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1.
Cancer Res Commun ; 3(5): 884-895, 2023 05.
Article in English | MEDLINE | ID: mdl-37377898

ABSTRACT

Purpose: Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed. Patients and Methods: We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival. Results: Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival. Conclusions: This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients. Significance: The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.


Subject(s)
Chemokine CCL21 , Melanoma , Humans , Chemokine CCL21/genetics , Chemokines/blood , Epigenesis, Genetic , Follow-Up Studies , Immunotherapy , Melanoma/drug therapy
2.
Cancers (Basel) ; 15(3)2023 Jan 20.
Article in English | MEDLINE | ID: mdl-36765608

ABSTRACT

Patients with metastatic melanoma have a historically poor prognosis, but recent advances in treatment options, including targeted therapy and immunotherapy, have drastically improved the outcomes for some of these patients. However, not all patients respond to available treatments, and around 50% of patients with metastatic cutaneous melanoma and almost all patients with metastases of uveal melanoma die of their disease. Thus, there is a need for novel treatment strategies for patients with melanoma that do not benefit from the available therapies. Chimeric antigen receptor-expressing T (CAR-T) cells are largely unexplored in melanoma. Traditionally, CAR-T cells have been produced by transducing blood-derived T cells with a virus expressing CAR. However, tumor-infiltrating lymphocytes (TILs) can also be engineered to express CAR, and such CAR-TILs could be dual-targeting. To this end, tumor samples and autologous TILs from metastasized human uveal and cutaneous melanoma were expanded in vitro and transduced with a lentiviral vector encoding an anti-HER2 CAR construct. When infused into patient-derived xenograft (PDX) mouse models carrying autologous tumors, CAR-TILs were able to eradicate melanoma, even in the absence of antigen presentation by HLA. To advance this concept to the clinic and assess its safety in an immune-competent and human-patient-like setting, we treated four companion dogs with autologous anti-HER2 CAR-TILs. We found that these cells were tolerable and showed signs of anti-tumor activity. Taken together, CAR-TIL therapy is a promising avenue for broadening the tumor-targeting capacity of TILs in patients with checkpoint immunotherapy-resistant melanoma.

3.
Cancers (Basel) ; 14(12)2022 Jun 10.
Article in English | MEDLINE | ID: mdl-35740548

ABSTRACT

The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs' capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

4.
Melanoma Res ; 32(4): 241-248, 2022 08 01.
Article in English | MEDLINE | ID: mdl-34753889

ABSTRACT

Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but around half of all patients develop resistance early or late during treatment. The situation is even worse for patients with metastatic uveal melanoma (UM). Here we hypothesized that the immunotherapy of therapy-resistant skin melanoma or UM can be enhanced by epigenetic inhibitors. Cultured B16F10 cells and human UM cells were treated with the histone deacetylase inhibitor (HDACi) entinostat or BETi JQ1. Entinostat-induced HLA expression and PD-L1, but JQ1 did not. A syngeneic mouse model carrying B16-F10 melanoma cells was treated with PD-1 and CTLA4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic melanoma resistant to PD-1 inhibition is warranted.


Subject(s)
Melanoma, Experimental , Skin Neoplasms , Animals , B7-H1 Antigen , Epigenesis, Genetic , Melanoma , Melanoma, Experimental/drug therapy , Mice , Melanoma, Cutaneous Malignant
5.
Cancers (Basel) ; 13(17)2021 Aug 26.
Article in English | MEDLINE | ID: mdl-34503115

ABSTRACT

Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.

6.
Nat Commun ; 12(1): 5155, 2021 08 27.
Article in English | MEDLINE | ID: mdl-34453044

ABSTRACT

Preclinical studies have suggested that epigenetic therapy could enhance immunogenicity of cancer cells. We report the results of the PEMDAC phase 2 clinical trial (n = 29; NCT02697630) where the HDAC inhibitor entinostat was combined with the PD-1 inhibitor pembrolizumab in patients with metastatic uveal melanoma (UM). The primary endpoint was objective response rate (ORR), and was met with an ORR of 14%. The clinical benefit rate at 18 weeks was 28%, median progression free survival was 2.1 months and the median overall survival was 13.4 months. Toxicities were manageable, and there were no treatment-related deaths. Objective responses and/or prolonged survival were seen in patients with BAP1 wildtype tumors, and in one patient with an iris melanoma that exhibited a UV signature. Longer survival also correlated with low baseline ctDNA levels or LDH. In conclusion, HDAC inhibition and anti-PD1 immunotherapy results in durable responses in a subset of patients with metastatic UM.Trial registration ClinicalTrials.gov registration number: NCT02697630 (registered 3 March 2016). EudraCT registration number: 2016-002114-50.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Benzamides/administration & dosage , Melanoma/drug therapy , Pyridines/administration & dosage , Uveal Neoplasms/drug therapy , Humans , Melanoma/pathology , Progression-Free Survival , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Uveal Neoplasms/pathology
7.
Epigenetics ; 16(1): 54-63, 2021 01.
Article in English | MEDLINE | ID: mdl-32603264

ABSTRACT

(1) Background: BET bromodomain proteins regulate transcription by binding acetylated histones and attracting key factors for, e.g., transcriptional elongation. BET inhibitors have been developed to block pathogenic processes such as cancer and inflammation. Despite having potent biological activities, BET inhibitors have still not made a breakthrough in clinical use for treating cancer. Multiple resistance mechanisms have been proposed but thus far no attempts to block this in glioma has been made. (2) Methods: Here, we have conducted a pharmacological synergy screen in glioma cells to search for possible combination treatments augmenting the apoptotic response to BET inhibitors. We first used HMBA, a compound that was developed as a differentiation therapy four decades ago but more recently was shown to primarily inhibit BET bromodomain proteins. Data was also generated using other BET inhibitors. (3) Results: In the synergy screen, we discovered that several MEK inhibitors can enhance apoptosis in response to HMBA in rat and human glioma cells in vitro as well as in vivo xenografts. The combination is not unique to HMBA but also other BET inhibitors such as JQ1 and I-BET-762 can synergize with MEK inhibitors. (4) Conclusions: Our findings validate a combination therapy previously demonstrated to exhibit anti-cancer activities in multiple other tumour types but which appears to have been lost in translation to the clinic.


Subject(s)
Acetamides/therapeutic use , Antineoplastic Agents/therapeutic use , Azepines/therapeutic use , Brain Neoplasms/drug therapy , Glioma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Triazoles/therapeutic use , Acetamides/administration & dosage , Acetamides/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Azepines/administration & dosage , Azepines/pharmacology , Cell Line, Tumor , Drug Synergism , Humans , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Rats , Transcription Factors/antagonists & inhibitors , Triazoles/administration & dosage , Triazoles/pharmacology
8.
Life Sci Alliance ; 3(8)2020 08.
Article in English | MEDLINE | ID: mdl-32571801

ABSTRACT

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.


Subject(s)
Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Animals , Cell Line, Tumor , Checkpoint Kinase 1/physiology , DNA Damage/drug effects , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics
9.
Nat Commun ; 11(1): 1894, 2020 04 20.
Article in English | MEDLINE | ID: mdl-32313009

ABSTRACT

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments. Here we genomically profile metastatic tumors and infiltrating lymphocytes. BAP1 alterations are overrepresented and found in 29/32 of cases. Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease. One outlier tumor has a high mutational burden associated with UV-damage. CDKN2A deletions also occur, which are rarely present in primaries. A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains. Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant. This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.


Subject(s)
Gene Expression Regulation, Neoplastic , Melanoma/genetics , Neoplasms, Second Primary/genetics , Uveal Neoplasms/genetics , Animals , Cyclin-Dependent Kinase Inhibitor p16/genetics , Female , Gene Dosage , Gene Knockdown Techniques , Humans , Lymphocytes , Lymphocytes, Tumor-Infiltrating/pathology , Melanoma/immunology , Melanoma/metabolism , Melanoma/pathology , Mice , Mutation , Neoplasms, Second Primary/immunology , Neoplasms, Second Primary/metabolism , Neoplasms, Second Primary/pathology , Prognosis , Sequence Analysis, DNA , Transcriptome , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Uveal Neoplasms/immunology , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathology
11.
Gut ; 69(8): 1472-1482, 2020 08.
Article in English | MEDLINE | ID: mdl-32001555

ABSTRACT

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) still carries a dismal prognosis with an overall 5-year survival rate of 9%. Conventional combination chemotherapies are a clear advance in the treatment of PDAC; however, subtypes of the disease exist, which exhibit extensive resistance to such therapies. Genomic MYC amplifications represent a distinct subset of PDAC with an aggressive tumour biology. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. The aim of the study was to find and to target MYC-associated dependencies. DESIGN: We analysed human PDAC gene expression datasets. Results were corroborated by the analysis of the small ubiquitin-like modifier (SUMO) pathway in a large PDAC cohort using immunohistochemistry. A SUMO inhibitor was used and characterised using human and murine two-dimensional, organoid and in vivo models of PDAC. RESULTS: We observed that MYC is connected to the SUMOylation machinery in PDAC. Components of the SUMO pathway characterise a PDAC subtype with a dismal prognosis and we provide evidence that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition. CONCLUSION: SUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.


Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins c-myc/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Aged , Animals , Apoptosis , Carcinoma, Pancreatic Ductal/drug therapy , Cell Line, Tumor , Cell Proliferation , Enzyme Inhibitors/pharmacology , Esters/pharmacology , Female , Gene Amplification , Gene Expression , Humans , Male , Mice , Middle Aged , Neoplasm Transplantation , Organoids/metabolism , Pancreatic Neoplasms/drug therapy , Prognosis , Proto-Oncogene Proteins c-myc/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Sulfonic Acids , Sumoylation/drug effects , Sumoylation/genetics , Transcriptome/drug effects , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitins/genetics , Ubiquitins/metabolism
12.
BMC Cancer ; 19(1): 415, 2019 05 02.
Article in English | MEDLINE | ID: mdl-31046743

ABSTRACT

BACKGROUND: While recent years have seen a revolution in the treatment of metastatic cutaneous melanoma, no treatment has yet been able to demonstrate any prolonged survival in metastatic uveal melanoma. Thus, metastatic uveal melanoma remains a disease with an urgent unmet medical need. Reports of treatment with immune checkpoint inhibitors have thus far been disappointing. Based on animal experiments, it is reasonable to hypothesize that the effect of immunotherapy may be augmented by epigenetic therapy. Proposed mechanisms include enhanced expression of HLA class I and cancer antigens on cancer cells, as well as suppression of myeloid suppressor cells. METHODS: The PEMDAC study is a multicenter, open label phase II study assessing the efficacy of concomitant use of the PD1 inhibitor pembrolizumab and the class I HDAC inhibitor entinostat in adult patients with metastatic uveal melanoma. Primary endpoint is objective response rate. Eligible patients have histologically confirmed metastatic uveal melanoma, ECOG performance status 0-1, measurable disease as per RECIST 1.1 and may have received any number of prior therapies, with the exception of anticancer immunotherapy. Twenty nine patients will be enrolled. Patients receive pembrolizumab 200 mg intravenously every third week in combination with entinostat 5 mg orally once weekly. Treatment will continue until progression of disease or intolerable toxicity or for a maximum of 24 months. DISCUSSION: The PEMDAC study is the first trial to assess whether the addition of an HDAC inhibitor to anti-PD1 therapy can yield objective anti-tumoral responses in metastatic UM. TRIAL REGISTRATION: ClinicalTrials.gov registration number: NCT02697630 . (Registered 3 March 2016). EudraCT registration number: 2016-002114-50.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Benzamides/administration & dosage , Melanoma/drug therapy , Pyridines/administration & dosage , Uveal Neoplasms/drug therapy , Administration, Intravenous , Administration, Oral , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamides/therapeutic use , Drug Administration Schedule , Female , Humans , Male , Prospective Studies , Pyridines/therapeutic use , Research Design , Treatment Outcome
13.
Cancer Res ; 79(5): 899-904, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30622115

ABSTRACT

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR-T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. SIGNIFICANCE: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.


Subject(s)
Immunotherapy, Adoptive/methods , Melanoma/therapy , Receptor, ErbB-2/immunology , T-Lymphocytes/transplantation , Uveal Neoplasms/therapy , Animals , Cell Line, Tumor , Humans , Interleukin Receptor Common gamma Subunit/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/transplantation , Melanoma/enzymology , Melanoma/immunology , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Receptor, ErbB-2/metabolism , Skin Neoplasms/enzymology , Skin Neoplasms/immunology , Skin Neoplasms/therapy , T-Lymphocytes/immunology , Uveal Neoplasms/enzymology , Uveal Neoplasms/immunology , Xenograft Model Antitumor Assays , Melanoma, Cutaneous Malignant
14.
Cell Death Dis ; 9(8): 810, 2018 07 24.
Article in English | MEDLINE | ID: mdl-30042422

ABSTRACT

Karonudib (TH1579) is a novel compound that exerts anti-tumor activities and has recently entered phase I clinical testing. The aim of this study was to conduct a pre-clinical trial in patient-derived xenografts to identify the possible biomarkers of response or resistance that could guide inclusion of patients suffering from metastatic melanoma in phase II clinical trials. Patient-derived xenografts from 31 melanoma patients with metastatic disease were treated with karonudib or a vehicle for 18 days. Treatment responses were followed by measuring tumor sizes, and the models were categorized in the response groups. Tumors were harvested and processed for RNA sequencing and protein analysis. To investigate the effect of karonudib on T-cell-mediated anti-tumor activities, tumor-infiltrating T cells were injected in mice carrying autologous tumors and the mice treated with karonudib. We show that karonudib has heterogeneous anti-tumor effect on metastatic melanoma. Thus, based on the treatment responses, we could divide the 31 patient-derived xenografts in three treatment groups: progression group (32%), suppression group (42%), and regression group (26%). Furthermore, we show that karonudib has anti-tumor effect, irrespective of major melanoma driver mutations. Also, we identify high expression of ABCB1, which codes for p-gp pumps as a resistance biomarker. Finally, we show that karonudib treatment does not hamper T-cell-mediated anti-tumor responses. These findings can be used to guide future use of karonudib in clinical use with a potential approach as precision medicine.


Subject(s)
Drug Resistance, Neoplasm , Enzyme Inhibitors/therapeutic use , Melanoma/drug therapy , Pyrimidines/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biomarkers, Tumor/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Humans , Melanoma/pathology , Mice , Pyrimidines/pharmacology , Transplantation, Heterologous , Tumor Cells, Cultured , Up-Regulation
15.
Article in English | MEDLINE | ID: mdl-32913988

ABSTRACT

PURPOSE: Cancer of unknown primary is a group of metastatic tumors in which the standard diagnostic workup fails to identify the site of origin of the tumor. The potential impact of precision oncology on this group of patients is large, because actionable driver mutations and a correct diagnosis could provide treatment options otherwise not available for patients with these fatal cancers. This study investigated if comprehensive genomic analyses could provide information on the origin of the tumor. PATIENTS AND METHODS: Here we describe a patient whose tumor was misdiagnosed at least three times. Next-generation sequencing, a patient-derived xenograft mouse model, and bioinformatics were used to identify an actionable mutation, predict resistance development to the targeted therapy, and correctly diagnose the origin of the tumor. Transcriptomic classification was benchmarked using The Cancer Genome Atlas (TCGA). RESULTS: Despite the lack of a known primary tumor site and the absence of diagnostic immunohistochemical markers, the origin of the patient's tumor was established using the novel bioinformatic workflow. This included a mutational signature analysis of the sequenced metastases and comparison of their transcriptomic profiles to a pan-cancer panel of tumors from TCGA. We further discuss the strengths and limitations of the latter approaches in the context of three potentially incorrectly diagnosed TCGA lung tumors. CONCLUSION: Comprehensive genomic analyses can provide information on the origin of tumors in patients with cancer of unknown primary.

17.
Nat Commun ; 8(1): 707, 2017 09 27.
Article in English | MEDLINE | ID: mdl-28955032

ABSTRACT

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma. To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 - a melanoma PDX model where tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in non-obese diabetic/severe combined immune-deficient/common gamma chain (NOG/NSG) knockout mouse. Key to T-cell survival/effect in this model is the continuous presence of interleukin-2 (IL-2). Tumors that grow in PDXv2.0 are eradicated if the autologous tumor cells and T cells come from a patient that exhibited an objective response to ACT in the clinic. However, T cells from patients that are non-responders to ACT cannot kill tumor cells in PDXv2.0. Taken together, PDXv2.0 provides the potential framework to further model genetically diverse human cancers for assessing the efficacy of immunotherapies as well as combination therapies.Combining different types of immune therapies might benefit certain patients. Here, the authors develop an autologous immune-humanized melanoma mouse model that allows the preclinical assessment of cancer cell-T cell interactions from each individual patient and the benefits of immunotherapies combinations.


Subject(s)
Interleukin-2/genetics , Lymphocytes, Tumor-Infiltrating/transplantation , Melanoma/therapy , Skin Neoplasms/therapy , T-Lymphocytes/transplantation , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Humans , Immunotherapy, Adoptive , Interleukin Receptor Common gamma Subunit/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/pathology , Melanoma/immunology , Melanoma/pathology , Mice, Knockout , Neoplasm Metastasis , Skin Neoplasms/immunology , Skin Neoplasms/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology
18.
Cell Death Dis ; 8(8): e2982, 2017 08 10.
Article in English | MEDLINE | ID: mdl-28796244

ABSTRACT

Metastatic malignant melanoma continues to be a challenging disease despite clinical translation of the comprehensive understanding of driver mutations and how melanoma cells evade immune attack. In Myc-driven lymphoma, efficacy of epigenetic inhibitors of the bromodomain and extra-terminal domain (BET) family of bromodomain proteins can be enhanced by combination therapy with inhibitors of the DNA damage response kinase ATR. Whether this combination is active in solid malignancies like melanoma, and how it relates to immune therapy, has not previously investigated. To test efficacy and molecular consequences of combination therapies cultured melanoma cells were used. To assess tumor responses to therapies in vivo we use patient-derived xenografts and B6 mice transplanted with B16F10 melanoma cells. Concomitant inhibition of BET proteins and ATR of cultured melanoma cells resulted in similar effects as recently shown in lymphoma, such as induction of apoptosis and p62, implicated in autophagy, senescence-associated secretory pathway and ER stress. In vivo, apoptosis and suppression of subcutaneous growth of patient-derived melanoma and B16F10 cells were observed. Our data suggest that ATRI/BETI combination therapies are effective in melanoma.


Subject(s)
Antineoplastic Agents/therapeutic use , Melanoma/drug therapy , Proteins/antagonists & inhibitors , Proteins/metabolism , Quinolines/therapeutic use , Animals , Apoptosis/drug effects , Apoptosis/genetics , Autophagy/drug effects , Autophagy/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Lymphoma/drug therapy , Lymphoma/genetics , Melanoma/genetics , Mice , Proteins/genetics , Signal Transduction/drug effects , Signal Transduction/genetics
19.
Biomaterials ; 102: 231-8, 2016 09.
Article in English | MEDLINE | ID: mdl-27344366

ABSTRACT

To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm.


Subject(s)
Cell Fractionation/methods , Exosomes/genetics , Gene Transfer Techniques , RNA Interference , RNA, Small Interfering/administration & dosage , Animals , Biomimetics/methods , Cell Line , Electroporation , Filtration/methods , Human Umbilical Vein Endothelial Cells , Humans , Mice , NIH 3T3 Cells , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering/genetics
20.
Cancer Res ; 76(8): 2376-83, 2016 04 15.
Article in English | MEDLINE | ID: mdl-26941288

ABSTRACT

Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376-83. ©2016 AACR.


Subject(s)
Acetamides/pharmacology , Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Cell Line, Tumor , Humans , Mice
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