KRAS is one of the most frequently mutated proteins in cancer, driving the excessive activation of the downstream RAF-MEK-ERK cascade, alongside additional pathways that promote cellular proliferation, survival and migration. KRAS traffics to the plasma membrane in order to be activated and bind to its effector proteins. Despite almost 40 years of research, there has been limited success in direct targeting of KRAS, as well as attempts to inhibit KRAS localisation to the plasma membrane. While inhibitors to downstream pathway components have been clinically approved, patients generally develop resistance to these within a few months, highlighting the need to develop novel strategies to inhibit KRAS-driven signalling. Here, we have characterised reporter cell lines where induction of KRAS^G12V or BRAF^V600E with Doxycycline drives the ERK1/2-dependent expression of GFP. GFP expression can be used as a reliable and quantitative readout for ERK1/2 pathway activation, allowing these cell lines to be used to identify novel regulators of the pathway.

In a collaboration between the Cook lab and PhoreMost, PROTEINi screens identified two novel peptides with high sequence similarity, OPX-385670 and HPX-119370, that interfered with KRAS-driven signalling. In this thesis, we show that cell penetrating peptides derived from these, TAT-OPX and TAT-HPX, significantly inhibited pathway activation and subsequent GFP expression driven by mutant KRAS, but not mutant BRAF.

Yeast-2-hybrid analysis revealed ORP9 to be a potential interactor of OPX-385670, and VAP proteins to be potential interactors of HPX-119370. ORP9 is a lipid transfer protein, and interacts with VAPs in the ER to transfer newly synthesised lipids out of the ER and into target organelles, such as the Golgi and late endosomes. ORP9 lipid cargoes include phosphatidylserine and cholesterol, which can be transferred out of the ER membrane in exchange for PI4P, which is then dephosphorylated by the ER-resident Sac1 phosphatase. TAT-OPX and TAT-HPX caused accumulation of PI4P on Rab7-positive endosomes, suggesting inhibition of ORP9-mediated lipid transfer. ORP9 knockdown reduced MEK1/2 phosphorylation, ERK1/2 phosphorylation and GFP expression downstream of mutant KRAS, but not mutant BRAF, highlighting ORP9 as a regulator of KRAS-driven signalling.

As well as PI4P, TAT-OPX and TAT-HPX caused actin to accumulate on Rab7-positive endosomes, suggesting defects in actin nucleation. These actin puncta also co-localised with YFP-VPS29, a component of the retromer. The retromer is a coat complex that interacts with specific internalised cargo, budding off from early/late endosomes to deliver cargo back to the plasma membrane. This recycling relies on WASH-mediated actin nucleation for membrane fission and vesicle trafficking. CPP-mediated defects in actin nucleation coincided with inhibition of the retromer complex, determined by missorting of the retromer cargo GLUT1 from the plasma membrane to lysosomes. TAT-OPX and TAT-HPX, as well as ORP9 knockdown, also caused mislocalisation of KRAS from the plasma membrane in multiple cell lines, suggesting that the retromer may be involved in KRAS trafficking. Knockdown of VPS35, a retromer subunit, significantly inhibited KRAS-driven activation of the downstream pathway and expression of GFP, suggesting KRAS is a potential cargo of the retromer.

The results presented in this thesis suggest that ORP9 is a novel regulator of KRAS driven signalling, potentially promoting its recycling to the plasma membrane. This may occur through both retromer-dependent and -independent routes, and suggests that affecting ORP9-mediated endosomal lipid levels may be a viable approach to inhibit KRAS localisation to the plasma membrane, and subsequent activation of downstream signalling pathways.