Abstract
Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. Therefore, molecules able to interfere with mutant KRAS protein are potentially important for wide-ranging tumour therapy. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. A KRAS-specific DARPin fused to the VHL E3 ligase is compared to a pan-RAS intracellular single domain antibody (iDAb) fused to the UBOX domain of the CHIP E3 ligase. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, however, affects proliferation irrespective of the RAS mutation. These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations.
Highlights
Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids
Mutations of the KRAS oncogene represent more than 85% of all RAS family mutations[1] and individual mutations occur at various codons giving rise to many forms of mutant KRAS protein[2]
A potential complementary direction in this context could be the addition of warheads to these macromolecules[25], such as single chain Fragment variable fused to proteasome targeting sequences[26] or E3 ligases engineered on intracellular single domains, called macrodrug degraders, that have been shown to invoke proteolysis of targets[27,28,29,30,31,32]
Summary
Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, affects proliferation irrespective of the RAS mutation These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations. Only a small portion of mutant KRAS tumours expresses a KRASG12C protein (around 12%, Cosmic database v91, https://cancer.sanger.ac.uk/cosmic) and can be targeted by these inhibitors. Macromolecule degraders induce the depletion of their target via the ubiquitinproteasome system They consist of a binder targeting a protein of interest (e.g. intracellular single domain), a linker and an E3 ligase domain. A similar protein target degradation strategy has been developed in which small molecules that bind proteins are linked to E3 ligase-binding ligands called proteolysis targeting chimeras (PROTACs) or small molecule degraders[33,34]
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