azd-6244 and dactolisib

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; MAP Kinase Kinase Kinases; Melanoma; Mice; Mice, Nude; Mice, SCID; Mutation; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Quinolines; Signal Transduction; Spheroids, Cellular; TOR Serine-Threonine Kinases

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells.. The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining.. A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested.. The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzimidazoles; Blotting, Western; Conjunctival Neoplasms; Female; Fluorescent Antibody Technique, Indirect; Humans; Imidazoles; Indazoles; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Quinolines; Sulfonamides; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution.

Topics: Antineoplastic Agents, Hormonal; Benzimidazoles; Cell Line, Tumor; Estradiol; Evolution, Molecular; Female; Fulvestrant; Humans; Imidazoles; Indoles; Male; Melanoma; Oximes; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; Quinolines; Signal Transduction; Sulfonamides

Although epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab are used widely to treat KRAS wild-type metastatic colorectal cancer (mCRC), patients become resistant by various mechanisms, including KRAS, BRAF, and PIK3CA mutations, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we investigated the mechanisms of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in a KRAS and PIK3CA mutation CRC xenograft model. HCT116 (KRAS (G13D) , PIK3CA (H1047R) mutant) cells were subcutaneously injected into the nude mice. Mice were randomly assigned to treatment with vehicle, cetuximab, AZD6244, BEZ235, or AZD6244 plus BEZ235, for up to 3 weeks; then, all mice were sacrificed, and tumor tissues were subjected to Western blot analysis and immunohistochemical staining. AZD6244 or BEZ235 slightly inhibit tumor growth of HCT116 xenografts, and the combination treatment markedly enhanced their antitumor effects. However, cetuximab had no effect on tumor growth. Western blot analysis and immunohistochemical staining revealed that treatment with AZD6244 or BEZ235 could significantly reduce the phosphorylation level of ERK1/2 or AKT in HCT116 tumor tissues. More interesting, the antiangiogenic effects were substantially enhanced when the agents were combined which may due to the reduced expression of VEGF and matrix metallopeptidase-9 (MMP-9) in tumor tissues. These results suggest that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in CRC harboring with KRAS and PIK3CA mutations. The mechanisms of synergistic antitumor effects may be due to antiangiogenesis.

Topics: Animals; Antibodies, Monoclonal, Humanized; Benzimidazoles; Cetuximab; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Imidazoles; MAP Kinase Kinase 1; Matrix Metalloproteinase 9; Mice; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinolines; ras Proteins; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Endometrial carcinoma (EC) is the most common gynecological cancer in the Western World. Treatment options are limited for advanced and recurrent disease. Therefore, new treatment options are necessary. Inhibition of the PI3K/AKT/mTOR and/or the Ras/Raf/MEK pathways is suggested to be clinically relevant. However, the knowledge about the effect of combination targeted therapy in EC is limited. The aim of this study was to investigate the effect of these therapies on primary endometrioid EC cell cultures in vitro and in vivo.. Primary endometrioid EC cell cultures were incubated with Temsirolimus (mTORC1 inhibitor), NVP-BKM120 (pan-PI3K inhibitor), NVP-BEZ235 (pan-PI3K/mTOR inhibitor), or AZD6244 (MEK1/2 inhibitor) as single treatment. In vitro, the effect of NVP-BEZ235 with or without AZD6244 was determined for cell viability, cell cycle arrest, apoptosis induction, and cell signaling. In vivo, the effect of NVP-BEZ35 was investigated for 2 subcutaneous xenograft models of the corresponding primary cultures.. NVP-BEZ235 was the most potent PI3K/AKT/mTOR pathway inhibitor. NVP-BEZ235 and AZD6244 reduced cell viability and induced cell cycle arrest and apoptosis, by reduction of p-AKT, p-S6, and p-ERK levels. Combination treatment showed a synergistic effect. In vivo, NVP-BEZ235 reduced tumor growth and inhibited p-S6 expression. The effects of the compounds were independent of the mutation profile of the cell cultures used.. A synergistic antitumor effect was shown for NVP-BEZ235 and AZD6244 in primary endometrioid EC cells in vitro. In addition, NVP-BEZ235 induced reduction of tumor growth in vivo. Therefore, targeted therapies seem an interesting strategy to further evaluate in clinical trials.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Carcinoma, Endometrioid; Drug Synergism; Endometrial Neoplasms; Female; Humans; Imidazoles; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; raf Kinases; ras Proteins; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The mitogen-activated protein kinases (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are intertwined on various levels and simultaneous inhibition reduces tumorsize and prolonges survival synergistically. Furthermore, inhibiting these pathways radiosensitized cancer cells in various studies. To assess, if phenotypic changes after perturbations of this signaling network depend on the genetic background, we integrated a time series of the signaling data with phenotypic data after simultaneous MAPK/ERK kinase (MEK) and PI3K/mTOR inhibition and ionizing radiation (IR).. The MEK inhibitor AZD6244 and the dual PI3K/mTOR inhibitor NVP-BEZ235 were tested in glioblastoma and lung carcinoma cells, which differ in their mutational status in the MAPK and the PI3K/mTOR pathways. Effects of AZD6244 and NVP-BEZ235 on the proliferation were assessed using an ATP assay. Drug treatment and IR effects on the signaling network were analyzed in a time-dependent manner along with measurements of phenotypic changes in the colony forming ability, apoptosis, autophagy or cell cycle.. Both inhibitors reduced the tumor cell proliferation in a dose-dependent manner, with NVP-BEZ235 revealing the higher anti-proliferative potential. Our Western blot data indicated that AZD6244 and NVP-BEZ235 perturbed the MAPK and PI3K/mTOR signaling cascades, respectively. Additionally, we confirmed crosstalks and feedback loops in the pathways. As shown by colony forming assay, the AZD6244 moderately radiosensitized cancer cells, whereas NVP-BEZ235 caused a stronger radiosensitization. Combining both drugs did not enhance the NVP-BEZ235-mediated radiosensitization. Both inhibitors caused a cell cycle arrest in the G1-phase, whereas concomitant IR and treatment with the inhibitors resulted in cell line- and drug-specific cell cycle alterations. Furthermore, combining both inhibitors synergistically enhanced a G1-phase arrest in sham-irradiated glioblastoma cells and induced apoptosis and autophagy in both cell lines.. Perturbations of the MEK and the PI3K pathway radiosensitized tumor cells of different origins and the combination of AZD6244 and NVP-BEZ235 yielded cytostatic effects in several tumor entities. However, this is the first study assessing, if the combination of both drugs also results in synergistic effects in terms of radiosensitivity. Our study demonstrates that simultaneous treatment with both pathway inhibitors does not lead to synergistic radiosensitization but causes cell line-specific effects.

Topics: Antineoplastic Agents; Benzimidazoles; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Enzyme Inhibitors; Humans; Imidazoles; Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Radiation Tolerance; Radiation-Sensitizing Agents; Signal Transduction; TOR Serine-Threonine Kinases

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models.. NCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining.. AZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9).. In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients.

Topics: Animals; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Female; Gefitinib; Humans; Imidazoles; Ki-67 Antigen; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet Endothelial Cell Adhesion Molecule-1; Protein Kinase Inhibitors; Protein Kinases; Quinazolines; Quinolines; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Imidazoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Targeted Therapy; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Quinolines; ras Proteins; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa

Prostate cancer is an ideal target for chemoprevention. To date, chemoprevention clinical trials with 5α-reductase inhibitors have yielded encouraging yet ultimately confounding results. Using a preclinical mouse model of high-grade prostatic intraepithelial neoplasia (HG-PIN) induced by PTEN loss, we observed unprecedented deteriorating effects of androgen deprivation, in which surgical castration or MDV3100 treatment accelerated disease progression of the otherwise stable HG-PIN to invasive castration-resistant prostate cancer (CRPC). As an alternative, targeting the phosphoinositide 3-kinase (PI3K) signaling pathway via either genetic ablation of genes encoding PI3K components or pharmacologic inhibition of the PI3K pathway reversed the PTEN loss-induced HG-PIN phenotype. Finally, concurrent inhibition of the PI3K and mitogen-activated protein kinase (MAPK) pathways was effective in blocking the growth of PTEN-null CRPC. Together, these data have revealed the potential adverse effects of antiandrogen chemoprevention in certain genetic contexts (such as PTEN loss) while showing the promise of targeted therapy in the clinical management of this complex and prevalent disease.. Chemoprevention with antiandrogen therapies is attractive for prostate cancer, given its prevalence and established hormonally mediated pathogenesis. However, because PTEN loss has been found in 9% to 45% of HG-PIN in the clinic, the current findings suggest that patients with PTEN-deficient prostate tumors might be better treated with PI3K-targeted therapies.

Topics: Aminopyridines; Androgen Receptor Antagonists; Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Castration; Imidazoles; Male; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Morpholines; Nitriles; Phenylthiohydantoin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Quinolines; Testosterone

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.

Topics: Benzamides; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphenylamine; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Humans; Imidazoles; Indazoles; Mitogen-Activated Protein Kinase Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Quinolines; Signal Transduction; Structure-Activity Relationship; Sulfonamides; TOR Serine-Threonine Kinases

Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.. We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).. Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the "AZD/BEZ" combination in the melanoma GEMM, we next tested this regimen in the "claudin-low" breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2(+) tumors.. These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Breast Neoplasms; Female; Humans; Imidazoles; Mammary Neoplasms, Animal; MAP Kinase Kinase Kinases; Melanoma; Mice; Neoplasms, Experimental; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases

Mutant KRAS is a feature of more than 25% of non-small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Female; Genes, ras; Humans; Imidazoles; Lung Neoplasms; Mice; Mice, Nude; Mutation; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Triazoles; Xenograft Model Antitumor Assays

Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K.. Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244.. Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC(50) values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage.. mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

Topics: Benzimidazoles; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Synergism; Humans; Imidazoles; Melanoma; Molecular Targeted Therapy; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Skin Neoplasms; Tissue Array Analysis; TOR Serine-Threonine Kinases