dactolisib and Melanoma

The PI3k pathway represents an attractive target for drug development in melanoma, as numerous studies have shown that this pathway is active in malignant melanocytes. In addition, previous work has shown that multi-level targeting of this pathway might be more effective than targeting the pathway at a single level. In this review, we discuss targeting different members of this pathway, potential escape mechanisms, classes of specific molecular inhibitors, and development of NVP-BEZ235, a novel dual PI3k/mTOR inhibitor.

Topics: Animals; Antineoplastic Agents; Clinical Trials as Topic; Humans; Imidazoles; Melanoma; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases

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

Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Lactams, Macrocyclic; Melanoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Skin; TOR Serine-Threonine Kinases

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

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

(V600E)B-RAF mutation is found in 50% to 60% of melanomas, and the novel agents PLX4032/vemurafenib and GSK2118436 that inhibit the (V600E)B-RAF kinase achieve a remarkable clinical response rate. However, as might be expected, acquired clinical resistance to these agents arises in most melanoma patients. PLX4032/vemurafenib resistance that arises in vivo in tumor matched short-term cultures or in vitro in melanoma cell lines is not caused by acquisition of secondary mutations in (V600E)B-RAF but rather is caused by upregulating platelet-derived growth factor receptor β (PDGFRβ) or N-RAS which results in resistance or sensitivity to mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK) kinase inhibitors, respectively. In this study, we define a targeted combinatorial strategy to overcome PLX4032/vemurafenib resistance in melanoma cell lines or short-term culture where the resistance is driven by PDGFRβ upregulation, achieving synergistic growth inhibition and cytotoxicity. PDGFRβ-upregulated, PLX4032-resistant (PPRM) cell lines show dual phospho (p)-ERK and p-AKT upregulation, and their growth inhibitory responses to specific small molecule inhibitors correlated with p-ERK, p-AKT, and p-p70S6K levels. Coordinate inhibition of (V600E)B-RAF inhibition and the RTK-PI3K-AKT-mTORC axis led to functionally significant rebound signaling, illustrating a robust and dynamic network connectivity. Combined B-RAF, phosphoinositide 3-kinase (PI3K), and mTORC1/2 inhibition suppressed both immediate early and delayed compensatory signaling, resulting in a highly synergistic growth inhibitory response but less efficient cytotoxic response. In contrast, the combination of MEK1/2, PI3K, and mTORC1/2 inhibitors consistently triggered apoptosis in a highly efficient manner. Together, our findings offer a rational strategy to guide clinical testing in preidentified subsets of patients who relapse during treatment with (V600E)B-RAF inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Imidazoles; Indoles; MAP Kinase Kinase Kinases; Mechanistic Target of Rapamycin Complex 1; Melanoma; Morpholines; Multiprotein Complexes; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Point Mutation; Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Quinolines; Receptor, Platelet-Derived Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Spheroids, Cellular; Sulfonamides; TOR Serine-Threonine Kinases; Transcription Factors; Vemurafenib

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