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DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.
Assuntos
Replicação do DNA , Imunidade Inata , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Transdução de Sinais , Arginina/metabolismo , Arginina/análogos & derivados , Estresse Fisiológico , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dano ao DNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.
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Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.
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Linfoma Difuso de Grandes Células B , Humanos , Prognóstico , Linfoma Difuso de Grandes Células B/patologia , Perfilação da Expressão Gênica , Transcriptoma , Centro Germinativo/patologia , Microambiente Tumoral/genéticaRESUMO
Deregulation of the DNA damage response (DDR) plays a critical role in the pathogenesis and progression of many cancers. The dependency of certain cancers on DDR pathways has enabled exploitation of such through synthetically lethal relationships e.g., Poly ADP-Ribose Polymerase (PARP) inhibitors for BRCA deficient ovarian cancers. Though lagging behind that of solid cancers, DDR inhibitors (DDRi) are being clinically developed for haematological cancers. Furthermore, a high proliferative index characterize many such cancers, suggesting a rationale for combinatorial strategies targeting DDR and replicative stress. In this review, we summarize pre-clinical and clinical data on DDR inhibition in haematological malignancies and highlight distinct haematological cancer subtypes with activity of DDR agents as single agents or in combination with chemotherapeutics and targeted agents. We aim to provide a framework to guide the design of future clinical trials involving haematological cancers for this important class of drugs.
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Rituximab-based chemo-immunotherapy is currently the standard first-line treatment for Waldenstrom macroglobulinaemia (WM), while ibrutinib has emerged as an alternative. In the absence of randomised trials (RCTs) comparing these regimens, the optimal first-line treatment for WM remains uncertain. In this systematic review and meta-analysis, we sought to assess the efficacy and safety of first-line treatment regimens for WM. We searched key databases from January 2007 to March 2023, including phase II and III trials, including treatment-naïve WM patients treated with rituximab-based regimens or ibrutinib. Response rates, progression-free survival (PFS), overall survival (OS), and toxicities were evaluated. Four phase III and seven phase II trials were included among 736 unique records. Pooled response rates from all comparative and non-comparative trials were 46%, 33% and 26% for bendamustine rituximab (BR), bortezomib-dexamethasone, cyclophosphamide, rituximab (BDRC) and ibrutinib rituximab (IR), respectively. Two-year pooled PFS was 89%, 81% and 82% with BR, BDRC and IR, respectively. Neuropathy was more frequent with bortezomib, while haematologic and cardiac toxicities were more common with chemo-immunotherapy and ibrutinib-based regimens respectively. Our findings suggest that BR yields higher response rates than bortezomib or ibrutinib-based combinations. RCTs comparing BR against emerging therapies, including novel Bruton Tyrosine Kinase Inhibitors, are warranted.
Assuntos
Macroglobulinemia de Waldenstrom , Humanos , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Rituximab/efeitos adversos , Bortezomib , Protocolos Clínicos , CiclofosfamidaRESUMO
BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.
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Transformação Celular Neoplásica , Linfoma Extranodal de Células T-NK , Humanos , Transformação Celular Neoplásica/metabolismo , Oncogenes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Interferente Pequeno/metabolismo , Células Matadoras Naturais/patologia , Linhagem Celular Tumoral , Proteínas HMGB/genética , Proteínas HMGB/metabolismoRESUMO
Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance. SIGNIFICANCE: Using single-cell-resolved multiplexed imaging, we show that selected subpopulations of cells expressing specific combinations of oncogenes influence clinical outcomes in lymphoma. We describe a probabilistic metric for the estimation of cellular oncogenic coexpression from IHC or bulk transcriptomes, with possible implications for prognostication and therapeutic target discovery in cancer. This article is highlighted in the In This Issue feature, p. 1027.
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Linfoma Difuso de Grandes Células B , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Oncogenes , Linfoma Difuso de Grandes Células B/patologiaRESUMO
DLBCL is the most common lymphoma with high tumor heterogeneity. Treatment refractoriness and relapse from R-CHOP therapy in patients remain a clinical problem. Activation of the non-canonical NF-κB pathway is associated with R-CHOP resistance. However, downstream targets of non-canonical NF-κB mediating R-CHOP-induced resistance remains uncharacterized. Here, we identify the common mechanisms underlying both intrinsic and acquired resistance that are induced by doxorubicin, the main cytotoxic component of R-CHOP. We performed global transcriptomic analysis of (1) a panel of resistant versus sensitive and (2) isogenic acquired doxorubicin-resistant DLBCL cell lines following short and chronic exposure to doxorubicin respectively. Doxorubicin-induced stress in resistant cells activates a distinct transcriptional signature that is enriched in metabolic reprogramming and oncogenic signalling. Selective and sustained activation of non-canonical NF-κB signalling in these resistant cells exacerbated their survival by augmenting glycolysis. In response to doxorubicin, p52-RelB complexes transcriptionally activated multiple glycolytic regulators with prognostic significance through increased recruitment at their gene promoters. Targeting p52-RelB and their targets in resistant cells increased doxorubicin sensitivity in vitro and in vivo. Collectively, our study uncovered novel molecular drivers of doxorubicin-induced resistance that are regulated by non-canonical NF-κB pathway. We reveal new avenues of therapeutic targeting for R-CHOP-treated refractory/relapsed DLBCL patients.
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Antineoplásicos , Linfoma Difuso de Grandes Células B , Humanos , NF-kappa B/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Transdução de Sinais , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Antineoplásicos/uso terapêutico , Rituximab/farmacologia , Rituximab/uso terapêutico , Ciclofosfamida/uso terapêutico , Vincristina/farmacologia , Vincristina/uso terapêutico , Prednisona/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Excessive genomic instability coupled with abnormalities in DNA repair pathways induces high levels of 'replication stress' when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention towards targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1, and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective ATR kinase inhibitor elimusertib (BAY 1895344) has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Dano ao DNA , Inibidores de Proteínas Quinases , Feminino , Humanos , Proliferação de Células , Linhagem Celular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Biomarcadores , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Although combination therapy is the standard of care for relapsed/refractory non-Hodgkin's lymphoma (RR-NHL), combination treatment chosen for an individual patient is empirical, and response rates remain poor in individuals with chemotherapy-resistant disease. Here, we evaluate an experimental-analytic method, quadratic phenotypic optimization platform (QPOP), for prediction of patient-specific drug combination efficacy from a limited quantity of biopsied tumor samples. In this prospective study, we enrolled 71 patients with RR-NHL (39 B cell NHL and 32 NK/T cell NHL) with a median of two prior lines of treatment, at two academic hospitals in Singapore from November 2017 to August 2021. Fresh biopsies underwent ex vivo testing using a panel of 12 drugs with known efficacy against NHL to identify effective single and combination treatments. Individualized QPOP reports were generated for 67 of 75 patient samples, with a median turnaround time of 6 days from sample collection to report generation. Doublet drug combinations containing copanlisib or romidepsin were most effective against B cell NHL and NK/T cell NHL samples, respectively. Off-label QPOP-guided therapy offered at physician discretion in the absence of standard options (n = 17) resulted in five complete responses. Among patients with more than two prior lines of therapy, the rates of progressive disease were lower with QPOP-guided treatments than with conventional chemotherapy. Overall, this study shows that the identification of patient-specific drug combinations through ex vivo analysis was achievable for RR-NHL in a clinically applicable time frame. These data provide the basis for a prospective clinical trial evaluating ex vivo-guided combination therapy in RR-NHL.
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Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma não Hodgkin , Humanos , Estudos Prospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma não Hodgkin/tratamento farmacológico , Combinação de MedicamentosRESUMO
BACKGROUND: Immune checkpoint inhibitors (ICI) are now standard-of-care treatment for patients with metastatic gastric cancer (GC). To guide patient selection for ICI therapy, programmed death ligand-1 (PD-L1) biomarker expression is routinely assessed via immunohistochemistry (IHC). However, with an increasing number of approved ICIs, each paired with a different PD-L1 antibody IHC assay used in their respective landmark trials, there is an unmet clinical and logistical need for harmonization. We investigated the interchangeability between the Dako 22C3, Dako 28-8 and Ventana SP-142 assays in GC PD-L1 IHC. METHODS: In this cross-sectional study, we scored 362 GC samples for PD-L1 combined positive score (CPS), tumor proportion score (TPS) and immune cells (IC) using a multiplex immunohistochemistry/immunofluorescence technique. Samples were obtained via biopsy or resection of gastric cancer. RESULTS: The percentage of PD-L1-positive samples at clinically relevant CPS ≥ 1, ≥ 5 and ≥ 10 cut-offs for the 28-8 assay were approximately two-fold higher than that of the 22C3 (CPS ≥ 1: 70.3 vs 49.4%, p < 0.001; CPS ≥ 5: 29.1 vs 13.4%, p < 0.001; CPS ≥ 10: 13.7 vs 7.0%, p = 0.004). The mean CPS score on 28-8 assay was nearly double that of the 22C3 (6.39 ± 14.5 vs 3.46 ± 8.98, p < 0.001). At the clinically important CPS ≥ 5 cut-off, there was only moderate concordance between the 22C3 and 28-8 assays. CONCLUSION: Our findings suggest that scoring PD-L1 CPS with the 28-8 assay may result in higher PD-L1 scores and higher proportion of PD-L1 positivity compared to 22C3 and other assays. Until stronger evidence of inter-assay concordance is found, we urge caution in treating the assays as equivalent.
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Antígeno B7-H1 , Imunoterapia , Neoplasias Gástricas , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Estudos Transversais , Humanos , Imuno-Histoquímica , Neoplasias Gástricas/tratamento farmacológicoRESUMO
One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes. Here, we demonstrate that a bio-functional polymer by itself is able to prevent retinal scarring in an experimental rabbit model of proliferative vitreoretinopathy. This is mediated primarily via clathrin-dependent internalisation of polymeric micelles, downstream suppression of canonical EMT transcription factors, reduction of RPE cell hyper-proliferation and migration. Nuclear factor erythroid 2-related factor 2 signalling pathway was identified in a genome-wide transcriptomic profiling as a key sensor and effector. This study highlights the potential of using synthetic bio-functional polymer to modulate RPE cellular behaviour and offers a potential therapy for retinal scarring prevention.
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Fator 2 Relacionado a NF-E2 , Epitélio Pigmentado da Retina , Animais , Linhagem Celular , Movimento Celular , Cicatriz/metabolismo , Transição Epitelial-Mesenquimal , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Polímeros/metabolismo , Coelhos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.
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Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Neoplasias , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Trifosfato de Adenosina/metabolismo , Apoptose , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Consumo de Oxigênio , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Quinase 1 Polo-LikeRESUMO
Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.
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Quinase 1 do Ponto de Checagem , DNA Glicosilases , Mieloma Múltiplo , Proteína Supressora de Tumor p53 , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/genética , Aberrações Cromossômicas , Deleção Cromossômica , DNA Glicosilases/genética , Instabilidade Genômica , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutações Sintéticas Letais , Proteína Supressora de Tumor p53/genéticaRESUMO
Primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of primary T-cell lymphoma not otherwise specified (PTCL-NOS). Herein, we analyzed copynumber aberrations (n=77) with a focus on global measures of genomic instability and homologous recombination deficiency and performed gene expression (n=84) and EBV miRNA expression (n=24) profiling as well as targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed that patients with PTCL-EBV had a significantly worse outcome compared to patients with PTCL-NOS (P=0.002) but not to those with ENKTL. Remarkably, PTCL-EBV exhibited significantly lower genomic instability and homologous recombination deficiency scores compared to ENKTL and PTCL-NOS. Gene set enrichment analysis revealed that many immune-related pathways, interferon α/γ response, and IL6_JAK_STAT3 signaling were significantly upregulated in PTCLEBV and correlated with lower genomic instability scores. We also identified that NFκB-associated genes, BIRC3, NFKB1 (P50) and CD27, and their proteins are upregulated in PTCL-EBV. Most PTCL-EBV demonstrated a type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNA compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are characterized by microsatellite stability. Overall, poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNA are distinctive features of PTCL-EBV. Our data support the concept that PTCL-EBV could be considered as a distinct entity, provide novel insights into the pathogenesis of the disease and offer potential new therapeutic targets for this tumor.
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Infecções por Vírus Epstein-Barr , Linfoma Extranodal de Células T-NK , Linfoma de Células T Periférico , MicroRNAs , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Instabilidade Genômica , Herpesvirus Humano 4/genética , Humanos , Linfoma Extranodal de Células T-NK/diagnóstico , Linfoma Extranodal de Células T-NK/genética , Linfoma de Células T Periférico/diagnóstico , Linfoma de Células T Periférico/genética , MicroRNAs/genética , Regulação para CimaRESUMO
B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.
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Gefitinibe/farmacologia , Linfoma Extranodal de Células T-NK/metabolismo , Linfoma de Células T Periférico/metabolismo , Regulação para Cima , Proteína-Tirosina Quinase ZAP-70/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Linfoma Extranodal de Células T-NK/genética , Linfoma de Células T Periférico/genética , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
OBJECTIVE: To date, there are no predictive biomarkers to guide selection of patients with gastric cancer (GC) who benefit from paclitaxel. Stomach cancer Adjuvant Multi-Institutional group Trial (SAMIT) was a 2×2 factorial randomised phase III study in which patients with GC were randomised to Pac-S-1 (paclitaxel +S-1), Pac-UFT (paclitaxel +UFT), S-1 alone or UFT alone after curative surgery. DESIGN: The primary objective of this study was to identify a gene signature that predicts survival benefit from paclitaxel chemotherapy in GC patients. SAMIT GC samples were profiled using a customised 476 gene NanoString panel. A random forest machine-learning model was applied on the NanoString profiles to develop a gene signature. An independent cohort of metastatic patients with GC treated with paclitaxel and ramucirumab (Pac-Ram) served as an external validation cohort. RESULTS: From the SAMIT trial 499 samples were analysed in this study. From the Pac-S-1 training cohort, the random forest model generated a 19-gene signature assigning patients to two groups: Pac-Sensitive and Pac-Resistant. In the Pac-UFT validation cohort, Pac-Sensitive patients exhibited a significant improvement in disease free survival (DFS): 3-year DFS 66% vs 40% (HR 0.44, p=0.0029). There was no survival difference between Pac-Sensitive and Pac-Resistant in the UFT or S-1 alone arms, test of interaction p<0.001. In the external Pac-Ram validation cohort, the signature predicted benefit for Pac-Sensitive (median PFS 147 days vs 112 days, HR 0.48, p=0.022). CONCLUSION: Using machine-learning techniques on one of the largest GC trials (SAMIT), we identify a gene signature representing the first predictive biomarker for paclitaxel benefit. TRIAL REGISTRATION NUMBER: UMIN Clinical Trials Registry: C000000082 (SAMIT); ClinicalTrials.gov identifier, 02628951 (South Korean trial).
Assuntos
Adenocarcinoma , Neoplasias Gástricas , Adenocarcinoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Intervalo Livre de Doença , Humanos , Aprendizado de Máquina , Paclitaxel/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.
Assuntos
Neoplasias Gastrointestinais , Neoplasias Gástricas , Animais , Epigênese Genética , Epigenômica , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/terapia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Camundongos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/terapia , Microambiente TumoralRESUMO
The implementation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems in mammalian cells has sparked an exciting new era in targeted gene editing. CRISPR-Cas9 technologies allow gene function to be interrogated by gene deletions, mutations, and truncations, and by epitope tagging and promoter activity modulation. Many robust protocols have been published to date on CRISPR-Cas9 techniques, however, most of these focus on adherent cell lines. Suspension cell lines, typically of hematolymphoid origin, such as Jurkat, Daudi, and TOLEDO, pose unique challenges to the setup of CRISPR experiments. Here, using B cell lymphoma cells as a primary model, we describe a comprehensive protocol for targeted gene manipulations using the CRISPR-Cas9 system in suspension cells. We also highlight necessary optimization steps to make this approach universal to other suspension cell lines. We first describe a detailed protocol for transient expression of the Cas9 nuclease and guide RNAs. We then suggest workflows for obtaining single-cell clones and for screening for successful homozygous knockout (KO) clones in suspension lines. This protocol aims to serve as a comprehensive resource to facilitate gene editing experiments for users starting CRISPR-Cas9 gene editing protocols on suspension cell lines or those looking to optimize their current workflows. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by NeonTM electroporation Support Protocol 1: Designing and preparing gene-specific gRNA Support Protocol 2: Preparation of conditioned medium and culture vessels for single-cell FACS Alternate Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by Nepagene electroporator Basic Protocol 2: FACS and single-cell clone generation Alternate Protocol 2: Manual cell dilution to obtain single-cell clones Basic Protocol 3: Confirming indels status in single-cell clones by PCR on genomic DNA and Sanger sequencing.