Narsis Attar

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Reversing Melanoma Cross-Resistance to BRAF and MEK Inhibitors by Co-Targeting the AKT/mTOR Pathway

Background The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway. Methodology/Principal Findings The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance. Conclusions/Significance Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

CTLA4 blockade increases Th17 cells in patients with metastatic melanoma

BackgroundTh17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature.MethodsPeripheral blood mononuclear cells (PBMC) were collected before and after receiving tremelimumab within two clinical trials, one with tremelimumab alone (21 patients) and another together with autologous dendritic cells (DC) pulsed with the melanoma epitope MART-126–35 (6 patients). Cytokines were quantified directly in plasma from patients and after in vitro stimulation of PBMC. We also quantified IL-17 cytokine-producing cells by intracellular cytokine staining (ICS).ResultsThere were no significant changes in 13 assayed cytokines, including IL-17, when analyzing plasma samples obtained from patients before and after administration of tremelimumab. However, when PBMC were activated in vitro, IL-17 cytokine in cell culture supernatant and Th17 cells, detected as IL-17-producing CD4 cells by ICS, significantly increased in post-dosing samples. There were no differences in the levels of Th17 cells between patients with or without an objective tumor response, but samples from patients with inflammatory and autoimmune toxicities during the first cycle of therapy had a significant increase in Th17 cells.ConclusionThe anti-CTLA4 blocking antibody tremelimumab increases Th17 cells in peripheral blood of patients with metastatic melanoma. The relation between increases in Th17 cells and severe autoimmune toxicity after CTLA4 blockade may provide insights into the pathogenesis of anti-CTLA4-induced toxicities.Trial RegistrationClinical trial registration numbers: NCT0090896 and NCT00471887

Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations

BackgroundA molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.Materials and methodsThe combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.ResultsSingle agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.ConclusionsThe combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Antitumor effects of the investigational selective MEK inhibitor TAK733 against cutaneous and uveal melanoma cell lines

BackgroundTAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.MethodsThe growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.ResultsFourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.ConclusionsThe MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

Endoplasmic reticulum–mitochondria junction is required for iron homeostasis

The endoplasmic reticulum (ER)–mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.

Exploitation of EP300 and CREBBP Lysine Acetyltransferases by Cancer

p300 and CREB-binding protein (CBP), two homologous lysine acetyltransferases in metazoans, have a myriad of cellular functions. They exert their influence mainly through their roles as transcriptional regulators but also via nontranscriptional effects inside and outside of the nucleus on processes such as DNA replication and metabolism. The versatility of p300/CBP as molecular tools has led to their exploitation by viral oncogenes for cellular transformation and by cancer cells to achieve and maintain an oncogenic phenotype. How cancer cells use p300/CBP in their favor varies depending on the cellular context and is evident by the growing list of loss- and gain-of-function genetic alterations in p300 and CBP in solid tumors and hematological malignancies. Here, we discuss the biological functions of p300/CBP and how disruption of these functions by mutations and alterations in expression or subcellular localization contributes to the cancer phenotype.

Histone deacetylase inhibitors provoke a tumor supportive phenotype in pancreatic cancer associated fibroblasts

Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACis in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACis with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.

The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

Ancestral histones were present in organisms with small genomes, no nucleus, and little evidence for epigenetic regulation, suggesting histones may have additional older functions. We report that the histone H3-H4 tetramer is an enzyme that catalyzes the reduction of Cu2+ to Cu1+ when assembled in vitro from recombinant histones. Mutations of residues in the putative active site at the interface of the apposing H3 proteins alter the enzymatic activity and cellular processes such as Sod1 function or mitochondrial respiration that depend on availability of reduced copper. These effects are not due to altered gene expression or copper abundance but are consistent with decreased levels of cuprous ions. We propose that the H3-H4 tetramer is an oxidoreductase that provides biousable copper for cellular and mitochondrial chemistry. As the emergence of eukaryotes coincided with the Great Oxidation Event and decreased biousability of metals, the histone enzymatic function may have facilitated eukaryogenesis.