[Show abstract][Hide abstract] ABSTRACT: Background
Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.Methods
Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.ResultsThe majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3ß and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.Conclusions
The combination of PRi¿+¿MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.
[Show abstract][Hide abstract] ABSTRACT: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short one-week manufacture protocol to determine the feasibility, safety and antitumor efficacy of this double cell therapy.
A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and re-infused with (n = 10) or without (n = 3) prior cryopreservation.
14 patients with metastatic melanoma were enrolled and nine out of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within two weeks of ACT indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared to cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using non-cryopreserved T cells.
Double cell therapy with ACT of TCR engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.
No preview · Article · Mar 2014 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cuSCCs) develop in 15-30% of patients with BRAF(V600E) metastatic melanoma treated with BRAF inhibitors (BRAFi). These lesions resemble mouse skin tumors induced by the two-stage DMBA/TPA skin carcinogenesis protocol; in this protocol BRAFi accelerates tumor induction. Since prior studies demonstrated cyclooxygenase 2 (COX-2) is necessary for DMBA/TPA tumor induction, we hypothesized that COX-2 inhibition might prevent BRAFi-accelerated skin tumors. Celecoxib, a COX-2 inhibitor, significantly delayed tumor acceleration by the BRAFi inhibitor PLX7420 and decreased tumor number by 90%. Tumor gene expression profiling demonstrated that celecoxib partially reversed the PLX4720-induced gene signature. In PDV cuSCC cells, vemurafenib (a clinically approved BRAFi) increased ERK phosphorylation and soft agar colony formation; both responses were greatly decreased by celecoxib. In clinical trials trametinib, a MEK inhibitor (MEKi) increases BRAFi therapy efficacy in BRAF(V600E) melanomas and reduces BRAFi-induced KA and cuSCC frequency. Trametinib also reduced vemurafenib-induced PDV soft agar colonies, but less efficiently than celecoxib. The trametinb/celecoxib combination was more effective than either inhibitor alone. In conclusion, celecoxib suppressed both BRAFi-accelerated skin tumors and soft-agar colonies, warranting its testing as a chemopreventive agent for non-melanoma skin lesions in patients treated with BRAFi alone or in combination with MEKi.
Full-text · Article · Dec 2013 · Molecular oncology
[Show abstract][Hide abstract] ABSTRACT: BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRβ), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.
[Show abstract][Hide abstract] ABSTRACT: Molecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers.
We studied the antitumor activity of 4-1BB mAb therapy using [(18) F]-labeled fluoro-2-deoxy-2-D-glucose ([(18) F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro.
The administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [(18) F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [(3)H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data.
Increased uptake of [(18) F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy.
A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.
[Show abstract][Hide abstract] ABSTRACT: Confined to one cell: A method to detect and isolate single circulating melanoma cells (CMCs; see figure) has been produced by integrating a polymer-nanofiber-embedded nanovelcro cell-affinity assay with a laser microdissection (LMD) technique. This method is able to separate CMCs from normal white blood cells (WBCs) and sequence individual cells for a specific mutation related to cancer progression, allowing for more personalized cancer therapy.
Full-text · Article · Mar 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: Transparente Polymer‐Nanofaser‐Substrate ergaben in Kombination mit Laser‐Mikrodissektion eine neue Technik zur Isolierung einzelner Zellen, wie H.‐R. Tseng et al. in ihrer Zuschrift auf S. 3463 ff. berichten. Mit diesem neuen Verfahren wurden einzelne zirkulierende Melanomzellen (CMCs) aus dem Blut von Patienten isoliert, um eine Einzelzell‐Genotypisierung durchzuführen und eine Mutation im Proto‐Onkogen BRAF zu identifizieren.
Full-text · Article · Mar 2013 · Angewandte Chemie
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma.
This study shows that cells harboring BRAF(L597R) mutants are sensitive to MEK inhibitor treatment, providing a rationale for routine screening and therapy of BRAF(L597R)-mutant melanoma.
[Show abstract][Hide abstract] ABSTRACT: Figure S1TAK733 MTS-based colorimetric cell proliferation assay curves in melanoma cell lines of cutaneous origin according to their BRAF (A) or NRAS (B) mutational status, WT (C) and of uveal origin (D). Modulation of the melanoma cell line viability at a range of different concentrations of TAK733. The effects of TAK733 on cell growth and viability were analyzed after 72 hours of treatment using an MTS assay.
[Show abstract][Hide abstract] ABSTRACT: Figure S2Time-course analyses of the effects of TAK733 on the signaling of the MAPK and PI3K/AKT pathways by Western blot. Two BRAFV600E melanoma cell lines were exposed for varying time points to TAK733. A) The sensitive BRAFV600E mutated cutaneous melanoma cell line M229; B) The resistant BRAFV600E mutated cutaneous melanoma cell line M233.
[Show abstract][Hide abstract] ABSTRACT: TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.
The 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.
Fourteen 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.
The 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.
[Show abstract][Hide abstract] ABSTRACT: The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor therapy for melanoma patients. Here we show (V600E)B-RAF copy-number gain as a mechanism of acquired B-RAF inhibitor resistance in 4 out of 20 (20%) patients treated with B-RAF inhibitor. In cell lines, (V600E)B-RAF overexpression and knockdown conferred B-RAF inhibitor resistance and sensitivity, respectively. In (V600E)B-RAF amplification-driven (versus mutant N-RAS-driven) B-RAF inhibitor resistance, extracellular signal-regulated kinase reactivation is saturable, with higher doses of vemurafenib down-regulating phosho-extracellular signal-regulated kinase and re-sensitizing melanoma cells to B-RAF inhibitor. These two mechanisms of extracellular signal-regulated kinase reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAF inhibitor vemurafenib. In contrast to mutant N-RAS-mediated (V600E)B-RAF bypass, which is sensitive to C-RAF knockdown, (V600E)B-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired B-RAF inhibitor resistance in melanoma.
Full-text · Article · Mar 2012 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Cleaved caspase-3 in sensitive and adaptive resistant cell lines treated with vemurafenib, AZD6244, rapamycin, AKTi. Cell lines were treated by the solvent (DMSO), 2 µM of vemurafenib, AZD6244, AKTi or 10 nM of rapamycin for 48 hours. Each sample was analyzed by Western blotting using a cleaved caspase-3 (CC3) specific antibody.
[Show abstract][Hide abstract] ABSTRACT: Effects of RAPTOR knockdown by siRNAs in combination with either vemurafenib or AZD6244. The efficiency of siRNA knockdowns and its effect on downstream signaling determined by Western blot analysis of protein lysates (a). M238 parental (b) and M238-AR2 resistant subline (c) were transfected with RAPTOR siRNAs and cultured in increasing concentrations of vemurafenib or AZD6244. The effect of raptor knockdown on resistance and growth inhibition was analyzed after 120 hours by an MTS assay. D in each graph refers to the un-transfected untreated cells and is used as the 100% reference point for all the conditions in the assays.