RESUMO
Research on non-coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature.
Assuntos
Genoma Humano/genética , RNA não Traduzido/classificação , Terminologia como Assunto , Humanos , RNA não Traduzido/genéticaRESUMO
BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.
Assuntos
Antígenos CD/imunologia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antígenos CD/metabolismo , Antígeno B7-H1 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1 , Linfócitos T , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
BACKGROUND: Schlafen 11 (SLFN11) has been linked with response to DNA-damaging agents (DDA) and PARP inhibitors. An in-depth understanding of several aspects of its role as a biomarker in cancer is missing, as is a comprehensive analysis of the clinical significance of SLFN11 as a predictive biomarker to DDA and/or DNA damage-response inhibitor (DDRi) therapies. METHODS: We used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting. RESULTS: SLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies. CONCLUSION: SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Padrão de Cuidado , Animais , Neoplasias da Mama/patologia , Feminino , Seguimentos , Humanos , Camundongos , Proteínas Nucleares/genética , Isoformas de Proteínas , Estudos Retrospectivos , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors. METHODS: We developed and validated a clinically applicable SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance. RESULTS: High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity. CONCLUSION: We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.
Assuntos
Dano ao DNA/genética , Proteínas Nucleares/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Clinical tissue specimens are often unscreened, and preparation of tissue sections for analysis by mass spectrometry imaging (MSI) can cause aerosolization of particles potentially carrying an infectious load. We here present a decontamination approach based on ultraviolet-C (UV-C) light to inactivate clinically relevant pathogens such as herpesviridae, papovaviridae human immunodeficiency virus, or SARS-CoV-2, which may be present in human tissue samples while preserving the biodistributions of analytes within the tissue. High doses of UV-C required for high-level disinfection were found to cause oxidation and photodegradation of endogenous species. Lower UV-C doses maintaining inactivation of clinically relevant pathogens to a level of increased operator safety were found to be less destructive to the tissue metabolome and xenobiotics. These doses caused less alterations of the tissue metabolome and allowed elucidation of the biodistribution of the endogenous metabolites. Additionally, we were able to determine the spatially integrated abundances of the ATR inhibitor ceralasertib from decontaminated human biopsies using desorption electrospray ionization-MSI (DESI-MSI).
Assuntos
Descontaminação/métodos , Raios Ultravioleta , Animais , Azetidinas/análise , Azetidinas/uso terapêutico , COVID-19/patologia , COVID-19/virologia , Neoplasias de Cabeça e Pescoço/química , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Metaboloma , Naftalenos/análise , Naftalenos/uso terapêutico , Fotólise/efeitos da radiação , Ratos , Ratos Wistar , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/efeitos da radiação , Espectrometria de Massas por Ionização por Electrospray/métodos , Terfenadina/química , Inativação de Vírus/efeitos da radiaçãoRESUMO
The Japanese sparrowhawk Accipiter gularis is a small raptor that breeds in Northeast Asia. The species consists of the widespread and mostly migratory subspecies A. g. gularis that is common in East Asia, including Japan, and the resident and endangered subspecies A. g. iwasakii which inhabits the Ryukyu and Yaeyama Islands in Okinawa, southern Japan. Given the minimal knowledge about the migration of the species, in this study we sought to compare the genetic variation of the populations breeding in Japan with those migrating through Southeast Asia. We sequenced 761 bp of mitochondrial DNA Control Region from each of 21 A. gularis collected during the breeding season in Japan and from 20 individuals intercepted on migration in Thailand. We detected 26 haplotypes among the 41 individuals which differed significantly between Japan and Thailand. Migrants in Thailand were presumed to have originated from a wide area in Eastern Eurasia. The phylogenetic and network analyses demonstrated that the haplotypes of all A. g. gularis detected in Japan were genetically close. Moreover, the Okinawa haplotypes of A. g. iwasakii were clustered with moderate genetic variation. The information presented here can be used towards implementing future conservation actions.
Assuntos
Distribuição Animal , Migração Animal , Falcões/genética , Animais , DNA Mitocondrial/análise , Plumas , Variação Genética , Haplótipos , Japão , Análise de Sequência de DNA/veterinária , TailândiaRESUMO
BACKGROUND: AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. METHODS: We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. RESULTS: We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers (n = 23), 99% colorectal cancers (n = 102), 95% triple-negative-breast cancers (TNBC) (n = 40) and 87.5% glioblastoma-multiformes (n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34-72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. CONCLUSION: Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Espectrometria de Massas/métodos , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Dano ao DNA , Humanos , Imuno-Histoquímica , Indóis , Irinotecano/farmacologia , Camundongos , Camundongos Nus , Morfolinas , Ftalazinas/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Transdução de Sinais , Sulfonamidas , Sulfóxidos/farmacologia , Sulfóxidos/uso terapêutico , Neoplasias de Mama Triplo Negativas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Histonas/metabolismo , Dobramento de Proteína , Multimerização Proteica , Linhagem Celular Tumoral , DNA/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Humanos , Ligação ProteicaRESUMO
Aim: Endogenous interferents can cause nonselectivity in ligand binding pharmacokinetic assays, leading to inaccurate quantification of drug concentrations. We describe the development of a Gyrolab immunoassay to quantify a new modality, CB307 and discuss strategies implemented to overcome matrix effects and achieve selectivity at the desired sensitivity. Results: Matrix effects were mitigated using strategies including increasing minimum required dilution (MRD) and lower limit of quantification, optimization of antibody orientation, assay buffer and solid phase. Conclusion: The strategies described resulted in a selective method for CB307 in disease state matrix that met bioanalytical method validation (BMV) guidance and is currently used to support clinical pharmacokinetic sample analysis in the first-in-human POTENTIA clinical study (NCT04839991) as a secondary clinical end point.
[Box: see text].
RESUMO
Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.
Assuntos
Antígenos CD , Imunoterapia , Proteína do Gene 3 de Ativação de Linfócitos , Receptor de Morte Celular Programada 1 , Proteínas Proto-Oncogênicas c-cbl , Transdução de Sinais , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Imunoterapia/métodos , Transdução de Sinais/efeitos dos fármacos , Antígenos CD/metabolismo , Antígenos CD/genética , Animais , Camundongos , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologiaRESUMO
PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Camundongos , Animais , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Imunoterapia/métodos , Microambiente TumoralRESUMO
The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos , Indóis , Morfolinas , Neoplasias , Pirimidinas , Sulfonamidas , Humanos , Animais , Camundongos , Antígeno B7-H1 , Microambiente Tumoral , Linhagem Celular Tumoral , Imunoterapia , Modelos Animais de Doenças , Proteínas Mutadas de Ataxia TelangiectasiaRESUMO
DNA double-stranded breaks (DSBs) are among the most severe forms of DNA damage and responsible for chromosomal translocations that may lead to gene fusions. The RAD51 family plays an integral role in preserving genome stability by homology directed repair of DSBs. From a proteomics screen, we recently identified SFPQ/PSF as an interacting partner with the RAD51 paralogs, RAD51D, RAD51C and XRCC2. Initially discovered as a potential RNA splicing factor, SFPQ was later shown to have homologous recombination and non-homologous end joining related activities and also to bind and modulate the function of RAD51. Here, we demonstrate that SFPQ interacts directly with RAD51D and that deficiency of both proteins confers a severe loss of cell viability, indicating a synthetic lethal relationship. Surprisingly, deficiency of SFPQ alone also leads to sister chromatid cohesion defects and chromosome instability. In addition, SFPQ was demonstrated to mediate homology directed DNA repair and DNA damage response resulting from DNA crosslinking agents, alkylating agents and camptothecin. Taken together, these data indicate that SFPQ association with the RAD51 protein complex is essential for homologous recombination repair of DNA damage and maintaining genome integrity.
Assuntos
Cromátides/ultraestrutura , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação a RNA/fisiologia , Alquilantes/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Instabilidade Cromossômica , Reagentes de Ligações Cruzadas/toxicidade , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Epistasia Genética , Deleção de Genes , Humanos , Camundongos , Mitomicina/toxicidade , Fator de Processamento Associado a PTB , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RecQ Helicases/metabolismo , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Linhagem Celular , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Família Multigênica , Fenótipo , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
BACKGROUND: The NCI-60 is a collection of tumor cell lines derived from a variety of human adult cancer tissue types and is commonly used for genetic analysis and screening of potential chemotherapeutic agents. We wanted to understand the contributions of specific mechanisms of genomic instability to the etiology of cancers represented by the NCI-60. RESULTS: We screened the NCI-60 for dysregulated homologous recombination by using the gene cluster instability (GCI) assay we pioneered, and for defects in base excision repair by sensitivity to 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). We identified subsets of the NCI-60 lines that either displayed the characteristic molecular signature of GCI or were sensitive to hmdUrd. With the exception of the NCI-H23 lung cancer line, these phenotypes were not found to overlap. None of the lines examined in either subset exhibited significant changes in the frequency of sister chromatid exchanges (SCE), neither did any of the lines in either subset exhibit microsatellite instability (MSI) indicative of defects in DNA mismatch repair. CONCLUSIONS: Gene cluster instability, sensitivity to hmdUrd and sister chromatid exchange are mechanistically distinct phenomena. Genomic instability in the NCI-60 appears to involve only one mechanism of instability for each individual cell line.
Assuntos
Antineoplásicos/farmacologia , Instabilidade Genômica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Timidina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Humanos , Família Multigênica , Fenótipo , Recombinação Genética , Troca de Cromátide Irmã , Timidina/farmacologiaRESUMO
Bloom syndrome confers strong predisposition to malignancy in multiple tissue types. The Bloom syndrome patient (BLM) protein defective in the disease biochemically functions as a Holliday junction dissolvase and human cells lacking functional BLM show 10-fold elevated rates of sister chromatid exchange. Collectively, these phenomena suggest that dysregulated mitotic recombination drives the genomic instability underpinning the development of cancer in these individuals. Here we use physical analysis of the highly repeated, highly self-similar human ribosomal RNA gene clusters as sentinel biomarkers for dysregulated homologous recombination to demonstrate that loss of BLM protein function causes a striking increase in spontaneous molecular level genomic restructuring. Analysis of single-cell derived sub-clonal populations from wild-type human cell lines shows that gene cluster architecture is ordinarily very faithfully preserved under mitosis, but is so unstable in cell lines derived from BLMs as to make gene cluster architecture in different sub-clonal populations essentially unrecognizable one from another. Human cells defective in a different RecQ helicase, the WRN protein involved in the premature aging Werner syndrome, do not exhibit the gene cluster instability (GCI) phenotype, indicating that the BLM protein specifically, rather than RecQ helicases generally, holds back this recombination-mediated genomic instability. An ataxia-telangiectasia defective cell line also shows elevated rDNA GCI, although not to the extent of BLM defective cells. Genomic restructuring mediated by dysregulated recombination between the abundant low-copy repeats in the human genome may prove to be an important additional mechanism of genomic instability driving the initiation and progression of human cancer.
Assuntos
Família Multigênica , RecQ Helicases/genética , Linhagem Celular , DNA Ribossômico/genética , Genoma Humano , Instabilidade Genômica , Humanos , Interferência de RNARESUMO
Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.
Assuntos
Biomarcadores/sangue , Leucócitos Mononucleares/imunologia , Plasma/imunologia , HumanosRESUMO
PURPOSE: This study reports the safety, tolerability, MTD, recommended phase II dose (RP2D), pharmacokinetic/pharmacodynamic profile, and preliminary antitumor activity of ceralasertib combined with carboplatin in patients with advanced solid tumors. It also examined exploratory predictive and pharmacodynamic biomarkers. PATIENTS AND METHODS: Eligible patients (n = 36) received a fixed dose of carboplatin (AUC5) with escalating doses of ceralasertib (20 mg twice daily to 60 mg once daily) in 21-day cycles. Sequential and concurrent combination dosing schedules were assessed. RESULTS: Two ceralasertib MTD dose schedules, 20 mg twice daily on days 4-13 and 40 mg once daily on days 1-2, were tolerated with carboplatin AUC5; the latter was declared the RP2D. The most common treatment-emergent adverse events (Common Terminology Criteria for Adverse Events grade ≥3) were anemia (39%), thrombocytopenia (36%), and neutropenia (25%). Dose-limiting toxicities of grade 4 thrombocytopenia (n = 2; including one grade 4 platelet count decreased) and a combination of grade 4 thrombocytopenia and grade 3 neutropenia occurred in 3 patients. Ceralasertib was quickly absorbed (tmax â¼1 hour), with a terminal plasma half-life of 8-11 hours. Upregulation of pRAD50, indicative of ataxia telangiectasia mutated (ATM) activation, was observed in tumor biopsies during ceralasertib treatment. Two patients with absent or low ATM or SLFN11 protein expression achieved confirmed RECIST v1.1 partial responses. Eighteen of 34 (53%) response-evaluable patients had RECIST v1.1 stable disease. CONCLUSIONS: The RP2D for ceralasertib plus carboplatin was established as ceralasertib 40 mg once daily on days 1-2 administered with carboplatin AUC5 every 3 weeks, with pharmacokinetic and pharmacodynamic studies confirming pharmacodynamic modulation and preliminary evidence of antitumor activity observed.
Assuntos
Neoplasias , Neutropenia , Trombocitopenia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Carboplatina , Humanos , Indóis , Dose Máxima Tolerável , Morfolinas , Neoplasias/etiologia , Neutropenia/induzido quimicamente , Proteínas Nucleares , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas , Sulfonamidas , Sulfóxidos , Trombocitopenia/induzido quimicamenteRESUMO
PURPOSE: Ceralasertib is a potent and selective oral inhibitor of the serine/threonine protein kinase ataxia telangiectasia and Rad3-related (ATR) protein. PATIENTS AND METHODS: Eligible patients with solid tumors, enriched for melanoma, received ceralasertib in combination with a fixed dose of paclitaxel (80 mg/m2 on D1, D8, D15) in 28-day cycles. The dose of ceralasertib was escalated to reach an MTD in a rolling 6 design. The starting dose of ceralasertib was 40 mg QD. Fifty-seven patients (33 patients with melanoma who failed prior PD1/L1 treatment) were enrolled in 7 dose cohorts ranging from 40 mg QD to 240 mg BD plus weekly paclitaxel. RESULTS: The RP2D was established as ceralasertib 240 mg BD days 1-14 plus paclitaxel 80 mg/m2 on D1, D8, D15 every 28 days. The most common toxicities were neutropenia (n = 39, 68%), anemia (n = 25, 44%), and thrombocytopenia (n = 21, 37%). In the full analysis set of 57 patients, the overall response rate (ORR) was 22.6% (95% CI, 12.5-35.3). In 33 patients with melanoma, resistant to prior anti-PD1 therapy, the ORR was 33.3% (95% CI, 18.0-51.8). In the melanoma subset, the mPFS was 3.6 months (95% CI, 2.0-5.8), the median duration of response was 9.9 months (95% CI, 3.7-23.2), and the mOS was 7.4 months (95% CI, 5.7-11.9). CONCLUSIONS: Ceralasertib in combination with paclitaxel was well tolerated in patients with advanced malignancies and showed evidence of antitumor activity. Durable responses were observed in patients with advanced cutaneous, acral, and mucosal melanoma resistant to anti-PD1/L1 treatment.See related commentary by Ashworth, p. 4667.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Indóis/administração & dosagem , Morfolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagem , Adulto , Idoso , Esquema de Medicação , Combinação de Medicamentos , Feminino , Humanos , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.