MEK Inhibitor PD98059 Research Articles

Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β

SummaryBackgroundThe C‐type lectin‐like receptor 2 (CLEC‐2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen‐activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC‐2 signaling is not known.ObjectivesWe sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC‐2.ResultsThe CLEC‐2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal‐related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC‐2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31‐8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors.ConclusionsThese results demonstrate that CLEC‐2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.

Combined inhibition of MEK and mTOR has a synergic effect on angiosarcoma tumorgrafts.

Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. Using tumorgraft models, we previously showed that AS is sensitive to small-molecule inhibitors that target mitogen-activated/extracellular-signal-regulated protein kinase kinases 1 and 2 (MEK). The objective of this study was to identify drugs that combine with MEK inhibitors to more effectively inhibit AS growth. We examined the in vitro synergy between the MEK inhibitor PD0325901 and inhibitors of eleven common cancer pathways in melanoma cell lines and canine angiosarcoma cell isolates. Combination indices were calculated using the Chou-Talalay method. Optimized combination therapies were evaluated in vivo for toxicity and efficacy using canine angiosarcoma tumorgrafts. Among the drugs we tested, rapamycin stood out because it showed strong synergy with PD0325901 at nanomolar concentrations. We observed that angiosarcomas are insensitive to mTOR inhibition. However, treatment with nanomolar levels of mTOR inhibitor renders these cells as sensitive to MEK inhibition as a melanoma cell line with mutant BRAF. Similar results were observed in B-Raf wild-type melanoma cells as well as in vivo, where treatment of canine AS tumorgrafts with MEK and mTOR inhibitors was more effective than monotherapy. Our data show that a low dose of an mTOR inhibitor can dramatically enhance angiosarcoma and melanoma response to MEK inhibition, potentially widening the field of applications for MEK-targeted therapy.

BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of Neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras-GAP protein causes increased Ras-GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model. We studied effects of MEK inhibition using 1.5 mg/kg/day PD-0325901 prior to neurofibroma onset in the Nf1 (flox/flox); Dhh-Cre mouse model. We also treated mice with established tumors at 0.5 and 1.5 mg/kg/day doses of PD-0325901. We monitored tumor volumes using MRI and volumetric measurements, and measured pharmacokinetic and pharmacodynamic endpoints. Early administration significantly delayed neurofibroma development as compared to vehicle controls. When treatment was discontinued neurofibromas grew, but no rebound effect was observed and neurofibromas remained significantly smaller than controls. Low dose treatment of mice with PD-0325901 resulted in neurofibroma shrinkage equivalent to that observed at higher doses. Tumor cell proliferation decreased, although less than at higher doses with drug. Tumor blood vessels per area correlated with tumor shrinkage. Neurofibroma development was not prevented by MEK inhibition, beginning at 1 month of age, but tumor size was controlled by early treatment. Moreover, treatment with PD-0325901 at very low doses may shrink neurofibromas while minimizing toxicity. These studies highlight how genetically engineered mouse models can guide clinical trial design.

Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2.

The PI3K/AKT/mTOR pathway, which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic ductal adenocarcinoma (PDAC) cells with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) BEZ235 blocked mTORC1/S6K activation (scored by S6 phosphorylation at Ser(240/244)), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr(37/46)), and mTORC2-mediated AKT phosphorylation at Ser(473), in a concentration-dependent manner. Strikingly, BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK overactivation coincided with complete inhibition of phosphorylation of AKT and 4E-BP1. ERK overactivation was induced by other PI3K/TOR-KIs, including PKI-587 and GDC-0980. The MEK inhibitors U126 or PD0325901 prevented ERK overactivation induced by PI3K/TOR-KIs. The combination of BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells indicate that PI3K/TOR-KIs act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated AKT phosphorylation on Ser(473), suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA markedly attenuated the enhancing effect of BEZ235 on ERK phosphorylation. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.

Physiological implications of biased signaling at histamine H2 receptors.

Histamine mediates numerous functions acting through its four receptor subtypes all belonging to the large family of seven transmembrane G-protein coupled receptors. In particular, histamine H2 receptor (H2R) is mainly involved in gastric acid production, becoming a classic pharmacological target to treat Zollinger–Ellison disease and gastric and duodenal ulcers. H2 ligands rank among the most widely prescribed and over the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands display biased agonism, selecting and triggering some, but not all, of the signaling pathways associated to the H2R. The aim of the present work is to study whether famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal levels, it mimics the effects of histamine and the agonist amthamine concerning receptor desensitization and internalization. Moreover, the treatment of HEK293T transfected cells with any of the three ligands lead to a concentration dependent pERK increment. Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this behavior could have pharmacological relevant implications. Based on that, histidine decarboxylase expression was studied by quantitative PCR in AGS cells and its levels were increased by famotidine as well as by histamine and amthamine. In all cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating that biased signaling toward ERK1/2 pathway is the responsible of such enzyme regulation. These results support that ligand bias is not only a pharmacological curiosity but has physiological and pharmacological implications on cell metabolism.

Autocrine prostaglandin E2signaling promotes promonocytic leukemia cell survival via COX-2 expression and MAPK pathway

The COX-2/PGE2 pathway has been implicated in the occurrence and progression of cancer. The underlying mechanisms facilitating the production of COX-2 and its mediator, PGE2, in cancer survival remain unknown. Herein, we investigated PGE2-induced COX-2 expression and signaling in HL-60 cells following menadione treatment. Treatment with PGE2 activated anti-apoptotic proteins such as Bcl-2 and Bcl-xL while reducing pro-apoptotic proteins, thereby enhancing cell survival. PGE2 not only induced COX-2 expression, but also prevented casapse-3, PARP, and lamin B cleavage. Silencing and inhibition of COX-2 with siRNA transfection or treatment with indomethacin led to a pronounced reduction of the extracellular levels of PGE2, and restored the menadione-induced cell death. In addition, pretreatment of cells with the MEK inhibitor PD98059 and the PKA inhibitor H89 abrogated the PGE2-induced expression of COX-2, suggesting involvement of the MAPK and PKA pathways. These results demonstrate that PGE2 signaling acts in an autocrine manner, and specific inhibition of PGE2 will provide a novel approach for the treatment of leukemia. [BMB Reports 2015; 48(2): 109-114]

Effect of PPARδ agonist on stearoyl-CoA desaturase 1 in human pancreatic cancer cells: role of MEK/ERK1/2 pathway.

The stearoyl-CoA desaturase 1 (SCD1), also known as Δ9-desaturase, is a regulatory enzyme in the cellular lipid modification process that has been linked to pancreatic cancer and diabetes. The aim of the present study was to investigate the effect of peroxisome proliferative-activated receptor δ (PPARδ) agonist and ERK1/2- and EGF receptor (EGFR)-dependent pathways on the expression of SCD1 in human pancreatic carcinoma cell line PANC-1. PANC-1 cells cultured in RPMI-1640 were exposed to the commonly used MEK inhibitor PD98059, EGFR-selective inhibitor AG1478, and PPARδ agonist GW0742. Changes in mRNA, protein expression and activity index of SCD1 were then determined using real-time reverse transcription polymerase chain reaction, Western blot and gas liquid chromatography, respectively. The activity index and expression of SCD1 (p<0.01) decreased following treatment with PPARδ agonist at both mRNA and protein levels, whereas significant increases were observed after treatment with MEK or EGFR inhibitor. It was also found that the activity index of SCD1 were lower (p<0.01) in the combined treatment compared to the incubation with either inhibitor alone. PPARδ and MEK/ERK1/2- and EGFR-dependent pathways affect the expression and activity of SCD1 in pancreatic cancer cells. Furthermore, the aforementioned kinase signalling pathways were involved in an inhibitory effect on the expression and activity of SCD1 in these cells, possibly via PPARδ activation.

MEK inhibitor PD-0325901 overcomes resistance to CK2 inhibitor CX-4945 and exhibits anti-tumor activity in head and neck cancer.

The serine-threonine kinase CK2 exhibits genomic alterations and aberrant overexpression in human head and neck squamous cell carcinomas (HNSCC). Here, we investigated the effects of CK2 inhibitor CX-4945 in human HNSCC cell lines and xenograft models. The IC50's of CX-4945 for 9 UM-SCC cell lines measured by MTT assay ranged from 3.4-11.9 μM. CX-4945 induced cell cycle arrest and cell death measured by DNA flow cytometry, and inhibited prosurvival mediators phospho-AKT and p-S6 in UM-SCC1 and UM-SCC46 cells. CX-4945 decreased NF-κB and Bcl-XL reporter gene activities in both cell lines, but upregulated proapoptotic TP53 and p21 reporter activities, and induced phospho-ERK, AP-1, and IL-8 activity in UM-SCC1 cells. CX-4945 exhibited modest anti-tumor activity in UM-SCC1 xenografts. Tumor immunostaining revealed significant inhibition of PI3K-Akt-mTOR pathway and increased apoptosis marker TUNEL, but also induced p-ERK, c-JUN, JUNB, FOSL1 and proliferation (Ki67) markers, as a possible resistance mechanism. To overcome the drug resistance, we tested MEK inhibitor PD-0325901 (PD-901), which inhibited ERK-AP-1 activation alone and in combination with CX-4945. PD-901 alone displayed significant anti-tumor effects in vivo, and the combination of PD-901 and CX-4945 slightly enhanced anti-tumor activity when compared with PD-901 alone. Immunostaining of tumor specimens after treatment revealed inhibition of p-AKT S129 and p-AKT T308 by CX-4945, and inhibition of p-ERK T202/204 and AP-1 family member FOSL-1 by PD-901. Our study reveals a drug resistance mechanism mediated by the MEK-ERK-AP-1 pathway in HNSCC. MEK inhibitor PD-0325901 is active in HNSCC resistant to CX-4945, meriting further clinical investigation.

Activation of Extracellular Signal-Regulated Protein Kinases 1 and 2 (ERK1/2) by Free Fatty Acid Receptor 1 (FFAR1/GPR40) Protects from Palmitate-Induced Beta Cell Death, but Plays no Role in Insulin Secretion

Aims: GPR40/FFAR1 mediates palmitate-induced stimulation of insulin secretion but its involvement in lipotoxicity is controversial. Our previous observations suggest that FFAR1/GPR40 agonists protect against lipotoxicity although the underlying mechanism remains elusive. The present study examines the role of ERK1/2 and GPR40/FFAR1 in palmitate-induced stimulation of insulin secretion and beta cell death. Methods: Insulin secretion of INS-1E cells was measured by radioimmunoassay. Protein phosphorylation was examined on Western blots. Apoptosis was assessed by TUNEL staining. Results: Palmitate and the GPR40/FFAR1 agonist TUG-469 increased phosphorylation of ERK1/2 at low (2.8 mmol/L) and high (12 mmol/L) glucose but stimulated insulin secretion only at high glucose. The MEK1 inhibitor PD98059 significantly reduced phosphorylation of ERK1/2 but did not reverse the stimulation of secretion induced by glucose, palmitate or TUG-469. PD98059 rather augmented glucose-induced secretion. Prolonged exposure to palmitate stimulated apoptosis, an effect counteracted by TUG-469. PD98059 accentuated palmitate-induced apoptosis and reversed TUG-469-mediated inhibition of cell death. Conclusions: Activation of ERK1/2 by palmitate and GPR40/FFAR1 agonist correlates neither with stimulation of insulin secretion nor with induction of apoptosis. The results suggest a significant anti-apoptotic role of ERK1/2 under conditions of lipotoxicity.

Personalized Drug Combinations for the Treatment of Acute Myeloid Leukemia (AML) Patients

Acute myeloid leukemia (AML) is a highly heterogeneous disorder characterized by the rapid clonal proliferation of blasts derived from hematopoietic progenitor cells, leading to failure of normal hematopoiesis. Although standard therapy, usually including idarubicin and cytarabine, has been used to achieve remission, the long-term survival rates remain low.To show that it is possible to improve existing AML standard therapy, we have used experimental algorithms we have recently developed (iterative algorithms that find effective combinations without a full combinatorial screening) to find selective drug combinations for AML cells. The first search was done using the AML cell line KG-1 for the initial steps and cells from an AML patient for the final step. A normal fibroblast cell line (IMR-90) and normal blood progenitor cells were used as controls for toxicity. Drugs were chosen from those already approved or in clinical trials for AML. As shown in Figure 1, this experimental search was able to achieve selective killing of AML patient cells with a ratio close to 100:1 with respect to non-cancer control cells.To refine the choice of drugs to be used in the search, we recently developed a network model of AML intracellular signal transduction (see www.leukemianetworks.org). The present version (AML 2.1) includes 5667 proteins and 22,218 interactions. The network can be used to integrate genomic and gene expression information from individual patients and to provide a shortlist of drugs to be tested further. Using literature data and our network model we identified three compounds as promising for AML drug combinations: the MEK inhibitor PD0325901, the FLT3 inhibitor Quizartinib and the CDK 4/6 inhibitor Palbociclib. For this experiment, normal progenitor blood cells were the controls. Figure 2 shows the results of 64 experimental tests of the response to different combinations of these drugs. The two AML patients clearly exhibited different drug sensitivities, further supporting the necessity of personalized drug combinations.To improve the clinical relevance of these in vitro studies, we investigated the correlation between clinical and in vitro response to standard AML therapy, using a set of media containing 16 cytokines/growth factors in different combinations. These molecules have been selected using three methods: ligands acting on receptors that are part of gene signatures with prognostic significance in AML; cytokines that promote hematopoietic differentiation of stem cells; and ligands of receptors identified as playing an important role in AML in a recent RNAi screen. During several iterations of our combinatorial search algorithm we studied 185 different media. Figure 3 shows the correlation between clinical response (residual blasts after 28 days of therapy) and response to the same therapy in vitro after 72 h achieved in the last iteration of the search, in a study of six patients. The medium with the highest correlation had a correlation coefficient of 0.99 (p=0.0002). The in vitro response in this medium is clearly able to distinguish patients that respond to standard therapy vs those that do not, as can be seen from the inset of Fig. 3. Some cytokines (BMP-4 and IL-4) were always absent in the group of 14 media with lower correlation shown on the right of Figure 3. The remarkable variation in correlation with clinical response of these media, spanning correlation coefficient values from 0.99 to 0.004, clearly show the importance of optimizing the in vitro microenvironment for prediction of patient drug response.In summary, we identified drug combinations that selectively kill AML primary cells and we optimized media that improve the in vitro prediction of clinical drug response. These methods can together assist in the personalization of AML therapy using patientÕs cells. [Display omitted] [Display omitted] [Display omitted] DisclosuresPiermarocchi:Salgomed Inc.: Equity Ownership. Paternostro:Salgomed Inc.: Equity Ownership.

BCL6 Enables RAS-Mediated Pre-B Cell Transformation in Childhood Acute Lymphoblastic Leukemia

Background & Hypothesis: The transcriptional repressor and proto-oncogene BCL6 has recently been identified as a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and as mediator of a novel for of drug-resistance in Ph+ acute lymphoblastic leukemia (ALL; Duy et al., Nature 2011). A previous senescence rescue screen identified BCL6 as a key factor that bypassed senescence and thereby enabled RAS-mediated transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev 2002). Since ~50% of pediatric cases of ALL carry genetic lesions that result in hyperactivation of the Ras-Erk pathway (Zhang et al., Blood 2012), we tested the role of Bcl6 in this large subgroup of childhood leukemia.Results: Mutations leading to hyperactivation of the Ras-Erk pathway are found in about 50% of childhood ALL cases (Zhang et al., 2012). Among 26 ALL xenografts, we found 9 cases with constitutive Erk-T202/Y204 phosphorylation, which was paralleled by elevated expression levels of BCL6 in these cases. Studying mouse pre-B cells that were engineered with a doxycycline-inducible NRASG12D mutant, we were able to directly measure the consequences of acute activation of the Ras-Erk pathway on BCL6 expression levels. First we incubated the cells with doxycycline to induce the expression of NRASG12D and then harvested the cells at different times point to test for BCL6 protein and mRNA expression levels. Interestingly, after 24h NRASG12D-TetO pre-B cells cells showed strong upregulation of BCL6 at the mRNA (352-fold) and protein level (15-fold). Upregulation of BCL6 in response to NRASG12D-activation was sensitive to treatment with the MEK kinase inhibitor PD325901, upstream of Erk, suggesting that BCL6 expression is a consequence of Erk activation in pre-B cells. Likewise, treatment of patient-derived pre-B ALL cells with the MEK inhibitor PD325901 reversed BCL6 expression, as demonstrated by quantitative RT-PCR and Western blot. From one patient, a diagnostic (KRAS wildtype) and a relapse sample with an acquired KRASG12V mutation were available. Consistent with specific expression of BCL6 in the KRASG12V relapse ALL sample, only KRASG12V ALL cells from the relapse but not wildtype cells from the diagnostic sample were sensitive to a retro inversoBCL6 peptide inhibitor (RI-BPI; Cerchietti et al., 2009).Development of a genetic mouse model for inducible ablation of Bcl6. These findings suggest an important role of BCL6 as a cofactor of RAS-driven pre-B cell transformation, comparable to previous findings in mouse embryonic fibroblasts. To directly test a mechanistic role of Bcl6 in RAS-mediated pre-B cell transformation, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Interestingly, Mb1-Cre x Bcl6fl/fl B cell lineage cells could be transduced with NRASG12D retroviral vectors, however these cells did not give rise to leukemia when injected into congenic recipients, whereas NRASG12D-transduced Bcl6fl/fl pro-B cells that retained Bcl6 function developed B cell lineage leukemia in all transplant recipients. In a second experiment, we transformed Bcl6fl/fl pro-B cells with NRASG12D and induced Cre with a second, tamoxifen-inducible vector in full-blown leukemia cells. Acute ablation of Bcl6 in NRASG12D ALL cells completely abrogated the ability of NRASG12DALL cells to form colonies in methylcellulose and resulted in rapid apoptosis and depletion from the cell culture. We conclude that BCL6 is not only required for the initiation of RAS-driven ALL in vivo but also for the maintenance of fully established RAS-driven leukemia.Conclusion: These findings provide genetic evidence for BCL6 function as a critical cofactor of RAS-mediated transformation in childhood ALL. Inhibition of BCL6 in RAS-driven ALL may be useful to prevent leukemia relapse after initial remission (Bcl6-dependent leukemia-initiation) and also to achieve profound remission by combining conventional cytotoxic therapies with BCL6 inhibition (e.g. RI-BPI). DisclosuresNo relevant conflicts of interest to declare.

Loss of Wild-Type Kras is Associated with Enhanced Fitness and Increased Mitogen-Activated Protein Kinase Pathway Addiction in K-RasG12D Acute Myeloid Leukemia

Functional analysis of therapeutic outlier responses in cancer therapy can identify unexpected synthetic lethal interactions and uncover biomarkers that predict enhanced sensitivity to targeted agents. We performed preclinical trials of the MEK inhibitor PD0325901 (PD901) in primary murine acute myeloid leukemias (AMLs) generated using retroviral insertional mutagenesis in KrasG12D “knock-in” mice. Similar to previous observations Nf1 and Nras mutant leukemias, treatment with PD901 prolonged survival in six independent leukemias harboring diverse retroviral integrations. Intriguingly, one outlier leukemia, clone Kras.101, demonstrated a greater than 4-fold improvement in median overall survival when treated with the MEK inhibitor. Importantly, this AML demonstrated a novel retroviral integration at relapse, indicating clonal evolution during treatment. The relapsed clone displayed phenotypic resistance upon transplantation into secondary recipients and retreatment with PD901. We performed a detailed analysis of the paired PD901-sensitive/resistant Kras.101 clones to identify the mediators of mitogen-activated protein kinase pathway (MAPK) addiction. Functional studies demonstrated that the novel dominant retroviral integration in the PD901 resistant clone did not directly alter drug response. However, genome-wide analysis of copy number alterations revealed a gain of chromosome 6 involving the Kras locus in the resistant clone, which was confirmed by spectral karyotyping and fluorescence in situ hybridization. Surprisingly, the PD901-sensitive leukemia harbored two copies of oncogenic KrasG12D due to somatic uniparental disomy, while the resistant clone harbored two mutant and one wild-type Kras alleles. This observation and paired single nucleotide polymorphism analysis indicated that the PD901-resistant leukemia was evolutionarily ancestral to the drug sensitive clone. Consistent with this hypothesis, competitive repopulation experiments with fluorescently labeled leukemia cells demonstrated increased fitness of the drug sensitive leukemia in the absence of the MEK inhibitor and over-expression of wild type K-Ras in the drug-sensitive leukemia reduced fitness in vivo. Finally, utilizing a tetracycline-inducible system, we found that wild-type Kras expression enhances the fitness of the Kras.101 clone only in the presence of the MEK inhibitor. Together, these data suggest that loss of wild-type Kras imparts a fitness advantage at the expense of increased MAPK pathway addiction, which results in enhanced sensitivity to MEK inhibition in leukemias driven by oncogenic K-Ras. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Efficacy of MEK Inhibition in Nras Mutant Acute Myeloid Leukemia

Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. DisclosuresNo relevant conflicts of interest to declare.

Abstract IA10: Signaling through individual Ras proteins in the absence of the Nf1-RASGAP

Abstract Neurofibromatosis type 1 (NF1) patients are predisposed to develop neurofibromas, benign peripheral nerve tumors resulting from loss of function mutations in the NF1 gene. NF1 (neurofibromin), the protein product of the NF1 gene, is a GTPase activating protein (GAP) that inactivates the Ras proteins H-, N-, K-, M-, and R-Ras, and R-Ras2/TC21 (TC21). We hypothesized that relevant Ras proteins would be activated in vivo by deletion of NF1, so that removing single Ras proteins in Nf1 mutant cells would reveal specific functions of individual Ras proteins. We found that activation of TC21 accounts for migration defects in NF1 mutant Schwann cells (Huang et al, 2004), through AKT and not ERK. Removing TC21 in Nf1 mutant Schwann cell precursors identified an Nf1/TGFβ/TGFβRII/AKT autocrine survival loop (Patmore et al, 2012). Implying that delayed benign tumor formation resulted from effects on tumor initiation, benign tumor formation was delayed in mice lacking Nf1 and TC21. In Schwann cell precursors, activation of P-ERK, not P-AKT, was blocked by dnH-Ras. To define the role of ERK signaling in neurofibroma, we used a DhhCre;Nf1fl/fl mouse model of neurofibroma formation. Preclinical testing of specific signaling pathways with Phase I drug candidates in the neurofibroma mouse model was read out neurofibroma growth by 7T MRI imaging, in conjunction with pharmacokinetic and pharmacodynamic readouts (Jessen et al., 2013). MEK inhibition was highly effective although not curative in this model; we established minimum effective doses of the MEK inhibitor PD0325901 and effects of early MEK inhibition on peripheral nerve tumor growth. The data support the idea that NF1 GAP loss can be used to identify roles of individual Ras proteins and downstream signaling pathways in specific cell types, and that combinations of Ras proteins can collaborate to drive tumorigenesis. Supported by R01-NS28840, P50-NS057531 and the Children's Tumor Foundation/ Neurofibromatosis Therapeutic Acceleration Program. Citation Format: Nancy Ratner. Signaling through individual Ras proteins in the absence of the Nf1-RASGAP. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA10. doi: 10.1158/1557-3125.RASONC14-IA10

Effects of AKT inhibition on HGF-mediated erlotinib resistance in non-small cell lung cancer cell lines.

Acquired resistance to erlotinib in patients with EGFR-mutant non-small cell lung cancer can result from aberrant activation of alternative receptor tyrosine kinases, such as the HGF-driven c-MET receptor. We sought to determine whether inhibition of AKT signaling could augment erlotinib activity and abrogate HGF-mediated resistance. The effects of MK-2206, a selective AKT inhibitor, were evaluated in combination with erlotinib on a large panel of 13 lung cancer cell lines containing different EGFR or KRAS abnormalities. The activity of the combination was assessed using proliferation assays, flow cytometry and immunoblotting. The MEK inhibitor PD0325901 was used to determine the role of the MAP kinase pathway in erlotinib resistance. The combination of MK-2206 and erlotinib resulted in synergistic growth inhibition independent of EGFR mutation status. In cell lines where HGF blocked the anti-proliferative and cytotoxic effects of erlotinib, MK-2206 could restore cell cycle arrest, but MEK inhibition was required for erlotinib-dependent apoptosis. Both AKT and MEK inhibition contributed to cell death independent of erlotinib in the T790M-containing H1975 and the EGFR-WT cell lines tested. These findings illustrate the potential advantages and challenges of combined signal transduction inhibition as a generalized strategy to circumvent acquired erlotinib resistance.

Abstract A2: Using plexiform neurofibroma preclinical therapeutics to help guide clinical trials

Abstract Background: Neurofibromatosis type 1 (NF1) patients are predisposed to develop benign plexiform neurofibromas (PNs) that cause substantial morbidity. Surgery, the only standard treatment, is not feasible for most tumors. The NF1 gene product accelerates Ras-GTP hydrolysis to Ras-GDP and thus functions as a potent negative regulator of Ras. We developed a genetically engineered mouse model of neurofibroma, DhhCre;Nf1fl/fl, for use in preclinical testing; response is monitored by MRI imaging and volumetric analysis in conjunction with pharmacokinetic and pharmacodynamic readouts. The most effective neurofibroma therapy in this neurofibroma mouse model to date is drug candidates targeting inhibition of MEK1/2 (Jessen, W. et al., J. Clin. Invest., 2013). Results: We have continued study of the allosteric MEK inhibitor PD-0325901 using the DhhCre;Nf1fl/fl model. To establish a minimum effective dose (MED) in this mouse model we administered doses of 0.5 – 10mg/kg/day for 2 months. Neurofibromas shrank at all dose levels, correlating with significant reductions in cell proliferation and tumor vasculature, despite significant differences in PD-0325901 blood levels. To test if early MEK inhibition prevents neurofibroma growth, we administered 1.5 mg/kg/day PD-0325901 before tumors formed in the mouse, for 3 months. Neurofibromas formed, but their size was reduced. There were no rebound effects off drug. Selumetinib (AZD6244; ARRY-142886), an orally bioavailable inhibitor of MEK1/2, is being tested in a Phase 1 trial in individuals with NF1 and inoperable PNs, with response monitored using MRI and volumetric analysis. Selumetinib is administered twice daily on a continuous dosing schedule (1 cycle=28 days); the maximum tolerated dose (MTD) is determined based on toxicities observed during the first 3 cycles. To date 12 subjects (median age 14 years, range, 5-18 years) have been enrolled. Six subjects each have been treated with 20 mg/m2/dose (50% of the dose recommended in adults with solid tumors) or 30 mg/m2/dose. The most frequent adverse events included acneiform rash, asymptomatic CPK elevation, and mild gastrointestinal toxicity. Dose limiting toxicities (DLT) in 2 of 6 patients at the 30 mg/m2 dose level were asymptomatic and reversible CPK elevation (n=1) and decrease in left ventricular ejection fraction (n=1), so this dose exceeded the MTD. Only 1 of 6 patients enrolled at the 20 mg/m2 dose level developed DLT (cellulitis); this dose has been identified as the MTD for this patient population. Partial responses (PN volume decrease ≥20%) have been observed at both dose levels in 5/9 subjects. Tumor shrinkage occurred slowly over time in progressive and non-progressive PNs, and progressive disease has not been observed to date. Enrollment on this study is ongoing, and a Phase 2 study is being planned. Conclusions: A genetically engineered mouse model of neurofibromatosis is predictive of response to therapy in human NF1, supporting use of a drug testing platform that integrates mouse models and human trials. MEK inhibition downstream of Ras-GTP may be effective as a single agent, at low doses, to treat pediatric patients with neurofibroma and warrants further study. Supported by NIHP50-NS057531 and the Children's Tumor Foundation Neurofibromatosis Therapeutic Consortium to NR. Citation Format: Jianqiang Wu, Edwin Jousma, Tilat Rizvi, Richard Scott Dunn, David R. Jones, Timothy P. Cripe, Mi-Ok Kim, Leigh Marcus, Eva Dombi, Andrea Baldwin, Lauren Meyerson Long, Gillespie Andrea, Patricia Whitcomb, Staci Martin Peron, Brigitte Widemann, David Janhofer, Nancy Ratner. Using plexiform neurofibroma preclinical therapeutics to help guide clinical trials. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A2.

Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

Abstract 2623: PI3K/mTOR dual inhibitor PF-5212384 inhibits aberrant NF-kB activation and exhibits activity in combination with MEK inhibitor PD-325901 and docetaxel in human head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and standard treatment with surgery or chemoradiation has significant morbidity, with 5 year survival of &amp;lt;50%. Personalized targeted therapies with small molecule inhibitors have gained interest following identification of genetic drivers from The Cancer Genome Atlas (TCGA). TCGA data shows &amp;gt;60% of head and neck tumors harbor genomic alterations in the RTK-PI3K-mTOR and MAPK pathways important in cell growth and survival. In this study, we determined the effects of the novel PI3K-mTOR inhibitor PF-5212384 (PF-384) on molecular targets and HNSCC growth in preclinical models. PF-384 IC50s of 0.75nM-133nM were found in 12 HNSCC lines by XTT cell density assays and treatment resulted in increased sub-G0 cell death and G0/G1 phase blockade by DNA flow cytometry. PF-384 strongly inhibited direct targets of PI3K-mTOR, aberrant NF-kB transactivation and induced cytokines, but only partially inhibited MEK pathway targets. Transient siRNA knockdown of PIK3CA inhibited NF-kB activity, supporting PI3K-mTOR as an important driver and target for inhibition of NF-kB prosurvival signaling in HNSCC. PF-384 partially inhibited UMSCC1 tumor xenografts in vivo. PF-384 with MEK inhibitor PD-325901 or docetaxel revealed combinatorial activity. Given the high proportion of HNSCC patients with aberrations in the PI3K pathway and combinatorial activity observed, PF-384 with PD-901 or docetaxel merit further investigation in HNSCC. Supported by NIH Medical Research Scholars Program (SM, RVB, DFE) and NIDCD intramural projects ZIA-DC-000016, 73 and 74 (AS, JC, ZC, CVW). Citation Format: Suresh Mohan, Robert J. Vander Broek, Anthony D. Saleh, Matthew L. Pierce, Jamie F. Coupar, Danielle F. Eytan, Zhong Chen, Carter Van Waes. PI3K/mTOR dual inhibitor PF-5212384 inhibits aberrant NF-kB activation and exhibits activity in combination with MEK inhibitor PD-325901 and docetaxel in human head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2014-2623

Abstract 2687: CK2 inhibitor CX-4945 modulates AKT, NF-kB, TP53 and MEK inhibitor PD-325901 targets AP1 mediated CK2 inhibitor drug resistance in head and neck cancer

Abstract We previously identified upregulated kinase CK2 as an activator of NF-B and repressor of TP53 in promoting the malignant phenotype of head and neck squamous cell carcinoma (HNSCC). Here, we investigate the anti-tumor effects of a small molecule CK2 inhibitor CX-4945, alone and in combination with MEK inhibitor PD-0325901 (PD-901), in human HNSCC models in vitro and in vivo. CX-4945 IC50s ranged from 3.4 µM to 11.9 µM in 9 UMSCC cell lines. CX-4945 caused S and G2/M cell cycle arrest, and induced sub-G0 DNA fragments, indicating cell death. CX-4945 inhibited NF-B and BcL-XL prosurvival reporter genes in wild type (wt) TP53 (UMSCC1) and mutant (mt) TP53 (UMSCC46) cell lines, and concurrently upregulated proapoptotic TP53, p21 and prosurvival AP-1 activity only in the wtTP53 cell line. Correspondingly, CK2 phosphorylation of AKT S129 as well as T308 and S473 prosurvival signaling was reduced in both cell lines, but AP-1 inducing p-Erk1/2Thr202/204 was increased in the wtTP53 cell line. In the UMSCC1 xenografts, CX-4945 treatment significantly decreased PI3K/Akt/mTOR pathway signaling by immunostaining. While CX-4945 increased TP53 and TUNEL apoptosis marker staining at early time points (13 days after CX4945 treatment), an opposing increase in p-Erk, FosL1, cJun, JunB, and proliferation (Ki67) were also observed. Consistent with these opposing effects, no significant tumor reduction or improvement in survival was observed by CX-4945 alone. However, combination of CX-4945 with MEK/ERK inhibitor PD-901 exhibited significant synergistic anti-proliferative effects in vitro. Significant anti-tumor effects were observed with MEK inhibitor alone and in combination with CX-4945 in vivo, further supporting a role for MEK/ERK/AP-1 in resistance to CX-4945 in this HNSCC model. Supported by NIH Medical Research Scholars Program and NIDCD intramural projects ZIA-DC-000016, 73 and 74. Citation Format: Yansong Bian, Jiawei Han, Vishnu Kannabiran, Suresh Mohan, Jay Friedman, Kenna Anderes, Zhong Chen, Carter Van Waes. CK2 inhibitor CX-4945 modulates AKT, NF-kB, TP53 and MEK inhibitor PD-325901 targets AP1 mediated CK2 inhibitor drug resistance in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2014-2687