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.This work was funded by the Biotechnology and Biological Sciences Research Council, a Medical Research Council CASE studentship with GSK, an MRC centenary award (A.G) and project grants from Bloodwise. DJH was supported by a Medical Research Council Clinician Scientist FellowshipThis is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via http://dx.doi.org/10.1126/science.aad597

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

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 (BRAFV600Evs. KRASG13D), further exemplifying the difficulties of targeting KRAS mutant tumour cells

Thrombin inhibits Bim (Bcl-2-interacting mediator of cell death) expression and prevents serum-withdrawal-induced apoptosis via protease-activated receptor 1.

To investigate the role of thrombin in regulating apoptosis, we have used CCl39 cells, a fibroblast cell line in which thrombin-induced cell proliferation has been extensively studied. Withdrawal of serum from CCl39 cells resulted in a rapid apoptotic response that was completely prevented by the inclusion of thrombin. The protective effect of thrombin was reversed by pertussis toxin, suggesting that cell-survival signalling pathways are activated via a G(i) or G(o) heterotrimeric GTPase. Serum-withdrawal-induced death required de novo gene expression and was preceded by the rapid de novo expression of the pro-apoptotic 'BH3-only' protein Bim (Bcl-2-interacting mediator of cell death). Thrombin strongly inhibited the up-regulation of both Bim protein and Bim mRNA. The ability of thrombin to repress Bim expression, and to protect cells from apoptosis, was reversed by U0126, a MEK1/2 [MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) 1/2] inhibitor, or LY294002, a phosphoinositide 3'-kinase (PI3K) inhibitor, suggesting that both the Raf-->MEK-->ERK1/2 and PI3K pathways co-operate to repress Bim and promote cell survival. A PAR1p (protease-activated receptor 1 agonist peptide) was also able to protect cells from serum-withdrawal-induced apoptosis, suggesting that thrombin acts via PAR1 to prevent apoptosis

ΔRaf-1:ER* Bypasses the Cyclic AMP Block of Extracellular Signal-Regulated Kinase 1 and 2 Activation but Not CDK2 Activation or Cell Cycle Reentry

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 ΔRaf-1:ER is insensitive to cAMP. Despite this, ΔRaf-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 ΔRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. ΔRaf-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 ΔRaf-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 ΔRaf-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 ΔRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1

ER stress-induced inhibition of cap-dependent translation and loss of MCL1 is PERK-dependent.

<p><b>(A)</b> HCT116 cells were pre-treated for 1 h with the indicated concentration of GSK2606414 before addition of 100 nM Tg for 6 h. Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. <b>(B)</b> HCT116 cells were transfected with a dual luciferase reporter construct for assay of CAP/IRES-dependent translation. 24 h post-transfection, cells were pre-treated for 1 h with 100 nM GSK2606414 (GSK) before addition of 2 μg ml^-1 Tm or 1 μM AZD8055 for 24 h. Results shown are the mean ± S.D. luciferase activity within the whole cell lysates of one experiment performed in technical triplicate and are representative of three independent experiments. Statistics shown are the results of Student’s unpaired <i>t</i>-tests; N.S., not significant; *, p < 0.05. <b>(C)</b> HCT116 cells were pre-treated for 1 h with 100 nM GSK2606414 prior to the addition of the indicated concentration of Tm for 24 h. Whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. Results in (A) and (C) are representative of 3 independent experiments.</p

The PERK inhibitor GSK2606414 exacerbates ER stress-induced apoptosis.

<p><b>(A)</b> HCT116 cells were pre-treated for 1 h with 100 nM GSK2606414 prior to addition of the indicated concentration of Tm for 48 h. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results shown are the means ± S.D. of 3 independent experiments each performed in technical triplicate. Statistics represent the results of two-way ANOVA and Bonferroni post-tests; ***, p < 0.001. <b>(B)</b> HCT116 cells were treated as in (A) for 6 h (top panel) or 24 h (bottom panel). Whole cell lysates were analysed by immunoblotting using the indicated antibodies following separation by SDS-PAGE. Results shown are representative of 3 independent experiments. <b>(C)</b> HCT116 and HCT116 BAK^-/-, BAX^-/- (DKO) cells were pre-treated for 1 h with 100 nM GSK2606414 (GSK) before 48 h treatment with 0.1 μg ml^-1 Tm. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. <b>(D)</b> HCT116 cells were pre-treated for 1 h with 100nM GSK2606414 prior to addition of 0.1 μg ml^-1 Tm and 10 μM QVD-oPh (QVD), as indicated, for 48 h. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results shown in (C) and (D) are the means ± S.D. of 3 independent experiments performed in technical triplicate. Statistics represent the results of Student’s unpaired <i>t</i>-tests; **, p < 0.01.</p

Activation of ERK1/2 protects against cell death arising from PERK inhibition.

<p><b>(A)</b> NIH3T3 ΔCRAF:ER cells were pre-treated with 100 nM 4-HT for 1 h before addition of 100 nM GSK2606414 (GSK) and 0.1 μg ml^-1 Tm alone or in combination as indicated for 24 h. Whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. Results are representative of two independent experiments. <b>(B)</b> NIH3T3 ΔCRAF:ER cells were pre-treated with 100 nM 4-HT for 1 h before addition of 100 nM GSK2606414 (GSK) and 0.1 μg ml^-1 Tm alone or in combination as indicated for 48 h. Cells were fixed and analysed by flow cytometry following propidium iodide staining. Results are means ± S.D. of three technical replicates from a single experiment and are representative of two independent experiments.</p

ER stress induces BAK/BAX-dependent, apoptotic cell death.

<p><b>(A)</b> HCT116 cells were treated with 100 nM Tg (top panel) or 2 μg ml^-1 Tm (bottom panel) for the indicated time, whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Results are representative of at least 3 independent experiments. <b>(B)</b> HCT116 cells were treated with 100 nM Tg in the presence or absence of 10 μM QVD-oPh for 48 h, fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results are the means ± S.D. of 3 experiments each performed in technical triplicate. Student’s unpaired <i>t</i>-test results are indicated as follows; ***, p < 0.001. <b>(C)</b> Whole cell lysates of HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies to confirm their genotype. <b>(D)</b> HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were treated with 2 μg ml^-1 Tm for 48 h, fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results are the means ± S.D. of 3 independent experiments each performed in technical triplicate. Statistics represent the results of two-way ANOVA and Bonferroni post-tests comparing each genotype to WT; ***, p < 0.001. <b>(E)</b> HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were treated with 2 μg ml^-1 Tm for 4 h (top panel) or 24 h (bottom panel). Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Results are representative of three independent experiments.</p