ABSTRACT
Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Neoplasm Proteins/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Stress, Physiological , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Line, Tumor , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Neoplasm Proteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
BACKGROUND: Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. METHODS: Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. RESULTS: Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. CONCLUSIONS: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease.
Subject(s)
Histone Deacetylases/adverse effects , Sarcoma, Ewing/pathology , Animals , Cell Line, Tumor , Cell Proliferation , Humans , MiceABSTRACT
BACKGROUND: Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9. METHODS: Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination. RESULTS: CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo. CONCLUSION: Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Osteoblasts are adherent cells, and under physiological conditions they attach to both mineralized and non-mineralized osseous surfaces. However, how exactly osteoblasts respond to these different osseous surfaces is largely unknown. Our hypothesis was that the state of matrix mineralization provides a functional signal to osteoblasts. To assess the osteoblast response to mineralized compared to demineralized osseous surfaces, we developed and validated a novel tissue surface model. We demonstrated that with the exception of the absence of mineral, the mineralized and demineralized surfaces were similar in molecular composition as determined, for example, by collagen content and maturity. Subsequently, we used the human osteoblastic cell line MG63 in combination with genome-wide gene set enrichment analysis (GSEA) to record and compare the gene expression signatures on mineralized and demineralized surfaces. Assessment of the 5 most significant gene sets showed on mineralized surfaces an enrichment exclusively of genes sets linked to protein synthesis, while on the demineralized surfaces 3 of the 5 enriched gene sets were associated with the matrix. Focusing on these three gene sets, we observed not only the expected structural components of the bone matrix, but also gene products, such as HMCN1 or NID2, that are likely to act as temporal migration guides. Together, these findings suggest that in osteoblasts mineralized and demineralized osseous surfaces favor intracellular protein production and matrix formation, respectively. Further, they demonstrate that the mineralization state of bone independently controls gene expression in osteoblastic cells.
Subject(s)
Bone Morphogenetic Proteins/genetics , Calcification, Physiologic/genetics , Extracellular Matrix Proteins/genetics , Extracellular Matrix/genetics , Osteoblasts/metabolism , Tibia/metabolism , Animals , Bone Density , Bone Morphogenetic Proteins/metabolism , Calcium-Binding Proteins , Cell Adhesion , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Movement , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Immunoglobulins/genetics , Immunoglobulins/metabolism , Osteoblasts/cytology , Primary Cell Culture , Protein Biosynthesis , Signal Transduction , Swine , Tibia/cytologyABSTRACT
Since publication of the article, the authors became aware that Figure 6(A,D) contained errors in the bands and loading controls. The newly compiled Figure 6A and 6D is given below.
ABSTRACT
In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.
ABSTRACT
Survival rates of pediatric sarcoma patients stagnated during the last two decades, especially in adolescents and young adults (AYAs). Targeted therapies offer new options in refractory cases. Gene expression profiling provides a robust method to characterize the transcriptome of each patient's tumor and guide the choice of therapy. Twenty patients with refractory pediatric sarcomas (age 8-35 years) were assessed with array profiling: ten had Ewing sarcoma, five osteosarcoma, and five soft tissue sarcoma. Overexpressed genes and deregulated pathways were identified as actionable targets and an individualized combination of targeted therapies was recommended. Disease status, survival, adverse events (AEs), and quality of life (QOL) were assessed in patients receiving targeted therapy (TT) and compared to patients without targeted therapy (non TT). Actionable targets were identified in all analyzed biopsies. Targeted therapy was administered in nine patients, while eleven received no targeted therapy. No significant difference in risk factors between these two groups was detected. Overall survival (OS) and progression free survival (PFS) were significantly higher in the TT group (OS: P=0.0014, PFS: P=0.0011). Median OS was 8.83 versus 4.93 months and median PFS was 6.17 versus 1.6 months in TT versus non TT group, respectively. QOL did not differ at baseline as well as at four week intervals between the two groups. TT patients had less grade 1 AEs (P=0.009). The frequency of grade 2-4 AEs did not differ. Overall, expression based targeted therapy is a feasible and likely beneficial approach in patients with refractory pediatric sarcomas that warrants further study.
ABSTRACT
Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.
Subject(s)
Genome, Human/genetics , Genomics , Mutation/genetics , Neoplasms/classification , Neoplasms/genetics , Adolescent , Adult , Child , Chromothripsis , Cohort Studies , DNA Copy Number Variations/genetics , Diploidy , Genetic Predisposition to Disease/genetics , Germ-Line Mutation/genetics , Humans , Molecular Targeted Therapy , Mutation Rate , Neoplasms/drug therapy , Tumor Suppressor Protein p53/genetics , Young AdultABSTRACT
Background: Chondromodulin-I (CHM1) sustains malignancy in Ewing sarcoma (ES). Refractory ES carries a dismal prognosis and patients with bone marrow (BM) metastases do not survive irrespective of therapy. We assessed HLA-A*02:01/CHM1-specific allorestricted T cell receptor (TCR) wild-type and transgenic cytotoxic (CD8+) T cells against ES. Patients and Methods: Three refractory HLA-A2+ ES patients were treated with HLA-A*02:01/peptide-specific allorepertoire-derived (i.e., allorestricted) CD8+ T cells. Patient #1 received up to 4.8 × 105/kg body weight HLA-A*02:01- allorestricted donor-derived wild-type CD8+ T cells. Patient #2 received up to 8.2 × 106/kg HLA-A*02:01- donor-derived and patient #3 up to 6 × 106/kg autologous allorestricted TCR transgenic CD8+ T cells. All patients were treated with the same TCR complementary determining region 3 allorecognition sequence for CHM1 peptide 319 (CHM1319). Results: HLA-A*02:01/CHM1319-specific allorestricted CD8+ T cells showed specific in vitro lysis of all patient-derived ES cell lines. Therapy was well tolerated and did not cause graft versus host disease (GvHD). Patients #1 and #3 showed slow progression, whereas patient #2, while having BM involvement, showed partial metastatic regression associated with T cell homing to involved lesions. CHM1319 TCR transgenic T cells could be tracked in his BM for weeks. Conclusions: CHM1319-TCR transgenic T cells home to affected BM and may cause partial disease regression. HLA-A*02:01/antigen-specific allorestricted T cells proliferate in vivo without causing GvHD.
ABSTRACT
Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by EWS-ETS translocations and early metastasis to lung and bone. In this study, we investigated the role of the BRICHOS chaperone domain-containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS-ETS translocation, EWS-FLI1. Using RNA interference, we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF. This was in line with the induction of the number of tartrate-resistant acid phosphatase (TRAP+ )-stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knockdown, indicating that CHM1-mediated inhibition of osteomimicry might play a role in homing, colonization, and invasion into bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated matrix metallopeptidase 9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, which need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES.
Subject(s)
Cell Differentiation , Intercellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/secondary , Membrane Proteins/metabolism , Sarcoma, Ewing/pathology , Animals , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation , Chondrocytes/metabolism , Chondrocytes/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/genetics , Lung Neoplasms/pathology , Membrane Proteins/genetics , Mice, Inbred BALB C , Neoplasm Invasiveness , Phenotype , Sarcoma, Ewing/geneticsABSTRACT
Pregnancy-associated plasma protein-A (PAPPA), also known as pappalysin, is a member of the insulin-like growth factor (IGF) family. PAPPA acts as a protease, cleaving IGF inhibitors, i.e., IGF binding proteins (IGFBPs), thereby setting free IGFs. The insulin/IGF-axis is involved in cancer in general and in Ewing sarcoma (ES) in particular. ES is a highly malignant bone tumor characterized by early metastatic spread. PAPPA is associated with various cancers. It is overexpressed and required for proliferation in ES. PAPPA also stimulates normal bone growth. We isolated HLA-A*02:01+/peptide-restricted T cells from A*02:01- healthy donors directed against PAPPA, generated by priming with A*02:01+ PAPPA peptide loaded dendritic cells. After TCR identification, retrovirally TCR transduced CD8+ T cells were assessed for their in vitro specificity and in vivo efficacy in human ES bearing Rag2-/-γc-/- mice. Engraftment in mice and tumor infiltration of TCR transgenic T cells in the mice was evaluated. The TCR transgenic T cell clone PAPPA-2G6 demonstrated specific reactivity toward HLA-A*02:01+/PAPPA+ ES cell lines. We furthermore detected circulating TCR transgenic T cells in the blood in Rag2-/-γc-/- mice and in vivo engraftment in bone marrow. Tumor growth in mice with xenografted ES was significantly reduced after treatment with PAPPA-2G6 TCR transgenic T cells in contrast to controls. Tumors of treated mice revealed tumor-infiltrating PAPPA-2G6 TCR transgenic T cells. In summary, we demonstrate that PAPPA is a first-rate target for TCR-based immunotherapy of ES.
ABSTRACT
Ewing sarcoma (ES) is the second most frequent childhood bone cancer driven by the EWS/FLI1 (EF) fusion protein. Genetically defined ES models are needed to understand how EF expression changes bone precursor cell differentiation, how ES arises and through which mechanisms of inhibition it can be targeted. We used mesenchymal Prx1-directed conditional EF expression in mice to study bone development and to establish a reliable sarcoma model. EF expression arrested early chondrocyte and osteoblast differentiation due to changed signaling pathways such as hedgehog, WNT or growth factor signaling. Mesenchymal stem cells (MSCs) expressing EF showed high self-renewal capacity and maintained an undifferentiated state despite high apoptosis. Blocking apoptosis through enforced BCL2 family member expression in MSCs promoted efficient and rapid sarcoma formation when transplanted to immunocompromised mice. Mechanistically, high BCL2 family member and CDK4, but low P53 and INK4A protein expression synergized in Ewing-like sarcoma development. Functionally, knockdown of Mcl1 or Cdk4 or their combined pharmacologic inhibition resulted in growth arrest and apoptosis in both established human ES cell lines and EF-transformed mouse MSCs. Combinatorial targeting of survival and cell cycle progression pathways could counteract this aggressive childhood cancer.
Subject(s)
Cell Cycle , Cell Transformation, Neoplastic/pathology , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/metabolism , Animals , Animals, Newborn , Apoptosis , Bone and Bones/pathology , Cell Cycle Checkpoints , Cell Differentiation , Cell Proliferation , Cell Survival , Cell Transformation, Neoplastic/metabolism , Extremities/pathology , Gene Expression Profiling , Gene Knockdown Techniques , Human Embryonic Stem Cells/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Mice , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Osteogenesis , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction , Transduction, GeneticABSTRACT
AIM: Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells. METHODS: We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells. RESULTS: Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319. CONCLUSION: Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells.
Subject(s)
Antigens, Neoplasm/metabolism , CD8-Positive T-Lymphocytes/cytology , Lysosomal Membrane Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adoptive Transfer , Annexins/metabolism , Cell Separation , Colon/metabolism , Flow Cytometry , Genotype , HLA-A2 Antigen/metabolism , Humans , Immunotherapy, Adoptive , K562 Cells , Leukocytes, Mononuclear/cytology , Lysosomal-Associated Membrane Protein 1/metabolism , Oligonucleotide Array Sequence Analysis , Receptors, Adrenergic, beta-3/metabolism , Retina/metabolism , Retroviridae/genetics , TransgenesABSTRACT
Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and ß-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.
ABSTRACT
This report summarizes the results of the 3rd Joint ENCCA-WP7, EuroSarc, EEC, PROVABES, and EURAMOS European Bone Sarcoma Network Meeting, which was held at the Children's Cancer Research Institute in Vienna, Austria on September 24-25, 2015. The joint bone sarcoma network meetings bring together European bone sarcoma researchers to present and discuss current knowledge on bone sarcoma biology, genetics, immunology, as well as results from preclinical investigations and clinical trials, to generate novel hypotheses for collaborative biological and clinical investigations. The ultimate goal is to further improve therapy and outcome in patients with bone sarcomas.
ABSTRACT
The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion.In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction.After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2-/-É£c-/- mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells.CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.
Subject(s)
CD8-Positive T-Lymphocytes/transplantation , Hematopoietic Stem Cell Transplantation/methods , Immunotherapy, Adoptive/methods , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Sarcoma, Ewing/therapy , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Coculture Techniques , DNA-Binding Proteins/genetics , Graft vs Host Disease/immunology , Granzymes/metabolism , HLA-A Antigens/genetics , HLA-A Antigens/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Immunotherapy, Adoptive/adverse effects , Interferon-gamma/metabolism , Liver/pathology , Lung/pathology , Mice , Mice, Inbred BALB C , Oncogene Addiction , Receptors, Antigen, T-Cell/genetics , Retroviridae/genetics , Sarcoma, Ewing/immunology , Sarcoma, Ewing/pathology , Transduction, Genetic , Transplantation, Homologous/adverse effects , Transplantation, Homologous/methodsABSTRACT
Small blue round cell tumors (SBRCTs) are a heterogenous group of tumors that are difficult to diagnose because of overlapping morphologic, immunohistochemical, and clinical features. About two-thirds of EWSR1-negative SBRCTs are associated with CIC-DUX4-related fusions, whereas another small subset shows BCOR-CCNB3 X-chromosomal paracentric inversion. Applying paired-end RNA sequencing to an SBRCT index case of a 44-year-old man, we identified a novel BCOR-MAML3 chimeric fusion, which was validated by reverse transcription polymerase chain reaction and fluorescence in situ hybridization techniques. We then screened a total of 75 SBRCTs lacking EWSR1, FUS, SYT, CIC, and BCOR-CCNB3 abnormalities for BCOR break-apart probes by fluorescence in situ hybridization to detect potential recurrent BCOR gene rearrangements outside the typical X-chromosomal inversion. Indeed, 8/75 (11%) SBRCTs showed distinct BCOR gene rearrangements, with 2 cases each showing either a BCOR-MAML3 or the alternative ZC3H7B-BCOR fusion, whereas no fusion partner was detected in the remaining 4 cases. Gene expression of the BCOR-MAML3-positive index case showed a distinct transcriptional profile with upregulation of HOX-gene signature, compared with classic Ewing's sarcoma or CIC-DUX4-positive SBRCTs. The clinicopathologic features of the SBRCTs with alternative BCOR rearrangements were also compared with a group of BCOR-CCNB3 inversion-positive cases, combining 11 from our files with a meta-analysis of 42 published cases. The BCOR-CCNB3-positive tumors occurred preferentially in children and in bone, in contrast to alternative BCOR-rearranged SBRCTs, which presented in young adults, with a variable anatomic distribution. Furthermore, BCOR-rearranged tumors often displayed spindle cell areas, either well defined in intersecting fascicles or blending with the round cell component, which appears distinct from most other fusion-positive SBRCTs and shares histologic overlap with poorly differentiated synovial sarcoma.
Subject(s)
DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Sarcoma, Small Cell/genetics , Transcription Factors/genetics , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Gene Expression Profiling , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators , Young AdultABSTRACT
Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor of the PI3K pathway. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.