RESUMO
BACKGROUND: We explored potential predictive biomarkers of immunotherapy response in patients with extensive-stage small-cell lung cancer (ES-SCLC) treated with durvalumab (D) + tremelimumab (T) + etoposide-platinum (EP), D + EP, or EP in the randomized phase 3 CASPIAN trial. METHODS: 805 treatment-naïve patients with ES-SCLC were randomized (1:1:1) to receive D + T + EP, D + EP, or EP. The primary endpoint was overall survival (OS). Patients were required to provide an archived tumor tissue block (or ≥ 15 newly cut unstained slides) at screening, if these samples existed. After assessment for programmed cell death ligand-1 expression and tissue tumor mutational burden, residual tissue was used for additional molecular profiling including by RNA sequencing and immunohistochemistry. RESULTS: In 182 patients with transcriptional molecular subtyping, OS with D ± T + EP was numerically highest in the SCLC-inflamed subtype (n = 10, median 24.0 months). Patients derived benefit from immunotherapy across subtypes; thus, additional biomarkers were investigated. OS benefit with D ± T + EP versus EP was greater with high versus low CD8A expression/CD8 cell density by immunohistochemistry, but with no additional benefit with D + T + EP versus D + EP. OS benefit with D + T + EP versus D + EP was associated with high expression of CD4 (median 25.9 vs. 11.4 months) and antigen-presenting and processing machinery (25.9 vs. 14.6 months) and MHC I and II (23.6 vs. 17.3 months) gene signatures, and with higher MHC I expression by immunohistochemistry. CONCLUSIONS: These findings demonstrate the tumor microenvironment is important in mediating better outcomes with D ± T + EP in ES-SCLC, with canonical immune markers associated with hypothesized immunotherapy mechanisms of action defining patient subsets that respond to D ± T. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03043872.
Assuntos
Biomarcadores Tumorais , Imunoterapia , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/terapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/mortalidade , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Feminino , Masculino , Imunoterapia/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pessoa de Meia-Idade , Idoso , Anticorpos Monoclonais/uso terapêutico , Resultado do Tratamento , Estadiamento de Neoplasias , Anticorpos Monoclonais Humanizados/uso terapêutico , Prognóstico , AdultoRESUMO
Many targeted cancer therapies rely on biomarkers assessed by scoring of immunohistochemically (IHC)-stained tissue, which is subjective, semiquantitative, and does not account for expression heterogeneity. We describe an image analysis-based method for quantitative continuous scoring (QCS) of digital whole-slide images acquired from baseline human epidermal growth factor receptor 2 (HER2) IHC-stained breast cancer tissue. Candidate signatures for patient stratification using QCS of HER2 expression on subcellular compartments were identified, addressing the spatial distribution of tumor cells and tumor-infiltrating lymphocytes. Using data from trastuzumab deruxtecan-treated patients with HER2-positive and HER2-negative breast cancer from a phase 1 study (NCT02564900; DS8201-A-J101; N = 151), QCS-based patient stratification showed longer progression-free survival (14.8 vs 8.6 months) with higher prevalence of patient selection (76.4 vs 56.9%) and a better cross-validated log-rank p value (0.026 vs 0.26) than manual scoring based on the American Society of Clinical Oncology / College of American Pathologists guidelines. QCS-based features enriched the HER2-negative subgroup by correctly predicting 20 of 26 responders.
Assuntos
Neoplasias da Mama , Seleção de Pacientes , Receptor ErbB-2 , Trastuzumab , Humanos , Feminino , Receptor ErbB-2/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Trastuzumab/uso terapêutico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Adulto , Imunoconjugados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Idoso , Imuno-Histoquímica , Camptotecina/análogos & derivadosRESUMO
The IL-6-gp130-STAT3 signaling axis is a major regulator of inflammation. Activating mutations in the gene encoding gp130 and germline gain-of-function mutations in STAT3 (STAT3GOF) are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis. To dissect crucial cellular subsets and disease biology involved in activated gp130 signaling, the gp130-JAK-STAT3 axis was constitutively activated using a transgene, L-gp130, specifically targeted to T cells. Activating gp130 signaling in T cells in vivo resulted in fatal, early onset, multi-organ autoimmunity in mice that resembled human STAT3GOF disease. Female mice had more rapid disease progression than male mice. On a cellular level, gp130 signaling induced the activation and effector cell differentiation of T cells, promoted the expansion of T helper type 17 (TH17) cells, and impaired the activity of regulatory T cells. Transcriptomic profiling of CD4+ and CD8+ T cells from these mice revealed commonly dysregulated genes and a gene signature that, when applied to human transcriptomic data, improved the segregation of patients with transcriptionally diverse STAT3GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to TH17-driven autoimmunity that phenotypically resembles human STAT3GOF disease.
Assuntos
Autoimunidade , Linfócitos T CD8-Positivos , Humanos , Masculino , Feminino , Camundongos , Animais , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Autoimunidade/genética , Linfócitos T CD8-Positivos/metabolismo , Transdução de Sinais , Inflamação , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
The DNA damage response (DDR) acts as a barrier to malignant transformation and is often impaired during tumorigenesis. Exploiting the impaired DDR can be a promising therapeutic strategy; however, the mechanisms of inactivation and corresponding biomarkers are incompletely understood. Starting from an unbiased screening approach, we identified the SMC5-SMC6 Complex Localization Factor 2 (SLF2) as a regulator of the DDR and biomarker for a B-cell lymphoma (BCL) patient subgroup with an adverse prognosis. SLF2-deficiency leads to loss of DDR factors including Claspin (CLSPN) and consequently impairs CHK1 activation. In line with this mechanism, genetic deletion of Slf2 drives lymphomagenesis in vivo. Tumor cells lacking SLF2 are characterized by a high level of DNA damage, which leads to alterations of the post-translational SUMOylation pathway as a safeguard. The resulting co-dependency confers synthetic lethality to a clinically applicable SUMOylation inhibitor (SUMOi), and inhibitors of the DDR pathway act highly synergistic with SUMOi. Together, our results identify SLF2 as a DDR regulator and reveal co-targeting of the DDR and SUMOylation as a promising strategy for treating aggressive lymphoma.
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Dano ao DNA , Linfoma de Células B , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B , Reparo do DNA , Linfoma de Células B/genéticaRESUMO
Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM cell lines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor-resistant MM.
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Mieloma Múltiplo , Inibidores de Proteassoma , Humanos , Animais , Camundongos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Sumoilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Apoptose , Enzimas Ativadoras de Ubiquitina/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/farmacologiaRESUMO
Aberrant activity of the SUMOylation pathway has been associated with MYC overexpression and poor prognosis in aggressive B-cell lymphoma (BCL) and other malignancies. Recently developed small-molecule inhibitors of SUMOylation (SUMOi) target the heterodimeric E1 SUMO activation complex (SAE1/UBA2). Here, we report that activated MYC signaling is an actionable molecular vulnerability in vitro and in a preclinical murine in vivo model of MYC-driven BCL. While SUMOi conferred direct effects on MYC-driven lymphoma cells, SUMO inhibition also resulted in substantial remodeling of various subsets of the innate and specific immunity in vivo. Specifically, SUMOi increased the number of memory B cells as well as cytotoxic and memory T cells, subsets that are attributed a key role within a coordinated anti-tumor immune response. In summary, our data constitute pharmacologic SUMOi as a powerful therapy in a subset of BCL causing massive remodeling of the normal B-cell and T-cell compartment.
Assuntos
Linfoma de Células B , Linfoma , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Linfoma/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Biomarcadores , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.
Assuntos
Apresentação de Antígeno , Neoplasias , Antígenos de Histocompatibilidade Classe I , Humanos , Evasão da Resposta Imune , Neoplasias/patologia , SumoilaçãoRESUMO
Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.
Assuntos
Apoptose/genética , Carcinoma Ductal Pancreático/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Expressão Gênica , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Mutações Sintéticas Letais , Carcinoma Ductal Pancreático/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Humanos , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.
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Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Mutação , Sumoilação/fisiologia , Animais , Biomarcadores Tumorais , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Cromatina , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Instabilidade Genômica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biomarkers that predict response to lenalidomide maintenance therapy in patients with multiple myeloma (MM) have remained elusive. We have shown that immunomodulatory drugs (IMiDs) exert anti-MM activity via destabilization of MCT1 and CD147. In this study, cell samples of 654 patients with MM who received lenalidomide (n = 455), thalidomide (n = 98), or bortezomib (n = 101) maintenance were assessed by gene expression profiling and RNA sequencing, followed by correlation of MCT1 and CD147 expression with data for progression-free survival (PFS) and overall survival (OS). Patients with high expression levels of MCT1 showed significantly reduced PFS (31.9 months vs 48.2 months in MCT1high vs MCT1low; P = .03) and OS (75.9 months vs not reached [NR] in MCT1high vs MCT1low; P = .001) in cases with lenalidomide maintenance, whereas MCT1 expression had no significant impact on PFS or OS in cases with bortezomib maintenance. We validated the predictive role of MCT1 for IMiD-based maintenance in an independent cohort of patients who received thalidomide (OS, 83.6 months vs NR in MCT1high vs MCT1low; P = .03). Functional validation showed that MCT1 overexpression in human MM cell lines significantly reduced the efficacy of lenalidomide, whereas no change was observed with bortezomib treatment, either in vitro or in a MM xenograft model. Our findings have established MCT1 expression as a predictive marker for response to lenalidomide-based maintenance in patients with MM.
Assuntos
Mieloma Múltiplo , Biomarcadores , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Humanos , Lenalidomida/uso terapêutico , Mieloma Múltiplo/terapia , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
BACKGROUND: Supply bottlenecks in midwifery care could intensify due to increasing birth rates in Bavaria. Midwives are already fully booked and can only take care of pregnant women by increasing their working hours. The situation of self-employed midwives is particularly precarious. Bavaria has been supporting self-employed midwives by granting the Bavarian midwife bonus of up to 1,000 per year since September 2018. This analysis aims to describe the current status of the unique Bavarian midwife bonus scheme and to describe current programmes to improve care in obstetrics in Bavaria. METHODS: All midwives who were self-employed, had their primary residence in Bavaria and had cared for at least four births in the application year were eligible for the Bavarian midwife bonus. We used data from application years 2017 and 2018 to describe the number of applications per year and the socio-demographic characteristics of the applicants and analyzed the regional distribution of the midwives and the regional supply density. RESULTS: In 2017, a total of 723 midwives received the bonus, in 2018 already 852 midwives. The average age of the applicants, who were exclusively female, was 44 years in the application year 2018. In 2018, 37 midwives per 100,000 women aged 15-44 were self-employed in obstetrics in Bavaria. A regional comparison showed an above-average supply density in the Upper Palatinate (52) and Lower Bavaria (54) and a below-average supply density in the Upper Franconia (13). CONCLUSION: The analysis of the Bavarian midwifery bonus enables a glance, albeit limited, into the supply situation of self-employed midwives in Bavaria. Whether the political instruments can improve the supply situation in Bavaria have to be evaluated using newly generated representative data. Central registration of midwives in Bavaria could be useful.
Assuntos
Gravidez , Feminino , Humanos , Adulto , AlemanhaRESUMO
Aberrant CXCR4 activity has been implicated in lymphoma pathogenesis, disease progression, and resistance to therapies. Using a mouse model with a gain-of-function CXCR4 mutation (CXCR4C1013G) that hyperactivates CXCR4 signaling, we identified CXCR4 as a crucial activator of multiple key oncogenic pathways. CXCR4 hyperactivation resulted in an expansion of transitional B1 lymphocytes, which represent the precursors of chronic lymphocytic leukemia (CLL). Indeed, CXCR4 hyperactivation led to a significant acceleration of disease onset and a more aggressive phenotype in the murine Eµ-TCL1 CLL model. Hyperactivated CXCR4 signaling cooperated with TCL1 to cause a distinct oncogenic transcriptional program in B cells, characterized by PLK1/FOXM1-associated pathways. In accordance, Eµ-TCL1;CXCR4C1013G B cells enriched a transcriptional signature from patients with Richter's syndrome, an aggressive transformation of CLL. Notably, MYC activation in aggressive lymphoma was associated with increased CXCR4 expression. In line with this finding, additional hyperactive CXCR4 signaling in the Eµ-Myc mouse, a model of aggressive B-cell cancer, did not impact survival. In summary, we here identify CXCR4 hyperactivation as a co-driver of an aggressive lymphoma phenotype.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Forkhead Box M1/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores CXCR4/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Progressão da Doença , Feminino , Proteína Forkhead Box M1/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores CXCR4/genética , Quinase 1 Polo-LikeRESUMO
An urgent medical need to develop novel treatment strategies for patients with pancreatic ductal adenocarcinoma (PDAC) exists. However, despite various efforts in the histopathological and molecular subtyping of PDAC, novel targeted or specific therapies have not been established. Posttranslational modifications (PTMs) with ubiquitin-like proteins, including small ubiquitin-like modifiers (SUMOs), mediate numerous processes that can contribute to the fitness and survival of cancer cells. The contribution of SUMOylation to transcriptional control, DNA repair pathways, mitotic progression, and oncogenic signalling has been described. Here we review functions of the SUMO pathway in PDAC, with a special focus on its connection to an aggressive subtype of the disease characterised by high MYC activity, and discuss SUMOylation inhibitors under development for precise PDAC therapies.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Carcinoma Ductal Pancreático/patologia , Sobrevivência Celular/fisiologia , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Reparo do DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Mitose/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína SUMO-1/antagonistas & inibidores , Proteína SUMO-1/metabolismo , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação/efeitos dos fármacos , Mutações Sintéticas Letais , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/metabolismoRESUMO
Multiple myeloma is a genetically heterogeneous plasma cell malignancy characterized by organ damage and a massive production of (in-)complete monoclonal antibodies. Coping with protein homeostasis and post-translational regulation is therefore essential for multiple myeloma cells to survive. Furthermore, post-translational modifications such as ubiquitination and SUMOylation play key roles in essential pathways in multiple myeloma, including NFκB signaling, epigenetic regulation, as well as DNA damage repair. Drugs modulating the ubiquitin-proteasome system, such as proteasome inhibitors and thalidomide analogs, are approved and highly effective drugs in multiple myeloma. In this review, we focus on ubiquitin and ubiquitin-like modifications in the biology and current developments of new treatments for multiple myeloma.
RESUMO
The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.
RESUMO
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.
Assuntos
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Idoso , Animais , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Ésteres/farmacologia , Feminino , Amplificação de Genes , Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Organoides/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Prognóstico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Ácidos Sulfônicos , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Transcriptoma/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Cisteína Endopeptidases/metabolismo , Genoma Humano , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cisteína Endopeptidases/química , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Sumoilação , Fatores de Transcrição/metabolismo , CoesinasRESUMO
How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.
Assuntos
Envelhecimento/patologia , Neoplasias do Sistema Nervoso Central/patologia , Quimiocina CCL19/metabolismo , Gliose/patologia , Linfoma/patologia , Adolescente , Adulto , Idoso , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/patologia , Linhagem Celular Tumoral/transplante , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Neoplasias do Sistema Nervoso Central/cirurgia , Quimiocina CCL19/genética , Quimiocina CXCL12 , Modelos Animais de Doenças , Feminino , Gliose/diagnóstico por imagem , Humanos , Microscopia Intravital , Linfoma/diagnóstico por imagem , Linfoma/cirurgia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Imagem com Lapso de Tempo , Adulto JovemRESUMO
Aberrant activity of the glycoprotein 130 130/JAK/STAT3 (gp130/JAK/STAT3) signaling axis is a recurrent event in inflammation and cancer. In particular, it is associated with a wide range of hematological malignancies, including multiple myeloma and leukemia. Novel targeted therapies have only been successful for some subtypes of these malignancies, underlining the need for developing robust mouse models to better dissect the role of this pathway in specific tumorigenic processes. Here, we investigated the role of selective gp130/JAK/STAT3 activation by generating a conditional mouse model. This model targeted constitutively active, cell-autonomous gp130 activity to B cells, as well as to the entire hematopoietic system. We found that regardless of the timing of activation in B cells, constitutively active gp130 signaling resulted in the formation specifically of mature B cell lymphomas and plasma cell disorders with full penetrance, only with different latencies, where infiltrating CD138+ cells were a dominant feature in every tumor. Furthermore, constitutively active gp130 signaling in all adult hematopoietic cells also led to the development specifically of largely mature, aggressive B cell cancers, again with a high penetrance of CD138+ tumors. Importantly, gp130 activity abrogated the differentiation block induced by a B cell-targeted Myc transgene and resulted in a complete penetrance of the gp130-associated, CD138+, mature B cell lymphoma phenotype. Thus, gp130 signaling selectively provides a strong growth and differentiation advantage for mature B cells and directs lymphomagenesis specifically toward terminally differentiated B cell cancers.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Receptor gp130 de Citocina/metabolismo , Linfoma/imunologia , Plasmocitoma/imunologia , Transdução de Sinais/imunologia , Animais , Carcinogênese/genética , Carcinogênese/imunologia , Diferenciação Celular/genética , Receptor gp130 de Citocina/genética , Modelos Animais de Doenças , Feminino , Humanos , Janus Quinases/metabolismo , Ativação Linfocitária/genética , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Plasmocitoma/genética , Plasmocitoma/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.