MEK/ERK activities are increased in many primary lung cancers, and MEK inhibitors have been tested clinically for treatment of non-small cell lung cancers. The molecular mechanisms of resistance to MEK inhibitors have not been clearly demonstrated, however, and no molecular biomarker that can predict lung cancer response to MEK inhibitors is available. By determining the dose-responses of 35 human lung cancer cell lines to MEK-specific inhibitor AZD6244, we identified subsets of lung cancer cell lines that are either sensitive or resistant to this agent. Subsequent molecular characterization showed that treatment with AZD6244 suppressed ERK phosphorylation in both sensitive and resistant cells, suggesting that resistance is not mediated by the activities of MEK/ERK themselves. Interestingly, we found that levels of phosphorylated AKT were dramatically higher in the resistant cancer cells than in the sensitive cells. Stable transfection of dominant-negative AKT into resistant cells by retroviral infection restored their susceptibility to AZD6244. These results indicate that phosphorylated AKT may be a biomarker of response to AZD6244 and that modulation of AKT activity may be a useful approach to overcome resistance to MEK inhibitors.
AZD6244; MEK inhibitor; resistance; AKT; lung cancer
The Mitogen-activated Protein Kinase (MAPK) pathway is important for cell proliferation, survival and differentiation and is frequently upregulated in cancers. The MAPK pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MEK1/2, on radiation response.
The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2 and DU145) was evaluated using clonogenic assays. DNA damage repair was evaluated using γH2AX and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity.
Exposure of each cell line to AZD6244 prior to irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors (DEF) at a surviving fraction of 0.1 ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of γH2AX foci after IR were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 3 hours after IR. Mitotic catastrophe was increased in cells receiving both AZD6244 and IR compared to the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (DEF of 3.38).
These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.
AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.
AZD6244 was tested against the PPTP in vitro panel (1 nM-10μM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily five days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting.
At the highest concentration used in vitro (10 μM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment.
At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.
Preclinical Testing; Developmental Therapeutics; AZD6244
The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 [ARRY-142886]) interacted with multiple CHK1 inhibitors (UCN-01 [7-hydroxystaurosporine], AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor-induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP 2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK−/− transformed fibroblasts and suppressed by overexpression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor-induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.
CHK1; SRC; apoptosis; breast cancer; kinase; therapeutics; intrinsic; caspase
The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression. All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.
melanoma; MEK; SRC; MMP-2; invasion; combination therapy
The present studies were initiated to determine whether inhibitors of MEK1/2 or SRC signaling, respectively, enhance CHK1 inhibitor lethality in primary human glioblastoma cells. Multiple MEK1/2 inhibitors (CI-1040 (PD184352); AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01, AZD7762) to kill multiple primary human glioma cell isolates that have a diverse set of genetic alterations typically found in the disease. Inhibition of SRC family proteins also enhanced CHK1 inhibitor lethality. Combined treatment of glioma cells with (MEK1/2 + CHK1) inhibitors enhanced radiosensitivity. Combined (MEK1/2 + CHK1) inhibitor treatment led to dephosphorylation of ERK1/2 and S6 ribosomal protein, whereas the phosphorylation of JNK and p38 was increased. MEK1/2 + CHK1 inhibitor-stimulated cell death was associated with the cleavage of pro-caspases 3 and 7 as well as the caspase substrate (PARP). We also observed activation of pro-apoptotic BCL-2 effector proteins BAK and BAX and reduced levels of pro-survival BCL-2 family protein BCL-XL. Overexpression of BCL-XL alleviated but did not completely abolish MEK1/2 + CHK1 inhibitor cytotoxicity in GBM cells. These findings argue that multiple inhibitors of the SRC-MEK pathway have the potential to interact with multiple CHK1 inhibitors to kill glioma cells.
Apoptosis; CHK 1 inhibitor; glioma; MEK1/2 inhibitors
To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer.
Patients and Methods
In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations.
Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated ≥ 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for ≥ 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles.
AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.
The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of MAP/ERK1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells ‥ Moreover, after treatment with 17-DMAG, the kinase activity of residual, un-degraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine (NAC), partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the anti-tumor effect of geldanamycin and its derivatives is also mediated though the production of ROS which may directly inactivate tumorigenic mutant BRAF(V600E).
BRAF; MAP kinase; geldanamycin; HSP90; ROS
The present studies determined in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin HSP90 inhibitors and MEK1/2 inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17AAG toxicity that was suppressed in cells deleted for mutant active RAS which were non-tumorigenic but was magnified in isogenic tumorigenic cells expressing H-RAS V12 or K-RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca2+ levels and reduced GRP78/BiP expression in a Ca2+ -dependent manner. GRP78/BiP over-expression, however, also suppressed drug-induced intracellular Ca2+ levels. MEK1/2 inhibitor and 17AAG treatment increased ROS levels that were blocked by quenching Ca2+ or over-expression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca2+ quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Over-expression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca2+ levels. Thus treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca2+ and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.
Geldanamycin; 17AAG; MEK1/2 inhibitor; CD95; c-FLIP-s; GRP78/BiP; autophagy; cell death; ASMase; de novo
Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of MEK1/2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC50 less than 1µM (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines, with a correlation between sensitivity and raf mutations in breast cancer cell lines (p= 0.022) and ras mutations in NSCLC cell lines (p= 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots demonstrated no clear association between MEK and PI3K pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies, including a small number of genes in which differential expression was common to both histologies. In total, these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations respectively.
AZD6244; MEK; Breast cancer; Lung Cancer
Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.
Selumetinib (AZD6244, ARRY-142886) is a potent, selective, uncompetitive inhibitor of MEK 1 / 2, part of the RAF/MEK/ERK protein kinase signal cascade, which is responsible for tumor. This pilot study was used to explore if 18F-fluoro-l-thymidine (FLT), a thymidine analogue positron emission tomography (PET) tracer and a surrogate marker for proliferation, can be used as an early predictor of response for patients with solid cancers treated with Selumetinib. FLT-PET scans were performed in four patients at baseline and after 2 weeks of treatment with Selumetinib. FLT uptake in tumors was analyzed qualitatively and quantitatively by measuring standard uptake value (SUV) max in regions of interest (ROI). Results were compared to computed tomography (CT) scans (baseline and after 8 weeks), which were evaluated using the response evaluation criteria in solid tumors (RECIST) 1.0 criteria. One patient with melanoma showed both a qualitative and quantitative decrease in FLT uptake associated with a decrease in sum of longest diameter of 12% RECIST on CT evaluation. Another patient who had colorectal carcinoma (CRC) showed a significant increase in FLT uptake with initially stable, but eventually progressive disease on CT. The other two patients (one with melanoma and one with CRC) showed no significant changes in FLT uptake, whereas CT evaluation showed progressive disease. This is the first report describing changes in FLT-PET in patients receiving Selumetinib. In two patients, changes in FLT uptake as early as after 2 weeks of treatment were consistent with CT results after 8 weeks. Biomarkers to predict and evaluate treatment the outcome of targeted therapies are highly warranted. These initial results need further investigation.
F-18 FLT; PET-CT; Selumetinib; treatment response
The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phosphor-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Anti-proliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.
Hsp90; MEK; ERK; 17-AAG
Current approaches to block KRAS oncogene function focus on inhibition of K-Ras downstream effector signaling. We evaluated the anti-tumor activity of selumetinib (AZD6244, ARRY-142886), a potent and selective MEK1/2 inhibitor, on a panel of colorectal carcinoma (CRC) cells and found no inhibition of KRAS mutant CRC cell anchorage-independent growth. While AKT activity was elevated in KRAS mutant cells, and PI3K inhibition did impair the growth of MEK inhibitor-insensitive CRC cell lines, concurrent treatment with selumetinib did not provide additional anti-tumor activity. Therefore, we speculated that inhibition of the Ral guanine exchange factor (RalGEF) effector pathway may be a more effective approach for blocking CRC growth. RalGEFs are activators of the related RalA and RalB small GTPases and we found activation of both in CRC cell lines and patient tumors. Interfering RNA stable suppression of RalA expression reduced CRC tumor cell anchorage-independent growth, but surprisingly, stable suppression of RalB greatly enhanced soft agar colony size and formation frequency. Despite their opposing activities, both RalA and RalB regulation of anchorage-independent growth required interaction with RalBP1/RLIP76 and components of the exocyst complex. Interestingly, RalA interaction with the Exo84 but not Sec5 exocyst component was necessary for supporting anchorage-independent growth, whereas RalB interaction with Sec5 but not Exo84 was necessary for inhibition of anchorage-independent growth. We suggest that anti-RalA-selective therapies may provide an effective approach for KRAS mutant CRC.
Ras; MEK; AKT; RalBP1/RLIP76; exocyst
The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.
The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.
Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.
AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors.
Molecular aberrations of the Ras/Raf/MEK/ERK and/or MDM2/p53 signaling pathways have been reported in 80% and 50% of primary AML samples and confer poor outcome. In this study, anti-leukemic effects of combined MEK inhibition by AZD6244 and non-genotoxic p53 activation by MDM2 antagonist Nutlin3a were investigated. Simultaneous blockade of MEK and MDM2 signaling by AZD6244 and Nutlin3a triggered synergistic proapoptotic responses in AML cell lines (CI = 0.06 ± 0.03 and 0.43 ± 0.03 in OCI/AML3 and MOLM13 cells, respectively) and in primary AML cells (CI = 0.52 ± 0.01). Mechanistically, the combination upregulated levels of BH3-only proteins Puma and Bim, in part via transcriptional up-regulation of the FOXO3a transcription factor. Suppression of Puma and Bim by short interfering RNA rescued OCI/AML3 cells from AZD/Nutlin-induced apoptosis. These results strongly indicate therapeutic potential of combined MEK/MDM2 blockade in AML and implicate Puma and Bim as major regulators of AML cell survival.
MEK inhibitor; MDM2 antagonist; Combination therapy; Apoptosis; Acute myeloid leukemia
Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines.
Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines.
All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations.
These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.
PI3K; MEK; combination; mTOR; colorectal
Selumetinib (AZD6244, ARRY-142886) is a selective, non–ATP-competitive inhibitor of mitogen-activated protein/extracellular signal–regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR–based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.
Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An in vivo analysis was conducted on pancreatic cancer xenografts. While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.
AZD6244 is a small molecule inhibitor of the MEK kinase pathway currently in clinical trials. However, the mechanisms mediating intrinsic resistance to MEK inhibition are not fully characterized. To define molecular mechanisms of MEK inhibitor resistance, we analyzed responses of 38 lung cancer cell lines following AZD6244 treatment and their genome-wide gene expression profiles and identified a panel of genes correlated with sensitivity or resistance to AZD6244 treatment. In particular, Ingenuity pathway analysis revealed that activation of the STAT3 pathway was associated with MEK inhibitor resistance. Inhibition of this pathway by JSI-124, a STAT3-specific small molecule inhibitor, or with STAT3-specific siRNA sensitized lung cancer cells to AZD6244 and induced apoptosis. Moreover, combining a STAT3 inhibitor with AZD6244 induced expression of BIM and polyADP-ribose polymerase (PARP) cleavage, whereas activation of the STAT3 pathway inhibited BIM expression and elicited resistance to MEK inhibitors. We found that the STAT3-regulated microRNA miR-17 played a critical role in MEK inhibitor resistance, such that miR-17 inhibition sensitized resistant cells to AZD6244 by inducing BIM and PARP cleavage. Together, these results indicated that STAT3-mediated overexpression of miR-17 blocked BIM expression and caused resistance to AZD6244. Our findings suggest novel approaches to overcome resistance to MEK inhibitors by combining AZD6244 with STAT3 or miR-17 inhibitors.
Gene expression profiling; MEK inhibitor resistance; AZD6244; STAT3 pathway; miR-17
The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy.
The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations.
Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib.
The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.
Selumetinib; barasertib; docetaxel; temozolomide; scheduling; apoptosis
Pulmonary fibrosis remains a significant public health burden with no proven therapies. The mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the MAPK/ERK pathway is detected in the lungs of human fibrosis samples; however, the effect of modulating the pathway in vivo is unknown. Overexpression of transforming growth factor (TGF)-α in the lung epithelium of transgenic mice causes a progressive pulmonary fibrosis associated with increased MEK/ERK activation localized primarily in mesenchymal cells. To determine the role of the MEK pathway in the induction of TGF-α–induced lung fibrosis, TGF-α was overexpressed for 4 weeks while mice were simultaneously treated with the specific MEK inhibitor, ARRY-142886 (ARRY). Treatment with ARRY prevented increases in lung cell proliferation and total lung collagen, attenuated production of extracellular matrix genes, and protected mice from changes in lung function. ARRY administered as a rescue treatment after fibrosis was already established inhibited fibrosis progression, as assessed by lung histology, changes in body weights, extracellular matrix gene expression, and lung mechanics. These findings demonstrate that MEK inhibition prevents progression of established fibrosis in the TGF-α model, and provides proof of concept of targeting the MEK pathway in fibrotic lung disease.
pulmonary fibrosis; transforming growth factor-α; epidermal growth factor receptor; mitogen-activated protein kinase/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase; ARRY-142886
Metastatic thyroid cancers that are refractory to radioiodine (iodine-131) are associated with a poor prognosis. In mouse models of thyroid cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists increase the expression of the sodium–iodide symporter and uptake of iodine. Their effects in humans are not known.
We conducted a study to determine whether the MAPK kinase (MEK) 1 and MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness to radioiodine in patients with metastatic thyroid cancer. After stimulation with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography (PET) was performed before and 4 weeks after treatment with selumetinib (75 mg twice daily). If the second iodine-124 PET study indicated that a dose of iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or lesions, therapeutic radioiodine was administered while the patient was receiving selumetinib.
Of 24 patients screened for the study, 20 could be evaluated. The median age was 61 years (range, 44 to 77), and 11 patients were men. Nine patients had tumors with BRAF mutations, and 5 patients had tumors with mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the 20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS mutations). Eight of these 12 patients reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8 patients treated with radioiodine, 5 had confirmed partial responses and 3 had stable disease; all patients had decreases in serum thyroglobulin levels (mean reduction, 89%). No toxic effects of grade 3 or higher attributable by the investigators to selumetinib were observed. One patient received a diagnosis of myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with progression to acute leukemia.
Selumetinib produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine; the effectiveness may be greater in patients with RAS-mutant disease. (Funded by the American Thyroid Association and others; ClinicalTrials.gov number, NCT00970359.)
AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC50 and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.