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.     

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors

Background

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies.

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.     

Interaction of LATS1 with SMAC links the MST2/Hippo pathway with apoptosis in an IAP-dependent manner

Metastatic malignant melanoma is the deadliest skin cancer, and it is characterised by its high resistance to apoptosis. The main melanoma driving mutations are part of ERK pathway, with BRAF mutations being the most frequent ones, followed by NRAS, NF1 and MEK mutations. Increasing evidence shows that the MST2/Hippo pathway is also deregulated in melanoma. While mutations are rare, MST2/Hippo pathway core proteins expression levels are often dysregulated in melanoma. The expression of the tumour suppressor RASSF1A, a bona fide activator of the MST2 pathway, is silenced by promoter methylation in over half of melanomas and correlates with poor prognosis. Here, using mass spectrometry-based interaction proteomics we identified the Second Mitochondria-derived Activator of Caspases (SMAC) as a novel LATS1 interactor. We show that RASSF1A-dependent activation of the MST2 pathway promotes LATS1-SMAC interaction and negatively regulates the antiapoptotic signal mediated by the members of the IAP family. Moreover, proteomic experiments identified a common cluster of apoptotic regulators that bind to SMAC and LATS1. Mechanistic analysis shows that the LATS1-SMAC complex promotes XIAP ubiquitination and its subsequent degradation which ultimately results in apoptosis. Importantly, we show that the oncogenic BRAF^V600E mutant prevents the proapoptotic signal mediated by the LATS1-SMAC complex while treatment of melanoma cell lines with BRAF inhibitors promotes the formation of this complex, indicating that inhibition of the LATS1-SMAC might be necessary for BRAF^V600E-driven melanoma. Finally, we show that LATS1-SMAC interaction is regulated by the SMAC mimetic Birinapant, which requires C-IAP1 inhibition and the degradation of XIAP, suggesting that the MST2 pathway is part of the mechanism of action of Birinapant. Overall, the current work shows that SMAC-dependent apoptosis is regulated by the LATS1 tumour suppressor and supports the idea that LATS1 is a signalling hub that regulates the crosstalk between the MST2 pathway, the apoptotic network and the ERK pathway.Subject terms: Protein-protein interaction networks, Extracellular signalling molecules     

Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival

Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro-apoptotic member of the BCL-2 family, is inhibited by BRAF-->MEK-->ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to elevated BIM expression. However, re-addition of trophic factors or activation of a conditional form of BRAF(V600E) leads to rapid inhibition of BIM expression. In both cases, inhibition of BIM expression was dependent on the activity of MEK1/2 and the proteasome. Consistent with these observations, pharmacological inhibition of BRAF(V600E) or MEK1/2 in human melanoma cells (using PLX4720 and CI-1040 respectively) led to a striking elevation of BIM expression. Re-activation of BRAF-->MEK-->ERK signaling led to phosphorylation of BIM-EL on serine 69 and its subsequent degradation. Interestingly, endogenous expression of BIM in melanoma cells was insufficient to induce apoptosis unless combined with serum deprivation. Under these circumstances, inhibition of BIM expression by RNA interference provided partial protection from apoptosis. These data suggest that regulation of BIM expression by BRAF-->MEK-->ERK signaling is one mechanism by which oncogenic BRAF(V600E) can influence the aberrant physiology of melanoma cells.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Dabrafenib; Preclinical Characterization,Increased Efficacy when Combined with Trametinib,while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF^V600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G₁ cell cycle arrest, followed by cell death. In a BRAF^V600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.     

Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-α-induced cell death in MCF-7 cells

The role of autophagy in cell death is under considerable debate. The process of autophagy has been shown to lead to either cell survival or cell death depending on cell type and stimulus. In the present study, we determined the contribution of ERK1/2 signalling to autophagy and cell death induced by tumour necrosis factor-α (TNF) in MCF-7 breast cancer cells. Treatment of MCF-7 cells with TNF caused a time-dependent increase in ERK1/2 activity. There was an induction of autophagy and cleavage of caspase-7, -8, -9 and PARP. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 resulted in a decrease in TNF-induced autophagy that was accompanied by an increase in cleavage of caspase-7, -8, -9 and PARP Furthermore, inhibition of ERK1/2 signalling resulted in decreased clonogenic capacity of MCF-7 cells. These data suggest that TNF-induces autophagy through ERK1/2 and that inhibition of autophagy increases cellular sensitivity to TNF.     

Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells.

The role of autophagy in cell death is under considerable debate. The process of autophagy has been shown to lead to either cell survival or cell death depending on cell type and stimulus. In the present study, we determined the contribution of ERK1/2 signalling to autophagy and cell death induced by tumour necrosis factor-alpha (TNF) in MCF-7 breast cancer cells. Treatment of MCF-7 cells with TNF caused a time-dependent increase in ERK1/2 activity. There was an induction of autophagy and cleavage of caspase-7, -8, -9 and PARP. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 resulted in a decrease in TNF-induced autophagy that was accompanied by an increase in cleavage of caspase-7, -8, -9 and PARP Furthermore, inhibition of ERK1/2 signalling resulted in decreased clonogenic capacity of MCF-7 cells. These data suggest that TNF-induces autophagy through ERK1/2 and that inhibition of autophagy increases cellular sensitivity to TNF.     

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS^G12C/G13D or BRAF^V600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.     

Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS‐mutant cells, and with oncogenic BRAF in BRAF^V600E‐mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS‐mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF^V600E‐mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.     

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.     

ERK5 and its role in tumour development

The MEK5 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 5]/ERK5 pathway is the least well studied MAPK signalling module. It has been proposed to play a role in the pathology of cancer. In the present paper, we review the role of the MEK5/ERK5 pathway using the 'hallmarks of cancer' as a framework and consider how this pathway is deregulated. As well as playing a key role in endothelial cell survival and tubular morphogenesis during tumour neovascularization, ERK5 is also emerging as a regulator of tumour cell invasion and migration. Several oncogenes can stimulate ERK5 activity, and protein levels are increased by a novel amplification at chromosome locus 17p11 and by down-regulation of the microRNAs miR-143 and miR-145. Together, these finding underscore the case for further investigation into understanding the role of ERK5 in cancer.     

Inhibition of urokinase-type plasminogen activator receptor induces apoptosis in melanoma cells by activation of p53

The urokinase-type plasminogen activator receptor (uPAR) is involved in several biological processes, including proteolysis, adhesion, migration and inflammation. Increased expression of uPAR is associated with metastasis in several tumor types. We studied the biological role of uPAR in melanoma and found that inhibition of uPAR via RNA interference induced massive death in three different metastatic cell lines. Annexin-V staining and caspase activation analysis revealed induction of the mitochondrial apoptotic pathway. The expression of members of the Bcl-2 family (Bax, Bcl-2, Bak and Bcl-x(L)) was changed in a pro-apoptotic manner. uPAR inhibition induced the expression of the tumor suppressor p53 and of its downstream target gene p21. Inhibition of p53 rescued cells from apoptosis indicating that p53 was critical for apoptosis induction. Apoptosis was observed in melanoma cells carrying activating BRAF mutations and occurred in the presence of extracellular signal-regulated kinase (ERK) phosphorylation. uPAR can activate focal adhesion kinase (FAK), which is implicated in adhesion-dependent tumor cell survival. However, inhibition of FAK did not induce apoptosis. Our data suggest a new function of uPAR acting as a survival factor for melanoma by downregulating p53. Inhibition of uPAR induces a pro-apoptotic signalling pathway via p53 that is independent of ERK or FAK signalling. These findings may offer new treatment strategies for metastatic melanoma.     

Oncogenic Ras in tumour progression and metastasis

The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.     

Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.     

Mechanisms of apoptosis regulation by viral oncogenes in infection and tumorigenesis

Apoptosis mediated by the proapoptotic BCL-2 family members BCL-2-associated X-protein (BAX) and BCL-2 antagonist/killer (BAK) is part of the antiviral response at the cellular level to limit virus replication. Viruses, in turn, have evolved to encode antiapoptotic BCL-2 homologs (v-BCL-2s) to prevent the premature death of the infected host cell to sustain virus replication. These same v-BCL-2 proteins cooperate with loss of retinoblastoma protein and p53 tumor suppressor function, by inactivating the BAX and BAK apoptotic pathway to promote epithelial solid tumor growth and resistance to chemotherapy. Analogously to infected cells, failure of apoptosis in tumors permits the survival of abnormal, damaged cells displaying chromosome instability that may further promote tumor progression. Thus, both infected cells and tumor cells require inhibition of the apoptotic host defense mechanism, the insights from which can be exploited for therapy development.     

Reticulon 2 promotes gastric cancer metastasis via activating endoplasmic reticulum Ca2+ efflux-mediated ERK signalling

Gastric cancer ranks fourth for mortality globally among various malignant tumours, and invasion and metastasis are the major reason leading to its poor prognosis. Recently, accumulating studies revealed the role of reticulon proteins in cell growth and transmigration. However, the expression and biological function of reticulon proteins in human gastric cancer remain largely unclear. Herein, we explored the potential role of reticulon 2 (RTN2) in the progression of gastric cancer. Tissue microarray was used to determine the expression levels of RTN2 in 267 gastric cancer patients by immunohistochemistry. Gastric cancer cell lines were utilised to examine the influences of RTN2 on cellular migration and invasion abilities, epithelial-to-mesenchymal transition (EMT) and signalling pathway. In vivo studies were also performed to detect the effect of RTN2 on tumour metastasis. We found that RTN2 expression was notably upregulated in tumour tissues compared to pericarcinomatous tissues. High RTN2 expression was positively correlated with patients’ age, vessel invasion, tumour invasion depth, lymph node metastasis and TNM stage. Besides, high RTN2 staining intensity was associated with adverse survival which was further identified as an independent prognostic factor for gastric cancer patients by multivariate analysis. And the predictive accuracy was also improved when incorporated RTN2 into the TNM-staging system. RTN2 could promote the proliferation, migration and invasion of gastric cancer cells in vitro and lung metastasis in vivo. Mechanistically, RTN2 interacted with IP3R, and activated ERK signalling pathway via facilitating Ca²⁺ release from the endoplasmic reticulum, and subsequently drove EMT in gastric cancer cells. These results proposed RTN2 as a novel promotor and potential molecular target for gastric cancer therapies.Subject terms: Gastric cancer, Calcium signalling     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.