critical roles in these molecular assemblies, and the possibility of using GAL1/3 inhibitors to treat KRAS-driven cancers has been proposed (2, 3). However, the mechanisms by which GAL1/3 participates in the EGFR and KRAS assemblies remain elusive, hindering the development of effective GAL1/3 inhibitors. This project aims to obtain molecular and structural insights into GAL1/3’s roles in the EGFR-KRAS signalling and to facilitate the development of small-molecule inhibitors targeting GAL1/3. Specific objectives are as follows.

Objective 1: Galectin localisation studies

Using CRISPR-Cas9 gene editing technology, we recently generated an isogenic set of human cell lines harbouring wildtype KRAS or three common oncogenic KRAS mutations, G12V, KRAS.G12C and KRAS.G12D, at the endogenous KRAS locus (4). Using these KRAS.G12X cell lines as a parental cell line, we will introduce SNAP and Halo-tagged galectin genes. SNAP and Halo-tags will allow us to conduct single-molecule localisation microscopy (SMLM) to observe individual galectin molecules in the cell (super-resolution microscopy). We will compare the spatio-temporal behaviour of the galectin molecules in the wildtype KRAS and oncogenic KRAS.G12X cells.

Objective 2: Assessment of galectin inhibitors

We will examine available galectin inhibitors (5, 6) for their effects on the EGFR/KRAS spatiotemporal localisation patterns through SMLM. We also use the KRAS.G12X cell lines to examine whether the oncogenic KRAS.G12X cells are more sensitive to the galectin inhibitors compared to the wildtype KRAS cells. This part of the project will be conducted in collaboration with Prof. Ulf Nilsson (Lunt University, Sweden, an expert on galectins and their inhibitor development).

Objective 3: Biochemical and structural analysis of galectins and KRAS

It has been proposed that GAL3 interacts with farnesylated KRAS (7). However, the precise structural insights remain elusive. We will prepare recombinant farnesylated KRAS and examine whether recombinant GAL1/3 directly interact with it in vitro. When interaction is confirmed, we will conduct SMLM and biochemical and structural analysis of the complex.

Training opportunities

The project involves a wide range of experimental approaches, maximising the research experience. The relevant techniques include CRISPR-Cas9 mediated gene editing of human culture cells, SMLM using total internal reflection fluorescence (TIRF) microscopy and High-Low (HiLo) microscopy, in vitro recombinant protein expression and purification, X-ray crystallography and NMR. The project involves national and international collaborations.

Outputs

The ultimate goal of this project is to establish galectin inhibitors as a combinational treatment strategy to enhance existing anti-KRAS therapeutics. Successful delivery of the project will facilitate the development of effective treatment options for KRAS-driven cancers.

The PhD student will present the project outcomes at international meetings/conferences and publish the results in a peer-reviewed journal.

Enquiries

Project Enquiries to kt96@le.ac.uk

To apply please refer to

https://le.ac.uk/study/research-degrees/research-subjects/molecular-and-cell-biology