David Oxley

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAF amplification whereas KRAS amplification promotes EMT-chemoresistance.
Sale MJ, Balmanno K, Saxena J, Ozono E, Wojdyla K, McIntyre RE, Gilley R, Woroniuk A, Howarth KD, Hughes G, Dry JR, Arends MJ, Caro P, Oxley D, Ashton S, Adams DJ, Saez-Rodriguez J, Smith PD, Cook SJ

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAF or KRAS to reinstate ERK1/2 signalling. Here we show that BRAF amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAF amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAF. p57 expression is required for loss of BRAF amplification and reversal of MEKi resistance. Thus, BRAF amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRAS amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRAS amplification.

Nature communications, 10, 2041-1723, 2030, 2019

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
Tsolakos N, Durrant TN, Chessa T, Suire SM, Oxley D, Kulkarni S, Downward J, Perisic O, Williams RL, Stephens L, Hawkins PT

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

Proceedings of the National Academy of Sciences of the United States of America, 115, 1091-6490, 12176-12181, 2018

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.
Olova N, Krueger F, Andrews S, Oxley D, Berrens RV, Branco MR, Reik W

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

Genome biology, 19, 1474-760X, 33, 2018