azd-6244 and Disease-Models--Animal

Patients with neurofibromatosis type 1 (NF1) are at increased risk for benign and malignant neoplasms. Recently, targeted therapy with the MEK inhibitor class has helped address these needs. We highlight recent successes with selumetinib while acknowledging ongoing challenges for NF1 patients and future directions.. MEK inhibitors have demonstrated efficacy for NF1-related conditions, including plexiform neurofibromas and low-grade gliomas, two common causes of NF1-related morbidity. Active investigations for NF1-related neoplasms have benefited from advanced understanding of the genomic and cell signaling alterations in these conditions and development of sound preclinical animal models. Selumetinib has become the first FDA-approved targeted therapy for NF1 following its demonstrated efficacy for inoperable plexiform neurofibroma. Investigations of combination therapy and the development of a representative NF1 swine model hold promise for translating therapies for other NF1-associated pathology.

Topics: Animals; Benzimidazoles; Clinical Trials as Topic; Disease Models, Animal; Humans; Mitogen-Activated Protein Kinase Kinases; Neurofibroma, Plexiform; Neurofibromatosis 1; Precision Medicine; Protein Kinase Inhibitors; Signal Transduction; Swine

Effective medical therapies are lacking for the treatment of neurofibromatosis type 1-related plexiform neurofibromas, which are characterized by elevated RAS-mitogen-activated protein kinase (MAPK) signaling.. We conducted a phase 1 trial of selumetinib (AZD6244 or ARRY-142886), an oral selective inhibitor of MAPK kinase (MEK) 1 and 2, in children who had neurofibromatosis type 1 and inoperable plexiform neurofibromas to determine the maximum tolerated dose and to evaluate plasma pharmacokinetics. Selumetinib was administered twice daily at a dose of 20 to 30 mg per square meter of body-surface area on a continuous dosing schedule (in 28-day cycles). We also tested selumetinib using a mouse model of neurofibromatosis type 1-related neurofibroma. Response to treatment (i.e., an increase or decrease from baseline in the volume of plexiform neurofibromas) was monitored by using volumetric magnetic resonance imaging analysis to measure the change in size of the plexiform neurofibroma.. A total of 24 children (median age, 10.9 years; range, 3.0 to 18.5) with a median tumor volume of 1205 ml (range, 29 to 8744) received selumetinib. Patients were able to receive selumetinib on a long-term basis; the median number of cycles was 30 (range, 6 to 56). The maximum tolerated dose was 25 mg per square meter (approximately 60% of the recommended adult dose). The most common toxic effects associated with selumetinib included acneiform rash, gastrointestinal effects, and asymptomatic creatine kinase elevation. The results of pharmacokinetic evaluations of selumetinib among the children in this trial were similar to those published for adults. Treatment with selumetinib resulted in confirmed partial responses (tumor volume decreases from baseline of ≥20%) in 17 of the 24 children (71%) and decreases from baseline in neurofibroma volume in 12 of 18 mice (67%). Disease progression (tumor volume increase from baseline of ≥20%) has not been observed to date. Anecdotal evidence of decreases in tumor-related pain, disfigurement, and functional impairment was observed.. Our early-phase data suggested that children with neurofibromatosis type 1 and inoperable plexiform neurofibromas benefited from long-term dose-adjusted treatment with selumetinib without having excess toxic effects. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01362803 .).

Topics: Adolescent; Animals; Benzimidazoles; Child; Child, Preschool; Disease Models, Animal; Disease Progression; Female; Humans; Magnetic Resonance Imaging; Male; Mice; Mitogen-Activated Protein Kinase Kinases; Neurofibroma, Plexiform; Neurofibromatosis 1; Protein Kinase Inhibitors

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 10

Topics: Animals; Antiviral Agents; Benzimidazoles; Cell Line; Cricetinae; Dengue Virus; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Interleukin-1beta; Mice; Severe Dengue; Signal Transduction; Tumor Necrosis Factor-alpha

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose.

Topics: Anaplastic Lymphoma Kinase; Animals; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Crizotinib; Disease Models, Animal; Drug Interactions; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Xenograft Model Antitumor Assays

Topics: Acrylonitrile; Aniline Compounds; Animals; Benzimidazoles; Biopsy, Needle; Disease Models, Animal; Gastric Mucosa; Gerbillinae; Helicobacter Infections; Helicobacter pylori; Immunohistochemistry; Male; Metaplasia; Random Allocation; Reference Values; Treatment Outcome

KRAS is one of the driver oncogenes in non-small-cell lung cancer (NSCLC) but remains refractory to current modalities of targeted pathway inhibition, which include inhibiting downstream kinase MEK to circumvent KRAS activation. Here, we show that pulsatile, rather than continuous, treatment with MEK inhibitors (MEKis) maintains T cell activation and enables their proliferation. Two MEKis, selumetinib and trametinib, induce T cell activation with increased CTLA-4 expression and, to a lesser extent, PD-1 expression on T cells in vivo after cyclical pulsatile MEKi treatment. In addition, the pulsatile dosing schedule alone shows superior anti-tumor effects and delays the emergence of drug resistance. Furthermore, pulsatile MEKi treatment combined with CTLA-4 blockade prolongs survival in mice bearing tumors with mutant Kras. Our results set the foundation and show the importance of a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy to optimally enhance tumor delay and promote long-term anti-tumor immunity.

Topics: Animals; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; CTLA-4 Antigen; Disease Models, Animal; Female; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Programmed Cell Death 1 Receptor; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Survival Rate; T-Lymphocytes

Spinal Muscular Atrophy (SMA) is a motoneuron disease caused by low levels of functional survival of motoneuron protein (SMN). Molecular disease mechanisms downstream of functional SMN loss are still largely unknown. Previous studies suggested an involvement of Rho kinase (ROCK) as well as the extracellular signal-regulated kinases (ERK) pathways in the pathomechanism. Both pathways are bi-directionally linked and inhibit each other. Thus, we hypothesize that both pathways regulate SMA pathophysiology in vivo in a combined manner rather than acting separately. Here, we applied the repurposed drugs, selumetinib, an ERK inhibitor, and the ROCK inhibitor fasudil to severe SMA mice. Thereby, separately applied inhibitors as well as a combination enabled us to explore the impact of the ROCK-ERK signaling network on SMA pathophysiology. ROCK inhibition specifically ameliorated the phenotype of selumetinib-treated SMA mice demonstrating an efficient ROCK to ERK crosstalk relevant for the SMA pathophysiology. However, ERK inhibition alone aggravated the condition of SMA mice and reduced the number of motoneurons indicating a compensatory hyper-activation of ERK in motoneurons. Taken together, we identified a regulatory network acting downstream of SMN depletion and upstream of the SMA pathophysiology thus being a future treatment target in combination with SMN dependent strategies.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Benzimidazoles; Cell Death; Cell Line; Cytoplasm; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Mice, Transgenic; Motor Neurons; Muscular Atrophy, Spinal; Random Allocation; rho-Associated Kinases; RNA, Small Interfering; Severity of Illness Index; Signal Transduction; Spinal Cord; Up-Regulation

Cancer cachexia is a multifactorial syndrome affecting the skeletal muscle. Previous clinical trials showed that treatment with MEK inhibitor selumetinib resulted in skeletal muscle anabolism. However, it is conflicting that MAPK/ERK pathway controls the mass of the skeletal muscle. The current study investigated the therapeutic effect and mechanisms of selumetinib in amelioration of cancer cachexia. The classical cancer cachexia model was established via transplantation of CT26 colon adenocarcinoma cells into BALB/c mice. The effect of selumetinib on body weight, tumor growth, skeletal muscle, food intake, serum proinflammatory cytokines, E3 ligases, and MEK/ERK-related pathways was analyzed. Two independent experiments showed that 30 mg/kg/d selumetinib prevented the loss of body weight in murine cachexia mice. Muscle wasting was attenuated and the expression of E3 ligases, MuRF1 and Fbx32, was inhibited following selumetinib treatment of the gastrocnemius muscle. Furthermore, selumetinib efficiently reduced tumor burden without influencing the cancer cell proliferation, cumulative food intake, and serum cytokines. These results indicated that the role of selumetinib in attenuating muscle wasting was independent of cancer burden. Detailed analysis of the mechanism revealed AKT and mTOR were activated, while ERK, FoxO3a, and GSK3β were inhibited in the selumetinib -treated cachexia group. These indicated that selumetinib effectively prevented skeletal muscle wasting in cancer cachexia model through ERK inhibition and AKT activation in gastrocnemius muscle via cross-inhibition. The study not only elucidated the mechanism of MEK/ERK inhibition in skeletal muscle anabolism, but also validated selumetinib therapy as an effective intervention against cancer cachexia. Mol Cancer Ther; 16(2); 334-43. ©2016 AACR.

Topics: Animals; Atrophy; Benzimidazoles; Biomarkers; Body Weight; Cachexia; Cell Line, Tumor; Colonic Neoplasms; Cytokines; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation Mediators; Mice; Models, Biological; Muscle, Skeletal; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Burden; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Recently, we had identified an unexplored pocket adjacent to the known binding site of allosteric MEK inhibitors which allowed us to design highly potent and in vivo efficacious novel inhibitors. We now report that our initial preclinical candidate, featuring a phenoxy side chain with a sulfamide capping group, displayed human carbonic anhydrase off-target activity and species-dependent blood cell accumulation, which prevented us from advancing this candidate further. Since this sulfamide MEK inhibitor displayed an exceptionally favorable PK profile with low brain penetration potential despite being highly oral bioavailable, we elected to keep the sulfamide capping group intact while taming its unwanted off-target activity by optimizing the structural surroundings. Introduction of a neighboring fluorine atom or installation of a methylene linker reduced hCA potency sufficiently, at the cost of MEK target potency. Switching to a higher fluorinated central core reinstated high MEK potency, leading to two new preclinical candidates with long half-lives, high bioavailabilities, low brain penetration potential and convincing efficacy in a K-Ras-mutated A549 xenograft model.

Topics: Allosteric Regulation; Animals; Antineoplastic Agents; Biological Availability; Brain; Carbonic Anhydrases; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Half-Life; Humans; Mice; Mitogen-Activated Protein Kinase Kinases; Molecular Structure; Protein Kinase Inhibitors; Structure-Activity Relationship; Sulfonamides; Xenograft Model Antitumor Assays

Although extracellular-regulated kinases (ERK) are a well-known central mediator in cardiac hypertrophy, no clinically available ERK antagonist has been tested for preventing cardiac hypertrophy. Selumetinib is a novel oral MEK inhibitor that is currently under Phase II and Phase III clinical investigation for advanced solid tumors. In this study, we investigated whether Selumetinib could inhibit the aberrant ERK activation of the heart in response to stress as well as prevent cardiac hypertrophy.. In an in vitro model of PE-induced cardiac hypertrophy, Selumetinib significantly inhibited the ERK activation and prevented enlargement of cardiomyocytes or reactivation of certain fetal genes. In the pathologic cardiac hypertrophy model of ascending aortic constriction, Selumetinib provided significant ERK inhibition in the stressed heart but not in the other organs. This selective ERK inhibition prevented left ventricular (LV) wall thickening, LV mass increase, fetal gene reactivation and cardiac fibrosis. In another distinct physiologic cardiac hypertrophy model of a swimming rat, Selumetinib provided a similar anti-hypertrophy effect, except that no significant fetal gene reactivation or cardiac fibrosis was observed.. Selumetinib, a novel oral anti-cancer drug with good safety records in a number of Phase II clinical trials, can inhibit ERK activity in the heart and prevent cardiac hypertrophy. These promising results indicate that Selumetinib could potentially be used to treat cardiac hypertrophy. However, this hypothesis needs to be validated in human clinical trials.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Aorta; Apoptosis; Benzimidazoles; Cardiomegaly; Constriction, Pathologic; Disease Models, Animal; Electrocardiography; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; MAP Kinase Signaling System; Myocytes, Cardiac; Proto-Oncogene Proteins c-akt; Rats; Stress, Physiological; Swimming

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Line, Tumor; Dexamethasone; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Therapy, Combination; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Ikaros Transcription Factor; Lenalidomide; Mice; Multiple Myeloma; Neoplasm Transplantation; Nerve Tissue Proteins; Plasmacytoma; Signal Transduction; Thalidomide; Trans-Activators

Improved therapeutic approaches are needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). As dual MEK and PI3K inhibition is presently being used in clinical trials for patients with PDAC, we sought to test the efficacy of combined targeting of these pathways in PDAC using both in vitro drug screens and genetically engineered mouse models (GEMM).. We performed high-throughput screening of >500 human cancer cell lines (including 46 PDAC lines), for sensitivity to 50 clinically relevant compounds, including MEK and PI3K inhibitors. We tested the top hit in the screen, the MEK1/2 inhibitor, AZD6244, for efficacy alone or in combination with the PI3K inhibitors, BKM120 or GDC-0941, in a Kras(G12D)-driven GEMM that recapitulates the histopathogenesis of human PDAC.. In vitro screens revealed that PDAC cell lines are relatively resistant to single-agent therapies. The response profile to the MEK1/2 inhibitor, AZD6244, was an outlier, showing the highest selective efficacy in PDAC. Although MEK inhibition alone was mainly cytostatic, apoptosis was induced when combined with PI3K inhibitors (BKM120 or GDC-0941). When tested in a PDAC GEMM and compared with the single agents or vehicle controls, the combination delayed tumor formation in the setting of prevention and extended survival when used to treat advanced tumors, although no durable responses were observed.. Our studies point to important contributions of MEK and PI3K signaling to PDAC pathogenesis and suggest that dual targeting of these pathways may provide benefit in some patients with PDAC. Clin Cancer Res; 21(2); 396-404. ©2014 AACR.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Benzimidazoles; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Disease Models, Animal; Drug Screening Assays, Antitumor; Drug Synergism; Erlotinib Hydrochloride; Humans; MAP Kinase Kinase Kinases; Mice, Transgenic; Morpholines; Pancreatic Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinazolines

Mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins cause dilated cardiomyopathy with variable muscular dystrophy. These mutations enhance mitogen-activated protein kinase signaling in the heart and pharmacological inhibition of extracellular signal-regulated kinase (ERK) 1 and 2 improves cardiac function in Lmna(H222P/H222P) mice. In the current study, we crossed mice lacking ERK1 to Lmna(H222P/H222P) mice and examined cardiac performance and survival. Male Lmna(H222P/H222P)/Erk1(-/-) mice lacking ERK1 had smaller left ventricular end systolic diameters and increased fractional shortening (FS) at 16 weeks of age than Lmna(H222P/H222P/)Erk1(+/+) mice. Their mean survival was also significantly longer. However, the improved cardiac function was abrogated at 20 weeks of age concurrent with an increased activity of ERK2. Lmna(H222P/H222P)/Erk1(-/-) mice treated with an inhibitor of ERK1/2 activation had smaller left ventricular diameters and increased FS at 20 weeks of age. These results provide genetic evidence that ERK1 and ERK2 contribute to the development of cardiomyopathy caused by LMNA mutations and reveal interplay between these isoenzymes in maintaining a combined pathological activity in heart.

Topics: Animals; Benzimidazoles; Cardiomyopathies; Disease Models, Animal; Female; Gene Expression Regulation; Heart Ventricles; Humans; Lamin Type A; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Autophagy; Benzimidazoles; Binding, Competitive; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Drug Synergism; Humans; Kinetics; Lung Neoplasms; Mice; Naphthyridines; Protein Binding; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; ras Proteins; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths.. The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations.. PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations.. Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Drug Synergism; Enzyme Activation; Female; Gene Knockdown Techniques; Humans; Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Rhabdomyosarcoma; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

MEK inhibition has clinical activity against biliary cancers and might therefore be successfully combined with gemcitabine, one of the most active chemotherapy agents for these cancers. As gemcitabine is active in S-phase, and the extracellular signal-regulated kinase (ERK) pathway has a major role driving cell-cycle progression, concurrent use of a MEK inhibitor could potentially antagonize the effect of gemcitabine. We therefore tested the sequence dependence of the combination of gemcitabine and the MEK inhibitor AZD6244 using a series of biliary cancer models.. Primary xenografts were established from patients with gallbladder and distal bile duct cancer and grown in severe combined immunodeficient (SCID) mice at the subcutaneous site. Plasma and tumor drug levels and the time course for recovery of ERK signaling and S-phase were measured in tumor-bearing mice treated for 48 hours with AZD6244 and then monitored for 48 hours off treatment. On the basis of these results, two different treatment schedules combining AZD6244 with gemcitabine were tested in four different biliary cancer models.. DNA synthesis was suppressed during treatment with AZD6244, and reentry into S-phase was delayed by approximately 48 hours after treatment. Strong schedule dependence was seen in all four biliary cancer models tested, suggesting that combined treatment with AZD6244 plus gemcitabine would be more active in patients with biliary cancer when gemcitabine is given following a 48-hour interruption in AZD6244 dosing, rather than concurrently.. The combination of AZD6244 plus gemcitabine is highly schedule dependent, and predicted to be more effective in the clinic using sequential rather than simultaneous dosing protocols.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Biliary Tract Neoplasms; Cell Cycle; Cell Line, Tumor; Deoxycytidine; Disease Models, Animal; Drug Therapy, Combination; Extracellular Signal-Regulated MAP Kinases; Gemcitabine; Humans; Male; Mice; Mice, SCID; Mitogen-Activated Protein Kinase Kinases; Tumor Burden; Xenograft Model Antitumor Assays

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.

Topics: Animals; Benzimidazoles; Cell Proliferation; Cells, Cultured; Collagen; Disease Models, Animal; Enzyme Activation; ErbB Receptors; Fibroblasts; Gene Expression Regulation; Humans; Lung; MAP Kinase Signaling System; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Protein Kinase Inhibitors; Pulmonary Fibrosis; Time Factors; Tumor Necrosis Factor-alpha

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Biomarkers, Tumor; Clinical Trials, Phase II as Topic; Disease Models, Animal; Docetaxel; Drug Evaluation, Preclinical; Fluorodeoxyglucose F18; Genes, p53; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Pharmacogenetics; Positron-Emission Tomography; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Randomized Controlled Trials as Topic; ras Proteins; Reproducibility of Results; Taxoids; Tomography, X-Ray Computed; Treatment Outcome

Topics: AMP-Activated Protein Kinases; Animals; Benzimidazoles; Clinical Trials as Topic; Disease Models, Animal; Drug Resistance, Neoplasm; Genes, p53; Humans; Mice; Neoplasms; Positron-Emission Tomography; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins

Topics: Animals; Benzimidazoles; Clinical Trials, Phase II as Topic; Disease Models, Animal; Docetaxel; Humans; Lung Neoplasms; Pharmacogenetics; Taxoids

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Topics: Animals; Antineoplastic Agents; Benzenesulfonates; Benzimidazoles; Breast Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Kinase 1; Mice; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Protein Kinases; Proteome; Proto-Oncogene Proteins c-myc; Pyridines; Receptor Protein-Tyrosine Kinases; Sorafenib

Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential.. We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry.. We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation.. Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Benzimidazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; Male; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Niacinamide; Phenylurea Compounds; Pyridines; raf Kinases; Signal Transduction; Sorafenib; Xenograft Model Antitumor Assays

Gastric cancer is a deadly disease for which current therapeutic options are extremely limited. Vascular endothelial growth factor receptors and platelet-derived growth factor receptors regulate gastric cancer cell proliferation, invasion, and tumor angiogenesis. In the present study, we report that sorafenib therapy effectively inhibited tumor growth and angiogenesis in tumor xenografts. These were associated with reduction in the phosphorylation of vascular endothelial growth factor receptor-2 Tyr951, c-Kit Tyr568/570, platelet-derived growth factor receptor-beta Tyr1021, and Akt Ser473 and Thr308, down-regulation of positive cell cycle regulators, increased apoptosis, and up-regulation of p27. Sorafenib treatment also caused up-regulation of p-c-Raf Ser338 and p-extracellular signal-regulated kinase (ERK) Thr202/Tyr204 in gastric cancer xenografts. The combination of sorafenib and MAP/ERK kinase inhibitor AZD6244 enhances the effectiveness of each compound alone. Potential effect of sorafenib/AZD6244 included increase in proapoptotic Bim. Our data show that MAP/ERK kinase inhibition enhances the antitumor activity of sorafenib in vivo, supporting a rationale for multitargeted suppression of the angiogenesis and ERK signaling network in gastric cancer therapy.

Topics: Animals; Antineoplastic Agents; Benzenesulfonates; Benzimidazoles; Blotting, Western; Disease Models, Animal; Drug Synergism; Humans; Mice; Mice, SCID; Niacinamide; Phenylurea Compounds; Pyridines; Sorafenib; Stomach Neoplasms