RESUMEN
Humans with inherited heterozygous BRCA2 mutations have an increased risk of developing cancer; however, what triggers carcinogenesis in these individuals is unclear. Tan et al. find that environmental and metabolic aldehydes pose a threat to these individuals by promoting degradation of wild-type BRCA2 protein, thereby predisposing them to genomic instability and perhaps to cancer.
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Aldehídos , Mutación , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Genes BRCA1 , Inestabilidad Genómica , Mutación de Línea Germinal , Heterocigoto , HumanosRESUMEN
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Pirazinas/farmacología , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/metabolismo , Pirazoles/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.
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Proteína BRCA1/deficiencia , Neoplasias de la Mama/enzimología , Replicación del ADN , ADN de Neoplasias/biosíntesis , Proteasas Ubiquitina-Específicas/metabolismo , Neoplasias del Cuello Uterino/enzimología , Animales , Proteína BRCA1/genética , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , ADN de Neoplasias/genética , Resistencia a Medicamentos , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones Desnudos , Mutación , Desnaturalización de Ácido Nucleico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.
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Anemia de Fanconi , Ratones , Animales , Anemia de Fanconi/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Espermatocitos/metabolismoRESUMEN
Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3'-5' superfamily 2 helicase HELQ in ICL repair in Drosophila melanogaster (MUS301 (ref. 3)) and Caenorhabditis elegans (HELQ-1 (ref. 4)). Although in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3' single-stranded DNA overhangs and also disrupts protein-DNA interactions while translocating along DNA, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with Helq heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals.
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Carcinogénesis , ADN Helicasas/metabolismo , Reparación del ADN , Células Germinativas/metabolismo , Células Germinativas/patología , Recombinasa Rad51/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Daño del ADN/genética , ADN Helicasas/deficiencia , ADN Helicasas/genética , Reparación del ADN/genética , Replicación del ADN/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Femenino , Eliminación de Gen , Células Germinativas/citología , Masculino , Ratones , Complejos Multiproteicos/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ovario/metabolismo , Ovario/patología , Reparación del ADN por Recombinación/genéticaRESUMEN
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Ciclina D1/metabolismo , Endopeptidasas/metabolismo , Linfoma de Células del Manto/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Ciclina D1/genética , Daño del ADN , Reparación del ADN , Endopeptidasas/genética , Humanos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/metabolismo , Proteínas de Neoplasias/genética , Inhibidores de Proteasas/química , Ubiquitina TiolesterasaRESUMEN
Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.
Asunto(s)
Anemia de Fanconi/genética , Terapia Genética/métodos , Hematopoyesis , Células Madre Pluripotentes Inducidas/citología , Animales , Células Cultivadas , Daño del ADN , Anemia de Fanconi/metabolismo , Anemia de Fanconi/terapia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Eliminación de Gen , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
It is unclear whether metabolic health corresponds to reduced oncogenesis or vice versa. We study Tudor-interacting repair regulator (TIRR), an inhibitor of p53 binding protein 1 (53BP1)-mediated p53 activation, and the physiological consequences of enhancing tumor suppressor activity. Deleting TIRR selectively activates p53, significantly protecting against cancer but leading to a systemic metabolic imbalance in mice. TIRR-deficient mice are overweight and insulin resistant, even under normal chow diet. Similarly, reduced TIRR expression in human adipose tissue correlates with higher BMI and insulin resistance. Despite the metabolic challenges, TIRR loss improves p53 heterozygous (p53HET) mouse survival and correlates with enhanced progression-free survival in patients with various p53HET carcinomas. Finally, TIRR's oncoprotective and metabolic effects are dependent on p53 and lost upon p53 deletion in TIRR-deficient mice, with glucose homeostasis and orexigenesis being primarily regulated by TIRR expression in the adipose tissue and the CNS, respectively, as evidenced by tissue-specific models. In summary, TIRR deletion provides a paradigm of metabolic deregulation accompanied by reduced oncogenesis.
Asunto(s)
Carcinogénesis , Proteínas de Unión al ARN , Proteína p53 Supresora de Tumor , Animales , Humanos , Masculino , Ratones , Tejido Adiposo/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Glucosa/metabolismo , Resistencia a la Insulina , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with the drug sensitivity. USP1 inhibition increased monoubiquitinated PCNA at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids therefore represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials.
RESUMEN
Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB1 and CB2) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB2 receptors are expressed in human and murine HSPCs. On ligand stimulation with CB2 agonists, CB2 receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB2 agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB2 antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB2/CB2 agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB2 agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation.
Asunto(s)
Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/fisiología , Receptor Cannabinoide CB2/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Cannabinoides/farmacología , Movimiento Celular/efectos de los fármacos , Ciclohexanoles/farmacología , Femenino , Citometría de Flujo , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Células del Estroma/metabolismoRESUMEN
A rapid, sensitive, and simple UHPLC-MS/MS method for the determination of the PARP inhibitor talazoparib in mouse plasma was developed and validated using [13C,2H4]-talazoparib as an internal standard (IS). The assay procedure involved extraction of talazoparib and the IS from plasma using a single-step deproteination and separation of the analytes was achieved on an ACQUITY UPLC RP18 HSS T3 column with a mobile phase gradient at a flow rate of 0.4 mL/min in a run time of 5 min. The calibration curve was linear (r2 > 0.99) over the concentration range of 0.5-100 ng/mL, and 10-fold dilution of samples could be accurately quantitated. The matrix effect and mean extraction recovery for talazoparib were between 93.7-109% and 87.7-105%, respectively. Precision and percent bias of quality control samples were always less than ±15%, indicating reproducibility and accuracy of the method. Talazoparib demonstrated bench-top stability at room temperature for 6 h, auto-sampler and reinjection stability at 4 °C for at least 24 h, and no significant degradation was observed after three freeze-thaw cycles. The developed method was successfully applied to pharmacokinetic studies involving serial blood sampling after oral administration of talazoparib to wild-type mice and animals with a genetic deficiency of the efflux transporters ABCB1 (P-gp) and ABCG2 (BCRP). Together, our results demonstrate the successful development of a suitable analytical method for talazoparib in mouse plasma and suggest that mice are a useful model to evaluate transporter-mediated drug-drug interactions involving therapy with talazoparib.
RESUMEN
PURPOSE: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. PATIENTS AND METHODS: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. RESULTS: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. CONCLUSIONS: No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Estudios de Cohortes , Desoxicitidina , Gemcitabina , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors. SIGNIFICANCE: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.
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Neoplasias , Mutaciones Letales Sintéticas , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Novobiocina , Piridazinas , Quinazolinas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.
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Células de la Médula Ósea/metabolismo , Moduladores de Receptores de Cannabinoides/biosíntesis , Endocannabinoides , Células Madre Hematopoyéticas/metabolismo , Células del Estroma/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/biosíntesis , Western Blotting , Células de la Médula Ósea/citología , Moduladores de Receptores de Cannabinoides/fisiología , Comunicación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Ciclohexanoles/farmacología , Femenino , Citometría de Flujo , Glicéridos/biosíntesis , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Alcamidas Poliinsaturadas , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Población Lateral/citología , Células de Población Lateral/metabolismo , Nicho de Células Madre/citología , Nicho de Células Madre/metabolismo , Células del Estroma/citologíaRESUMEN
Fanconi anemia (FA) is a human genetic disease characterized by a DNA repair defect and progressive bone marrow failure. Central events in the FA pathway are the monoubiquitination of the Fancd2 protein and the removal of ubiquitin by the deubiquitinating enzyme, Usp1. Here, we have investigated the role of Fancd2 and Usp1 in the maintenance and function of murine hematopoietic stem cells (HSCs). Bone marrow from Fancd2-/- mice and Usp1-/- mice exhibited marked hematopoietic defects. A decreased frequency of the HSC populations including Lin-Sca-1+Kit+ cells and cells enriched for dormant HSCs expressing signaling lymphocyte activation molecule (SLAM) markers, was observed in the bone marrow of Fancd2-deficient mice. In addition, bone marrow from Fancd2-/- mice contained significantly reduced frequencies of late-developing cobblestone area-forming cell activity in vitro compared to the bone marrow from wild-type mice. Furthermore, Fancd2-deficient and Usp1-deficient bone marrow had defective long-term in vivo repopulating ability. Collectively, our data reveal novel functions of Fancd2 and Usp1 in maintaining the bone marrow HSC compartment and suggest that FA pathway disruption may account for bone marrow failure in FA patients.
Asunto(s)
Endopeptidasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Proteínas de Arabidopsis , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular , Supervivencia Celular , Endopeptidasas/deficiencia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Células Madre Hematopoyéticas/patología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Proteasas Ubiquitina-EspecíficasRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow and supply blood cells. Efficient methods for isolation of HSPCs are required. Here, we present protocols for the isolation of human and murine HSPCs using manual and FACS-assisted techniques. Isolated HSPCs can be used for downstream applications, including colony forming unit assays and DNA damage and repair assays. For complete details on the use and execution of this protocol, please refer to Rodríguez et al. (2021a) and (2021b).
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Animales , Ensayo de Unidades Formadoras de Colonias , Daño del ADN/genética , Reparación del ADN , Humanos , RatonesRESUMEN
PURPOSE: Checkpoint kinase 1 (CHK1) plays a central role in the response to replication stress through modulation of cell-cycle checkpoints and homologous recombination (HR) repair. In BRCA-deficient cancers with de novo or acquired PARP inhibitor resistance, the addition of the CHK1 inhibitor prexasertib to the PARP inhibitor olaparib compromises replication fork stability, as well as HR proficiency, allowing for sensitization to PARP inhibition. PATIENTS AND METHODS: This study followed a 3+3 design with a 7-day lead-in of olaparib alone, followed by 28-day cycles with prexasertib administered on days 1 and 15 in combination with an attenuated dose of olaparib on days 1-5 and 15-19. Pharmacokinetic blood samples were collected after olaparib alone and following combination therapy. Patients enrolled to the expansion phase of the study underwent paired tumor biopsies for pharmacodynamic (PD) assessments. RESULTS: Twenty-nine patients were treated. DLTs included grade 3 neutropenia and grade 3 febrile neutropenia. The MTD/recommended phase 2 dose (RP2D) was prexasertib at 70 mg/m2 i.v. with olaparib at 100 mg by mouth twice daily. Most common treatment-related adverse events included leukopenia (83%), neutropenia (86%), thrombocytopenia (66%), and anemia (72%). Four of 18 patients with BRCA1-mutant, PARP inhibitor-resistant, high-grade serous ovarian cancer (HGSOC) achieved partial responses. Paired tumor biopsies demonstrated reduction in RAD51 foci and increased expression of γ-H2AX, pKAP1, and pRPA after combination exposure. CONCLUSIONS: Prexasertib combined with olaparib has preliminary clinical activity in BRCA-mutant patients with HGSOC who have previously progressed on a PARP inhibitor. PD analyses show that prexasertib compromises HR with evidence of induction of DNA damage and replication stress.
Asunto(s)
Cistadenocarcinoma Seroso/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirazinas/administración & dosificación , Pirazoles/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Cistadenocarcinoma Seroso/patología , Combinación de Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patologíaRESUMEN
Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor ß (TGFß) pathway, regulated by the TGFß1, TGFß2, and TGFß3 ligands. Accordingly, the TGFß pathway is an attractive therapeutic target for FA. While inhibition of TGFß1 and TGFß3 promotes blood cell expansion, inhibition of TGFß2 is known to suppress hematopoiesis. Here, we report the effects of AVID200, a potent TGFß1- and TGFß3-specific inhibitor, on FA hematopoiesis. AVID200 promoted the survival of murine FA HSPCs in vitro. AVID200 also promoted in vitro the survival of human HSPCs from patients with FA, with the strongest effect in patients progressing to severe aplastic anemia or myelodysplastic syndrome (MDS). Previous studies have indicated that the toxic upregulation of the nonhomologous end-joining (NHEJ) pathway accounts, at least in part, for the poor growth of FA HSPCs. AVID200 downregulated the expression of NHEJ-related genes and reduced DNA damage in primary FA HSPC in vitro and in in vivo models. Collectively, AVID200 exhibits activity in FA mouse and human preclinical models. AVID200 may therefore provide a therapeutic approach to improving BMF in FA.
Asunto(s)
Anemia de Fanconi/tratamiento farmacológico , Hematopoyesis/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta3/antagonistas & inhibidores , Adolescente , Adulto , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Anemia de Fanconi/metabolismo , Anemia de Fanconi/fisiopatología , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Masculino , Ratones , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.