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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.

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-xL and Mcl-1. Upon serum withdrawal, newly expressed BimEL associates with Bcl-xL 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 BimEL/Mcl-1 and BimEL/Bcl-xL complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for BimEL and requires ERK1/2-dependent phosphorylation of BimEL at Ser65. Finally, ERK1/2-dependent dissociation of BimEL from Mcl-1 and Bcl-xL may play a role in regulating BimEL degradation, since mutations in the BimEL BH3 domain that disrupt binding to Mcl-1 cause increased turnover of BimEL. These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway. [ABSTRACT FROM AUTHOR]

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of ?MEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38a but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21CIP1 expression is silenced by methylation, ?MEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G1 arrest. Furthermore, p21CIP1-/- 3T3 cells were defective for the ?MEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated ?MEKK3:ER* inhibits the G1 ? S progression by two kinetically distinct mechanisms, with expression of p21CIP1 being required to ensure a sustained G1 cell cycle arrest. The ERK1/2 and p38aß pathways cooperated to induce p21CIP1 expression and inhibition of p38aß caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by ?Raf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by ?MEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G1 ? S transition.Oncogene (2004) 23, 3284-3295. doi:10.1038/sj.onc.1207467 Published online 23 February 2004 [ABSTRACT FROM AUTHOR]

CC139 fibroblasts are one of several model systems in which the Raf?MEK?ERK1/2 pathway can inhibit apoptosis independently of the PI3K pathway; however, the precise mechanism for this protective effect is not known. Serum withdrawal from CC139 fibroblasts resulted in the rapid onset of apoptosis, which was prevented by actinomycin D or cycloheximide. Serum withdrawal promoted the rapid, de novo accumulation of BimEL, a proapoptotic 'BH3-only' member of the Bcl-2 protein family. BimEL expression was an early event, occurring several hours prior to caspase activation. In contrast to studies in neurons, activation of the JNK?c-Jun pathway was neither necessary nor sufficient to induce BimEL expression. Selective inhibition of either the ERK pathway (with U0126) or the PI3K pathway (with LY294002) caused an increase in the expression of BimEL. Furthermore, selective activation of the ERK1/2 pathway by ?Raf-1:ER* substantially reduced BimEL expression, abolished conformational changes in Bax and blocked the appearance of apoptotic cells. The ability of ?Raf-1:ER* to repress BimEL expression required the ERK pathway but was independent of the PI3K?PDK?PKB pathway. Thus, serum withdrawal-induced expression of BimEL occurs independently of the JNK?c-Jun pathway and can be repressed by the ERK pathway independently of the PI3K pathway. This may contribute to Raf- and Ras-induced cell survival at low serum concentrations.Oncogene (2003) 22, 1281-1293. doi:10.1038/sj.onc.1206261 [ABSTRACT FROM AUTHOR]

Whilst many studies have examined the role of the MAP Kinases in regulating the G1→S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant ΔMEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of ΔMEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of ΔMEKK3:ER* resulted in the up-regulation of p21CIP1 and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. ΔMEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21CIP1, since both events were observed in Rat-1 cells in which p21CIP1 is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38α/β2 pathway can promote a G2 cell cycle arrest.Oncogene (2002) 21, 8089–8104. doi:10.1038/sj.onc.1206000 [ABSTRACT FROM AUTHOR]

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21Cip1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent... [ABSTRACT FROM AUTHOR]

The pharmacokinetics and pharmacodynamics of prolonged oral etoposide chemotherapy were investigated in 15 women with metastatic breast cancer who received oral etoposide 100 mg as a single daily dose for up to 15 days. There was considerable interpatient variability in the day 1 pharmacokinetic parameters: area under the plasma concentration time curve (AUC) (0-24 h) 1.95±0.87 mg/ml per min (mean ± SD), apparent oral clearance 60.9±21.7 ml/min per 1.73 m, peak plasma concentration 5.6±2.5 μg/ml, time to peak concentration 73±35 min and half-life 220±83 min. However, intrapatient variability in systemic exposure to etoposide was much less with repeated doses. The intrapatient coefficient of variation (CV) of AUC for day 8 relative to day 1 was 20% and for day 15 relative to day 1 was 15%, compared to the day 1 interpatient CV of 45%. Neutropenia was the principal toxicity. Day 1 pharmacokinetic parameters were related to the percentage decrease in absolute neutrophil count using the sigmoidal E equation. A good fit was found between day 1 AUC and neutrophil toxicity ( R=0.77). All patients who had a day 1 AUC>2.0 mg/ml per min had WHO grade III or IV neutropenia. The predictive performance of the models for neutrophil toxicity was better for AUC (percentage mean predictive error 5%, percentage root mean square error 18.1%) than apparent oral clearance, peak plasma concentration, or daily dose (mg/m). A limited sampling strategy was developed to predict AUC using a linear regression model incorporating a patient effect. Data sets were divided into training and test sets. The AUC could be estimated using a model utilizing plasma etoposide concentration at only two time points, 4 h and 6 h after oral dosing ( R=98.9%). The equation AUC=−0.376+0.631×C+0.336×C was validated on the test set with a relative mean predictive error of −0.88% and relative root mean square error of 6.4%. These results suggest monitoring of AUC to predict subsequent myelosuppression as a strategy for future trials with oral etoposide. [ABSTRACT FROM AUTHOR]

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. Colorectal cancer cells can acquire resistance to MEK inhibition due to BRAF or KRAS amplification. Here, the authors show that while MEK inhibitor withdrawal in BRAF mutant cells restores sensitivity to the inhibitor through the loss of BRAF amplification mediated by a p57-dependent mechanism, drug withdrawal from KRAS mutant cells does not restore sensitivity but results in EMT and chemoresistance. [ABSTRACT FROM AUTHOR]

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-xL and Mcl-1. Upon serum withdrawal, newly expressed BimEL associates with Bcl-xL 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 BimEL/Mcl-1 and BimEL/Bcl-xL complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for BimEL and requires ERK1/2-dependent phosphorylation of BimEL at Ser65. Finally, ERK1/2-dependent dissociation of BimEL from Mcl-1 and Bcl-xL may play a role in regulating BimEL degradation, since mutations in the BimEL BH3 domain that disrupt binding to Mcl-1 cause increased turnover of BimEL. These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway. [ABSTRACT FROM AUTHOR]

Bim, a ‘BH3-only’ protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK½ pathway promotes phosphorylation of BimEL, targeting it for degradation via the proteasome. However, the nature of the kinase responsible for BimEL phosphorylation remained unclear. We now show that BimEL is phosphorylated on at least three sites in response to activation of the ERK½ pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK½-dependent phosphorylation of BimEL occurs at (S/T)P motifs. ERK½ phosphorylates BimEL, but not BimS or BimL, in vitro, and mutation of Ser65 to alanine blocks the phosphorylation of BimEL by ERK½ in vitro and in vivo and prevents the degradation of the protein following activation of the ERK½ pathway. We also find that ERK½, but not JNK, can physically associate with GST-BimEL, but not GST-BimL or GST-BimS, in vitro. ERK½ also binds to full-length BimEL in vivo, and we have localized a potential ERK½ ‘docking domain’ lying within a 27-amino acid stretch of the BimEL protein. Our findings provide new insights into the post-translational regulation of BimEL and the role of the ERK½ pathway in cell survival signaling. [ABSTRACT FROM AUTHOR]

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.

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, 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.

The conditional protein kinase DeltaMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of DeltaMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of Bim(EL) and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of Bim(EL) but caused only a partial reversion of the cyto-protective effect of DeltaMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing DeltaMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IkappaB kinase. Activation of DeltaMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death.

Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by growth factors, we now show that activation of ERK1/2 by DeltaRaf-1:ER is insensitive to cAMP. Despite this, DeltaRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK1/2. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit DeltaRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. DeltaRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21(CIP1). cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27(KIP1); however, loss of p27(KIP1) in response to DeltaRaf-1:ER was less sensitive in CCl39 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBS- and DeltaRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK1/2 activation may operate early in G(1), but when this early block is bypassed by DeltaRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.