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1.
Cancer Immunol Immunother ; 65(6): 753-63, 2016 06.
Article in English | MEDLINE | ID: mdl-27160390

ABSTRACT

T cell checkpoint blockade with antibodies targeting programmed cell death (ligand)-1 (PD-1/PD-L1) and/or cytotoxic T lymphocyte-antigen 4 (CTLA-4) has improved therapy outcome in melanoma patients. However, a considerable proportion of patients does not benefit even from combined α-CTLA-4 and α-PD-1 therapy. We therefore examined to which extent T cell (co)stimulation and/or stereotactic body radiation therapy (SBRT) could further enhance the therapeutic efficacy of T cell checkpoint blockade in a genetically engineered mouse melanoma model that is driven by PTEN-deficiency, and BRAFV600 mutation, as in human, but lacks the sporadic UV-induced mutations. Tumor-bearing mice were treated with different combinations of immunomodulatory antibodies (α-CTLA-4, α-PD-1, α-CD137) or interleukin-2 (IL-2) alone or in combination with SBRT. None of our immunotherapeutic approaches (alone or in combination) had any anti-tumor efficacy, while SBRT alone delayed melanoma outgrowth. However, α-CD137 combined with α-PD-1 antibodies significantly enhanced the anti-tumor effect of SBRT, while the anti-tumor effect of SBRT was not enhanced by interleukin-2, or the combination of α-CTLA-4 and α-PD-1. We conclude that α-CD137 and α-PD-1 antibodies were most effective in enhancing SBRT-induced tumor growth delay in this mouse melanoma model, outperforming the ability of IL-2, or the combination of α-CTLA-4 and α-PD-1 to synergize with SBRT. Given the high mutational load and increased immunogenicity of human melanoma with the same genotype, our findings encourage testing α-CD137 and α-PD-1 alone or in combination with SBRT clinically, particularly in patients refractory to α-CTLA-4 and/or α-PD-1 therapy.


Subject(s)
Codon , Melanoma/genetics , Mutation , Programmed Cell Death 1 Receptor/metabolism , Proto-Oncogene Proteins B-raf/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Biomarkers , Combined Modality Therapy , Disease Models, Animal , Humans , Immunomodulation/drug effects , Immunomodulation/radiation effects , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma/drug therapy , Melanoma/pathology , Melanoma/radiotherapy , Mice , Mice, Transgenic , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor Necrosis Factor Receptor Superfamily, Member 9/antagonists & inhibitors
2.
Int J Mol Sci ; 17(12)2016 Dec 20.
Article in English | MEDLINE | ID: mdl-27999416

ABSTRACT

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies.


Subject(s)
Carcinoma, Squamous Cell/genetics , Integrases/genetics , Keratoacanthoma/genetics , Melanoma/genetics , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Animals , Carcinoma, Squamous Cell/pathology , Cell Line , Disease Models, Animal , Gene Transfer Techniques , HEK293 Cells , Humans , Keratoacanthoma/pathology , Melanoma/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , PTEN Phosphohydrolase/genetics , Promoter Regions, Genetic/genetics
3.
Elife ; 102021 08 17.
Article in English | MEDLINE | ID: mdl-34399888

ABSTRACT

The molecular basis underlying glioblastoma (GBM) heterogeneity and plasticity is not fully understood. Using transcriptomic data of human patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a key regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the neurofibromatosis type 1 gene (NF1), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature and reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations.


Subject(s)
Brain Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Neoplastic Stem Cells/pathology , Neurofibromin 1/genetics , Proto-Oncogene Proteins c-fos/genetics , Cell Line, Tumor , Humans , Neurofibromin 1/metabolism , Proto-Oncogene Proteins c-fos/metabolism
4.
Nat Commun ; 11(1): 3883, 2020 08 04.
Article in English | MEDLINE | ID: mdl-32753598

ABSTRACT

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Subject(s)
Brain Neoplasms/drug therapy , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Drug Resistance, Neoplasm/genetics , Gene Rearrangement , Glioma/drug therapy , Neoplasm Recurrence, Local/genetics , Temozolomide/pharmacology , Tumor Suppressor Proteins/genetics , Adolescent , Adult , Aged , Animals , Brain Neoplasms/genetics , Cell Line, Tumor , DNA Adducts/drug effects , DNA Adducts/metabolism , DNA Methylation , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Female , Gene Expression Regulation, Neoplastic , Glioma/genetics , Humans , Male , Mice , Middle Aged , Neoplasm Recurrence, Local/prevention & control , Promoter Regions, Genetic/genetics , RNA-Seq , Temozolomide/therapeutic use , Tumor Suppressor Proteins/metabolism , Up-Regulation , Whole Genome Sequencing , Xenograft Model Antitumor Assays , Young Adult
5.
Cancer Immunol Res ; 7(4): 670-682, 2019 04.
Article in English | MEDLINE | ID: mdl-30782666

ABSTRACT

To increase cancer immunotherapy success, PD-1 blockade must be combined with rationally selected treatments. Here, we examined, in a poorly immunogenic mouse breast cancer model, the potential of antibody-based immunomodulation and conventional anticancer treatments to collaborate with anti-PD-1 treatment. One requirement to improve anti-PD-1-mediated tumor control was to promote tumor-specific cytotoxic T-cell (CTL) priming, which was achieved by stimulating the CD137 costimulatory receptor. A second requirement was to overrule PD-1-unrelated mechanisms of CTL suppression in the tumor microenvironment (TME). This was achieved by radiotherapy and cisplatin treatment. In the context of CD137/PD-1-targeting immunotherapy, radiotherapy allowed for tumor elimination by altering the TME, rather than intrinsic CTL functionality. Combining this radioimmunotherapy regimen with low-dose cisplatin improved CTL-dependent regression of a contralateral tumor outside the radiation field. Thus, systemic tumor control may be achieved by combining immunotherapy protocols that promote T-cell priming with (chemo)radiation protocols that permit CTL activity in both the irradiated tumor and (occult) metastases.


Subject(s)
Antineoplastic Agents/therapeutic use , Cisplatin/therapeutic use , Neoplasms/therapy , Radioimmunotherapy , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Female , Mice, Inbred C57BL , Neoplasms/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor Microenvironment , Tumor Necrosis Factor Receptor Superfamily, Member 9/agonists
6.
Oncoimmunology ; 5(12): e1238557, 2016.
Article in English | MEDLINE | ID: mdl-28123875

ABSTRACT

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAFV600E/PTEN-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8+ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade.

7.
Cancer Res ; 73(16): 5288-98, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23824741

ABSTRACT

Interleukin (IL)-6 overexpression and constitutive STAT3 activation occur in many cancers, including prostate cancer. However, their contribution to prostate stem and progenitor cells has not been explored. In this study, we show that stem-like cells from patients with prostate cancer secrete higher levels of IL-6 than their counterparts in non-neoplastic prostate. Tumor grade did not influence the levels of expression or secretion. Stem-like and progenitor cells expressed the IL-6 receptor gp80 with concomitant expression of pSTAT3. Blockade of activated STAT3, by either anti-IL-6 antibody siltuximab (CNTO 328) or LLL12, a specific pSTAT3 inhibitor, suppressed the clonogenicity of the stem-like cells in patients with high-grade disease. In a murine xenograft model used to determine the in vivo effects of pSTAT3 suppression, LLL12 treatment effectively abolished outgrowth of a patient-derived castrate-resistant tumor. Our results indicate that the most primitive cells in prostate cancer require pSTAT3 for survival, rationalizing STAT3 as a therapeutic target to treat advanced prostate cancer.


Subject(s)
Janus Kinases/antagonists & inhibitors , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , Aged , Aged, 80 and over , Animals , Anthraquinones/pharmacology , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Male , Mice , Middle Aged , Neoplastic Stem Cells/drug effects , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism , Sulfonamides/pharmacology
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