ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.

Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.

IKKα plays a major role in canonical NF-κB signalling in colorectal cells.

Inhibitor of kappa B (IκB) kinase ß (IKKß) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKß in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKß inhibitors. IKKα and IKKß were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKß alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKß was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKß. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKß inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKß contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKß to date.

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAFV600E amplification whereas KRASG13D amplification promotes EMT-chemoresistance.

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E 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 p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D 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 KRASG13D amplification.

Resistance to ERK1/2 pathway inhibitors; sweet spots, fitness deficits and drug addiction.

MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma, where they are used in combination with BRAF inhibitors, and are undergoing evaluation in other malignancies. Acquired resistance to MEK1/2 inhibitors, including selumetinib (AZD6244/ARRY-142866), can arise through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. We have found that BRAFV600E amplification and selumetinib resistance are fully reversible following drug withdrawal. This is because resistant cells with BRAFV600E amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling (2%-3% of total ERK1/2 active), that is optimal for cell proliferation and survival. Selumetinib withdrawal drives ERK1/2 activation outside of this critical "sweet spot" (~20%-30% of ERK1/2 active) resulting in a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy; these terminal responses select against cells with amplified BRAFV600E. ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of selumetinib resistance. Growth of selumetinib-resistant cells with BRAFV600E amplification as tumour xenografts also requires the presence of selumetinib to "clamp" ERK1/2 activity within the sweet spot. Thus, BRAFV600E amplification confers a selective disadvantage or "fitness deficit" during drug withdrawal, providing a rationale for intermittent dosing to forestall resistance. Remarkably, selumetinib resistance driven by KRASG13D amplification/upregulation is not reversible. In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents. Our results reveal that the emergence of drug-addicted, MEKi-resistant cells, and the opportunity this may afford for intermittent dosing schedules ("drug holidays"), may be determined by the nature of the amplified driving oncogene (BRAFV600E vs. KRASG13D), further exemplifying the difficulties of targeting KRAS mutant tumour cells.

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-µ at the pre-BCR checkpoint.

Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome.

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B(S421A) mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B(D239A) mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS(G12V). In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

Adaptation to mTOR kinase inhibitors by amplification of eIF4E to maintain cap-dependent translation.

The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2-ERK1/2 pathway.

Adaptation to chronic mTOR inhibition in cancer and in aging.

The mTOR [mammalian (or mechanistic) target of rapamycin] protein kinase co-ordinates catabolic and anabolic processes in response to growth factors and nutrients and is a validated anticancer drug target. Rapamycin and related allosteric inhibitors of mTORC1 (mTOR complex 1) have had some success in specific tumour types, but have not exhibited broad anticancer activity, prompting the development of new ATP-competitive mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2. In common with other targeted kinase inhibitors, tumours are likely to adapt and acquire resistance to mTOR inhibitors. In the present article, we review studies that describe how tumour cells adapt to become resistant to mTOR inhibitors. mTOR is a central signalling hub which responds to an array of signalling inputs and activates a range of downstream effector pathways. Understanding how this signalling network is remodelled and which pathways are invoked to sustain survival and proliferation in the presence of mTOR inhibitors can provide new insights into the importance of the various mTOR effector pathways and may suggest targets for intervention to combine with mTOR inhibitors. Finally, since chronic mTOR inhibition by rapamycin can increase lifespan and healthspan in nematodes, fruitflies and mice, we contrast these studies with tumour cell responses to mTOR inhibition.

Regulation of MEK/ERK pathway output by subcellular localization of B-Raf.

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.

Tumour cell responses to MEK1/2 inhibitors: acquired resistance and pathway remodelling.

The Raf/MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2]/ERK1/2 signalling pathway is frequently activated in human tumours due to mutations in BRAF or KRAS. B-Raf and MEK1/2 inhibitors are currently undergoing clinical evaluation, but their ultimate success is likely to be limited by acquired drug resistance. We have used colorectal cancer cell lines harbouring mutations in B-Raf or K-Ras to model acquired resistance to the MEK1/2 inhibitor selumetinib (AZD6244). Selumetinib-resistant cells were refractory to other MEK1/2 inhibitors in cell proliferation assays and exhibited a marked increase in MEK1/2 and ERK1/2 activity and cyclin D1 abundance when assessed in the absence of inhibitor. This was driven by a common mechanism in which resistant cells exhibited an intrachromosomal amplification of their respective driving oncogene, B-Raf V600E or K-RasG13D. Despite the increased signal flux from Raf to MEK1/2, resistant cells maintained in drug actually exhibited the same level of ERK1/2 activity as parental cells, indicating that the pathway is remodelled by feedback controls to reinstate the normal level of ERK1/2 signalling that is required and sufficient to maintain proliferation in these cells. These results provide important new insights into how tumour cells adapt to new therapeutics and highlight the importance of homoeostatic control mechanisms in the Raf/MEK1/2/ERK1/2 signalling cascade.

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL.

The pro-apoptotic BH3 only protein BIM(EL) is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM(EL) to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM(EL), which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5-/- fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM(EL) occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM(EL) drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM(EL) and may be relevant to the therapeutic use of agents such as paclitaxel.

A correction to the research article titled: "Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells" by A. S. Little, K. Balmanno, M. J. Sale, S. Newman, J. R. Dry, M. Hampson, P. A. W. Edwards, P. D. Smith, S. J. Cook.

The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal­regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal­regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF600E or KRAS13D, due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF600E. Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF600E or KRAS13D) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS13D leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.

Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells.

The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF(600E) or KRAS(13D), due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF(600E). Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF(600E) or KRAS(13D)) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS(13D) leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.

Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics.

The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.

Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY-142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines.

Mutations in KRAS or BRAF frequently manifest in constitutive activation of the MEK1/2-ERK1/2 signalling pathway. The MEK1/2-selective inhibitor, AZD6244 (ARRY-142886), blocks ERK1/2 activation and is currently undergoing clinical evaluation. Tumour cells can vary markedly in their response to MAPK or ERK kinase (MEK) inhibitors, and the presence of a BRAF mutation is thought to predict sensitivity, with the RAS mutations being associated with intrinsic resistance. We analysed cell proliferation in a panel of 19 colorectal cancer cell lines and found no simple correlation between BRAF or KRAS mutation and sensitivity to AZD6244, though cells that harbour neither mutation tended to be resistant. Cells that were sensitive arrested in G(1) and/or underwent apoptosis and the presence of BRAF or KRAS mutation was not sufficient to predict either fate. Cell lines that were resistant to AZD6244 exhibited low or no ERK1/2 activation or exhibited coincident activation of ERK1/2 and protein kinase B (PKB), the latter indicative of activation of the PI3K pathway. In cell lines with coincident ERK1/2 and PKB activation, sensitivity to AZD6244 could be re-imposed by any of the 3 distinct PI3K/mTOR inhibitors. We conclude that AZD6244 is effective in colorectal cancer cell lines with BRAF or KRAS mutations. Sensitivity to MEK1/2 inhibition correlates with a biochemical signature; those cells with high ERK1/2 activity (whether mutant for BRAF or KRAS) evolve a dependency upon that pathway and tend to be sensitive to AZD6244 but this can be offset by high PI3K-dependent signalling. This may have implications for the use of MEK inhibitors in combination with PI3K inhibitors.

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.