One of the keys to understanding lifelong health is to understand the signalling pathways that operate inside cells and govern key fate decisions such as cell death, cell survival, cell division or cell senescence (collectively cell longevity). These signalling pathways involve enzymes called ‘protein kinases’ that attach phosphate groups to specific cellular proteins, thereby controlling their activity, location or abundance. In this way protein kinases orchestrate the cellular response to growth factors, nutrient availability or stress and damage.
Ageing results in part from the imbalance between cellular damage, accrued throughout life, and the progressive decline in stress response and repair pathways. We are interested in how protein kinases function in stress responses, the removal of damaged cellular components (e.g. autophagy, see also Nicholas Ktistakis and Oliver Florey) and the control of cellular lifespan. We believe this will enhance our understanding of how the normal declines in these processes drive ageing.
Signalling pathways are frequently de-regulated in certain age-related diseases – notably in cancer, inflammation and neurodegeneration – and many protein kinases are attractive drug targets. Consequently we translate our basic knowledge of signalling through collaborations with charities and pharmaceutical companies (e.g. AstraZeneca and MISSION Therapeutics).
Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing are poorly understood. Here, we investigate amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells, a well-characterized model for reproducible emergence of drug resistance, and show that amplifications acquired are the primary cause of resistance. Selumetinib causes long-term G1 arrest accompanied by reduced expression of DNA replication and repair genes, but cells stochastically re-enter the cell cycle during treatment despite continued repression of pERK1/2. Most DNA replication and repair genes are re-expressed as cells enter S and G2; however, mRNAs encoding a subset of factors important for error-free replication and chromosome segregation, including TIPIN, PLK2 and PLK3, remain at low abundance. This suggests that DNA replication following escape from G1 arrest in drug is more error prone and provides a potential explanation for the DNA damage observed under long-term RAF-MEK-ERK1/2 pathway inhibition. To test the hypothesis that escape from G1 arrest in drug promotes amplification, we exploited the combination of palbociclib and selumetinib. Combined treatment with selumetinib and a dose of palbociclib sufficient to reinforce G1 arrest in selumetinib-sensitive cells, but not to impair proliferation of resistant cells, delays the emergence of resistant colonies, meaning that escape from G1 arrest is critical in the formation of resistant clones. Our findings demonstrate that acquisition of MEK inhibitor resistance often occurs through gene amplification and can be suppressed by impeding cell cycle entry in drug.
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is frequently de-regulated in human cancer. Melanoma in particular exhibits a high incidence of activating BRAF and NRAS mutations and such cells are addicted to the activity of these mutant oncoproteins. As a result three different BRAF inhibitors (BRAFi) have now been approved for BRAFV600E/K- mutant melanoma and have transformed the treatment of this disease. Despite this, clinical responses are typically transient as tumour cells develop resistance. These resistance mechanisms frequently involve reinstatement of ERK1/2 signalling and BRAFi are now deployed in combination with one of three approved MEK1/2 inhibitors (MEKi) to provide more durable, but still transient, clinical responses. Furthermore, inhibitors to ERK1/2 (ERK1/2i) have also been developed to counteract ERK1/2 signalling. However, recent studies have suggested that BRAFi/MEKi and ERK1/2i resistance can arise through activation of a parallel signalling pathway leading to activation of ERK5, an unusual protein kinase that contains both a kinase domain and a transcriptional transactivation domain. Here we review the evidence supporting ERK5 as a mediator of BRAFi/MEKi and ERK1/2i resistance. We also review the challenges in targeting ERK5 signalling with small molecules, including paradoxical activation of the transcriptional transactivation domain, and discuss new therapeutic modalities that could be employed to target ERK5.
The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
DNA REPLICATION DURING ACUTE MEK INHIBITION DRIVES ACQUISITION OF RESISTANCE THROUGH AMPLIFICATION OF THE BRAF ONCOGENE Prasanna Channathodiyil, Anne Segonds-Pichon, Paul D. Smith, Simon J. Cook, Jonathan Houseley bioRxiv 2021.03.23.436572https://doi.org/10.1101/2021.03.23.436572