RESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (â¼80%) exhibit activation of GCK, this therapy may be applicable to most patients.
Assuntos
Apoptose , Pontos de Checagem do Ciclo Celular , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/etnologia , Proteínas de Neoplasias/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Linhagem Celular Tumoral , Quinases do Centro Germinativo , Xenoenxertos , Humanos , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Transplante de NeoplasiasRESUMO
Ig class switching requires cell proliferation and is division linked, but the detailed mechanism is unknown. By analyzing the first switching cells early in the kinetics, our analysis suggested that proliferating B cells had a very short G1 phase (<3.5 h), a total cell cycle time of â¼ 11 h, and that Ig class switching preferentially occurred in the late G1 or early S phase. Inhibition of cyclin-dependent kinases (CDKs) caused dramatic reduction of switching rate within 6 h. This was associated with less targeting of activation-induced cytidine deaminase (AID) to the Igh locus. Interestingly, ectopically expressed nuclear AID in HeLa cells was preferentially found in the early S phase. Furthermore, in CDK2 hypomorphic cells there was reduced nuclear AID accumulation. Thus, our data are compatible with the idea that division-linked Ig class switching is in part due to CDK2-regulated AID nuclear access at the G1/S border.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Citidina Desaminase/metabolismo , Switching de Imunoglobulina , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ciclo Celular/genética , Linhagem Celular , Quinase 2 Dependente de Ciclina/metabolismo , Replicação do DNA , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Isotipos de Imunoglobulinas/genética , Camundongos , Ligação Proteica , Transporte ProteicoRESUMO
Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.
Assuntos
Citidina Desaminase/metabolismo , Quebras de DNA , Reparo do DNA por Junção de Extremidades/fisiologia , Switching de Imunoglobulina/genética , Análise de Variância , Animais , Linfócitos B/imunologia , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Reparo do DNA por Junção de Extremidades/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Translocação Genética/genética , Uracila-DNA Glicosidase/genéticaRESUMO
We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G â A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies.
Assuntos
Citidina Desaminase/genética , Modelos Animais de Doenças , Síndrome de Imunodeficiência com Hiper-IgM/genética , Síndrome de Imunodeficiência com Hiper-IgM/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Mutação , Hipermutação Somática de Imunoglobulina , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Citidina Desaminase/metabolismo , Análise Mutacional de DNA , Feminino , Centro Germinativo/imunologia , Síndrome de Imunodeficiência com Hiper-IgM/metabolismo , Imunofenotipagem , Padrões de Herança , Contagem de Linfócitos , Masculino , Camundongos , Linhagem , Fenótipo , Característica Quantitativa HerdávelRESUMO
Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.
Assuntos
Linfócitos B/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Exodesoxirribonucleases/metabolismo , Switching de Imunoglobulina/genética , Oxirredutases do Álcool/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Citidina Desaminase/genética , Proteínas de Ligação a DNA/metabolismo , Desaminação/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS/metabolismo , Ligação Proteica , Transporte Proteico , Reparo de DNA por Recombinação/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Introduction: Cancer-associated cachexia (CC) is a progressive syndrome characterized by unintentional weight loss, muscle atrophy, fatigue, and poor outcomes that affects most patients with pancreatic ductal adenocarcinoma (PDAC). The ability to identify and classify CC stage along its continuum early in the disease process is challenging but critical for management. Objectives: The main objective of this study was to determine the prevalence of CC stage overall and by sex and race and ethnicity among treatment-naïve PDAC cases using clinical, nutritional, and functional criteria. Secondary objectives included identifying the prevalence and predictors of higher symptom burden, supportive care needs, and quality of life (QoL), and examining their influence on overall survival (OS). Materials and methods: A population-based multi-institutional prospective cohort study of patients with PDAC was conducted between 2018 and 2021 by the Florida Pancreas Collaborative. Leveraging patient-reported data and laboratory values, participants were classified at baseline into four stages [non-cachexia (NCa), pre-cachexia (PCa), cachexia (Ca), and refractory cachexia (RCa)]. Multivariate regression, Kaplan Meier analyses, and Cox regression were conducted to evaluate associations. Results: CC stage was estimated for 309 PDAC cases (156 females, 153 males). The overall prevalence of NCa, PCa, Ca, and RCa was 12.9%, 24.6%, 54.1%, and 8.4%, respectively. CC prevalence across all CC stages was highest for males and racial and ethnic minorities. Criteria differentiated NCa cases from other groups, but did not distinguish PCa from Ca. The most frequently reported symptoms included weight loss, fatigue, pain, anxiety, and depression, with pain significantly worsening over time. The greatest supportive care needs included emotional and physical domains. Males, Black people, and those with RCa had the worst OS. Conclusions: Using clinical, nutritional, and functional criteria, nearly one-quarter of the PDAC cases in our diverse, multi-institutional cohort had PCa and 62.5% had Ca or RCa at the time of diagnosis. The PCa estimate is higher than that reported in prior studies. We recommend these criteria be used to aid in CC classification, monitoring, and management of all incident PDAC cases. Findings also highlight the recommendation for continued emotional support, assistance in alleviating pain, and supportive care needs throughout the PDAC treatment journey.
RESUMO
Wastewater-based epidemiology (WBE) has been utilized to track community infections of Coronavirus Disease 2019 (COVID-19) by detecting RNA of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), within samples collected from wastewater. The correlations between community infections and wastewater measurements of the RNA can potentially change as SARS-CoV-2 evolves into new variations by mutating. This study analyzed SARS-CoV-2 RNA, and indicators of human waste in wastewater from two sewersheds of different scales (University of Miami (UM) campus and Miami-Dade County Central District wastewater treatment plant (CDWWTP)) during five internally defined COVID-19 variant dominant periods (Initial, Pre-Delta, Delta, Omicron and Post-Omicron wave). SARS-CoV-2 RNA quantities were compared against COVID-19 clinical cases and hospitalizations to evaluate correlations with wastewater SARS-CoV-2 RNA. Although correlations between documented clinical cases and hospitalizations were high, prevalence for a given wastewater SARS-CoV-2 level varied depending upon the variant analyzed. The correlative relationship was significantly steeper (more cases per level found in wastewater) for the Omicron-dominated period. For hospitalization, the relationships were steepest for the Initial wave, followed by the Delta wave with flatter slopes during all other waves. Overall results were interpreted in the context of SARS-CoV-2 virulence and vaccination rates among the community.
RESUMO
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater has been used to track community infections of coronavirus disease-2019 (COVID-19), providing critical information for public health interventions. Since levels in wastewater are dependent upon human inputs, we hypothesize that tracking infections can be improved by normalizing wastewater concentrations against indicators of human waste [Pepper Mild Mottle Virus (PMMoV), ß-2 Microglobulin (B2M), and fecal coliform]. In this study, we analyzed SARS-CoV-2 and indicators of human waste in wastewater from two sewersheds of different scales: a University campus and a wastewater treatment plant. Wastewater data were combined with complementary COVID-19 case tracking to evaluate the efficiency of wastewater surveillance for forecasting new COVID-19 cases and, for the larger scale, hospitalizations. Results show that the normalization of SARS-CoV-2 levels by PMMoV and B2M resulted in improved correlations with COVID-19 cases for campus data using volcano second generation (V2G)-qPCR chemistry (r s = 0.69 without normalization, r s = 0.73 with normalization). Mixed results were obtained for normalization by PMMoV for samples collected at the community scale. Overall benefits from normalizing with measures of human waste depend upon qPCR chemistry and improves with smaller sewershed scale. We recommend further studies that evaluate the efficacy of additional normalization targets.
RESUMO
Standardized protocols for wastewater-based surveillance (WBS) for the RNA of SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, are being developed and refined worldwide for early detection of disease outbreaks. We report here on lessons learned from establishing a WBS program for SARS-CoV-2 integrated with a human surveillance program for COVID-19. We have established WBS at three campuses of a university, including student residential dormitories and a hospital that treats COVID-19 patients. Lessons learned from this WBS program address the variability of water quality, new detection technologies, the range of detectable viral loads in wastewater, and the predictive value of integrating environmental and human surveillance data. Data from our WBS program indicated that water quality was statistically different between sewer sampling sites, with more variability observed in wastewater coming from individual buildings compared to clusters of buildings. A new detection technology was developed based upon the use of a novel polymerase called V2G. Detectable levels of SARS-CoV-2 in wastewater varied from 102 to 106 genomic copies (gc) per liter of raw wastewater (L). Integration of environmental and human surveillance data indicate that WBS detection of 100 gc/L of SARS-CoV-2 RNA in wastewater was associated with a positivity rate of 4% as detected by human surveillance in the wastewater catchment area, though confidence intervals were wide (ß ~ 8.99 ∗ ln(100); 95% CI = 0.90-17.08; p < 0.05). Our data also suggest that early detection of COVID-19 surges based on correlations between viral load in wastewater and human disease incidence could benefit by increasing the wastewater sample collection frequency from weekly to daily. Coupling simpler and faster detection technology with more frequent sampling has the potential to improve the predictive potential of using WBS of SARS-CoV-2 for early detection of the onset of COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , RNA Viral , Águas ResiduáriasRESUMO
Background: Well-annotated, high-quality biorepositories provide a valuable platform to support translational research. However, most biorepositories have poor representation of minority groups, limiting the ability to address health disparities. Methods: We describe the establishment of the Florida Pancreas Collaborative (FPC), the first state-wide prospective cohort study and biorepository designed to address the higher burden of pancreatic cancer (PaCa) in African Americans (AA) compared to Non-Hispanic Whites (NHW) and Hispanic/Latinx (H/L). We provide an overview of stakeholders; study eligibility and design; recruitment strategies; standard operating procedures to collect, process, store, and transfer biospecimens, medical images, and data; our cloud-based data management platform; and progress regarding recruitment and biobanking. Results: The FPC consists of multidisciplinary teams from fifteen Florida medical institutions. From March 2019 through August 2020, 350 patients were assessed for eligibility, 323 met inclusion/exclusion criteria, and 305 (94%) enrolled, including 228 NHW, 30 AA, and 47 H/L, with 94%, 100%, and 94% participation rates, respectively. A high percentage of participants have donated blood (87%), pancreatic tumor tissue (41%), computed tomography scans (76%), and questionnaires (62%). Conclusions: This biorepository addresses a critical gap in PaCa research and has potential to advance translational studies intended to minimize disparities and reduce PaCa-related morbidity and mortality.
RESUMO
Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCLs) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2-positive DLBCLs and T cell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Furthermore, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2-positive tumors. Together, our results reveal that LMO2 expression predicts HR deficiency and the potential therapeutic use of PARP inhibitors in DLBCL and T-ALL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Domínio LIM/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Mutações Sintéticas Letais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína BRCA1/metabolismo , Biópsia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Tonsila Palatina/patologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Reparo de DNA por Recombinação/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma is a plasma cell neoplasm with an extremely variable clinical course. Animal models are needed to better understand its pathophysiology and for preclinical testing of potential therapeutic agents. Hematopoietic cells expressing the hypermorphic Rad50(s) allele show hematopoietic failure, which can be mitigated by the lack of a transcription factor, Mef/Elf4. However, we find that 70% of Mef(-/-)Rad50(s/s) mice die from multiple myeloma or other plasma cell neoplasms. These mice initially show an abnormal plasma cell proliferation and monoclonal protein production, and then develop anemia and a decreased bone mineral density. Tumor cells can be serially transplanted and according to array CGH and whole exome sequencing, the pathogenesis of plasma cell neoplasms in these mice is not linked to activation of a specific oncogene, or inactivation of a specific tumor suppressor. This model recapitulates the systemic manifestations of human plasma cell neoplasms, and implicates cooperativity between the Rad50(s) and Mef/Elf4 pathways in initiating myelomagenic mutations that promote plasma cell transformation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Ligação a DNA/genética , Mieloma Múltiplo/genética , Fenótipo , Fatores de Transcrição/genética , Hidrolases Anidrido Ácido , Animais , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Células Sanguíneas/fisiologia , Células Sanguíneas/transplante , Densidade Óssea , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Exoma , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/patologiaRESUMO
Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG). In contrast, in the absence of UNG activity, deleterious processing by mismatch repair leads to telomere loss and defective cell proliferation. Indeed, we show that UNG deficiency reduces B cell clonal expansion in the germinal center in mice and blocks the proliferation of tumor B cells expressing AID. We propose that AID-induced damage at telomeres acts as a fail-safe mechanism to limit the tumor promoting activity of AID when it overwhelms uracil excision repair.