HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

Purpose: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed.

Experimental Design: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays.

Results: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP–specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF-mutant colorectal cancer cell lines.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor Vemurafenib (PLX4032) has shown significant increases in response rates and overall survival, only minor responses to Vemurafenib treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MEK inhibition in BRAFMT CRC. Methods: Paired BRAFMT/WT RKO and VACO432 CRC cells and non-isogenic BRAFMT LIM2405, WiDR, HT-29 and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interactions between MEK1/2 and JAK1/2 or c-MET inhibition were assessed using the MTT cell viability assays and Flow Cytometry. Apoptosis was measured using Western Blotting for PARP, cleaved caspase 3, 8 and 9, and caspase 3/7 and 8 activity assays. Results: Treatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in synergy and significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or trametinib in BRAFMT CRC cells. The RTK c-MET is activated upstream of STAT3 following MEK1/2 inhibition. Inhibition of c-MET and MEK1/2, using pharmacological inhibitors (crizotinib and AZD6244), results in synergy and increased cell death in BRAFMT CRC cells. Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition in vitro. Combinations of JAK/MEKi or MET/MEKi can be a potential novel treatment strategy for poor prognostic BRAFMT advanced CRC patients.

Abstract B64: The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal cancer.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor PLX4032 has shown significant increases in response rates and overall survival compared to standard Dacarbazine treatment, only minor responses to PLX4032 treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumors has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MAPK inhibition in BRAFMT CRC.

Methods: Paired BRAFMT/WT RKO and VACO432 CRC cell line models and non-isogenic BRAFMT LIM2405, WiDR and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interaction between MEK1/2 and JAK1/2 inhibition was assessed using the MTT cell viability assays and flow cytometry. Apoptosis was measured using Western blotting for PARP, cleaved caspase 3/8 and caspase 8, 3/7 activity assays.

Results: Treatment with MEK1/2 inhibitors AZD6244, GSK1120212, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or GSK1120212 in BRAFMT CRC cells. In addition, combination of MEK1/2 and JAK/STAT3 inhibition resulted in strong synergy with CI values between 0.3 and 0.7 in BRAFMT CRC cells.

Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition. These data provide a strong rationale for further investigation of combination of MEK1/2 and JAK/STAT3 inhibition in BRAFMT in vivo models.

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

BACKGROUND: Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. RESULTS: The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1. CONCLUSIONS: Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Sinalização da MAPK/ERK na diferenciaçãao da oligodendroglia: efeitos de inibidores da MEK sobre a morfologia e distribuição de proteínas de oligodendrócitos/mielina in vitro

A via de sinalização da cinase regulada por fatores extracelulares, da família das proteínas cinases ativadas por mitógenos (MAPK/ERK) é importante tanto para a sobrevivência como para a progressão da diferenciação de oligodendrócitos. Neste trabalho, a via da MAPK/ERK foi avaliada na oligodendroglia in vitro com a utilização de inibidores da MEK. A morfologia celular, assim como a distribuição de proteínas foram analisadas em diferentes estágios de maturação da oligodendroglia. Culturas primárias de oligodendrócitos foram tratadas com os inibidores da MEK PD98059 ou U0126, aos 5 ou 11dias in vitro (div), por 30min, 24 ou 48h. A oligodendroglia foi distinguida com marcadores estágio-específicos: A2B5, 23nucleotídeo cíclico 3 fosfodiesterase (CNPase) e proteína básica de mielina (MBP), e classificada de acordo com sua morfologia em diferentes estágios de desenvolvimento. O tratamento aumentou significativamente o número de células com morfologia mais imatura e diminuiu o número de células maduras. Além disso, aumentou o número de células redondas e sem prolongamentos as quais não puderam ser classificadas em nenhum dos estágios de desenvolvimento da oligodendroglia. Os efeitos mais evidentes foram observados logo após o menor tempo de tratamento. Células redondas eram positivas para CNPase e MBP, porém não foram marcadas com A2B5 ou com NG2, indicando que seriam células maduras incapazes de estender ou manter seus prolongamentos. De fato, estas mudanças foram acompanhadas por alterações na distribuição de proteínas de oligodendrócitos como a MBP e a CNPase, assim como alterações em proteínas de citoesqueleto, como actina, tubulina e na cinase de adesão focal (FAK). A MBP foi observada nas células tratadas em um padrão de distribuição desorganizado e disperso, oposto ao padrão contínuo que é observado nas células das culturas controle. Além disso, o tratamento causou uma desorganização na distribuição da CNPase, actina e tubulina. Nas células das culturas controle, estas proteínas apresentam um padrão organizado compondo as estruturas de citoqueleto semelhantes a nervuras. Após um pequeno período de tratamento (30min), actina e tubulina apresentaram o mesmo padrão de marcação puntiforme que a CNPase apresentou. O tratamento também reduziu os pontos de adesão focal demonstrados pela FAK. Com o decorrer do tratamento, após 24 e 48h, actina e tubulina aparentavam estar se reorganizando em um padrão filamentar. Estes resultados indicam um efeito importante da via da MAPK/ERK na ramificação e alongamento dos prolongamentos dos oligodendrócitos, com possíveis consequências para a formação da bainha de mielina.

ADAM17-Dependent c-MET-STAT3 Signaling Mediates Resistance to MEK Inhibitors in KRAS Mutant Colorectal Cancer

There are currently no approved targeted therapies for advanced KRAS mutant (KRASMT) colorectal cancer (CRC). Using a unique systems biology approach, we identified JAK1/2-dependent activation of STAT3 as the key mediator of resistance to MEK inhibitors in KRASMT CRC in vitro and in vivo. Further analyses identified acute increases in c-MET activity following treatment with MEK inhibitors in KRASMT CRC models, which was demonstrated to promote JAK1/2-STAT3-mediated resistance. Furthermore, activation of c-MET following MEK inhibition was found to be due to inhibition of the ERK-dependent metalloprotease ADAM17, which normally inhibits c-MET signaling by promoting shedding of its endogenous antagonist, soluble "decoy" MET. Most importantly, pharmacological blockade of this resistance pathway with either c-MET or JAK1/2 inhibitors synergistically increased MEK-inhibitor-induced apoptosis and growth inhibition in vitro and in vivo in KRASMT models, providing clear rationales for the clinical assessment of these combinations in KRASMT CRC patients.

Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis.

Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Combination of MEK-ERK inhibitor and hyaluronic acid has a synergistic effect on anti-hypertrophic and pro-chondrogenic activities in osteoarthritis treatment

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of V600EBRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of V600EBRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by V600EBRAF/MEK signalling in melanoma cells.

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.

MEK/ERK-mediated phosphorylation of Bim is required to ensure survival of T and B lymphocytes during mitogenic stimulation

Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.

Dsh homolog DVL3 mediates resistance to IGFIR inhibition by regulating IGF-RAS signaling

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.