RESUMO
BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structureactivity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.
Assuntos
Anti-Inflamatórios não Esteroides , Antineoplásicos , Anticoncepcionais Masculinos , Descoberta de Drogas , Proteínas Nucleares , Bibliotecas de Moléculas Pequenas , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Anticoncepcionais Masculinos/química , Anticoncepcionais Masculinos/isolamento & purificação , Anticoncepcionais Masculinos/farmacologia , DNA/genética , Humanos , Masculino , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Domínios Proteicos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Defects in spermatogenesis are an important cause of male infertility. Multiple aspects of spermatogenesis are controlled by chromatin remodellers, including regulating transcription. We previously described mutations in chromatin remodelling gene Cecr2 that resulted in the lethal neural tube defect exencephaly in most mutant mice and subfertility in mice that were non-penetrant for exencephaly. Here, we show that the severity of male subfertility is dependent on age. Cecr2GT/Del males contain two mutant alleles, one of which is hypomorphic and therefore produces a small amount of protein. These males sire the fewest pups just after sexual maturity (88% fewer than Cecr2+/+ at P42-60) but improve with age (49% fewer than Cecr2+/+ at P81-100), although never completely recovering to Cecr2+/+(wild type) levels. When young, they also have defects in testis histology, in vivo fertilization frequency, sperm number and motility, and testis weight that show similar improvement with age. Immunostaining of staged seminiferous tubules showed CECR2 in type A, intermediate and B spermatogonia, and less in preleptotene and leptotene spermatocytes. Histological defects were first apparent in Cecr2GT/Del testes at P24, and RNA-seq analysis revealed 387 differentially expressed genes. This included 66 genes on the X chromosome (almost double the number on any other chromosome), all more highly expressed in Cecr2GT/Del testes. This inappropriate expression of X chromosome genes could be caused by a failure of effective meiotic sex chromosome inactivation. We identify several abnormally expressed genes that may contribute to defects in spermatogenesis at P24. Our results support a role for Cecr2 in juvenile spermatogenesis.
Assuntos
Cromatina , Infertilidade Masculina , Espermatogênese , Fatores de Transcrição , Animais , Montagem e Desmontagem da Cromatina , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Espermatogênese/genética , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)-rearranged samples were also distinct from non-KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE- samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE- patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.
Assuntos
Leucemia Mieloide Aguda , Animais , Criança , Estudos de Coortes , Regulação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , CamundongosRESUMO
Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of â¼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Edição de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous work has suggested androgen receptor (AR) signaling mediates prostate cancer progression in part through the modulation of autophagy. However, clinical trials testing autophagy inhibition using chloroquine derivatives in men with castration-resistant prostate cancer (CRPC) have yet to yield promising results, potentially due to the side effects of this class of compounds. We hypothesized that identification of the upstream activators of autophagy in prostate cancer could highlight alternative, context-dependent targets for blocking this important cellular process during disease progression. Here, we used molecular, genetic, and pharmacological approaches to elucidate an AR-mediated autophagy cascade involving Ca2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2; a kinase with a restricted expression profile), 5'-AMP-activated protein kinase (AMPK), and Unc-51 like autophagy activating kinase 1 (ULK1), but independent of canonical mechanistic target of rapamycin (mTOR) activity. Increased CAMKK2-AMPK-ULK1 signaling correlated with disease progression in genetic mouse models and patient tumor samples. Importantly, CAMKK2 disruption impaired tumor growth and prolonged survival in multiple CRPC preclinical mouse models. Similarly, an inhibitor of AMPK-ULK1 blocked autophagy, cell growth, and colony formation in prostate cancer cells. Collectively, our findings converge to demonstrate that AR can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote prostate cancer by increasing autophagy. Thus, this pathway may represent an alternative autophagic target in CRPC.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas Quinases/genética , Receptores Androgênicos/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Eosinophils are a myeloid cell subpopulation that mediates type 2 T helper cell immune responses. Unexpectedly, we identified a rapid accumulation of eosinophils in 22 human liver grafts after hepatic transplantation. In contrast, no eosinophils were detectable in healthy liver tissues before transplantation. Studies with two genetic mouse models of eosinophil deficiency and a mouse model of antibody-mediated eosinophil depletion revealed exacerbated liver injury after hepatic ischemia and reperfusion. Adoptive transfer of bone marrow-derived eosinophils normalized liver injury of eosinophil-deficient mice and reduced hepatic ischemia and reperfusion injury in wild-type mice. Mechanistic studies combining genetic and adoptive transfer approaches identified a critical role of suppression of tumorigenicity (ST2)-dependent production of interleukin-13 by eosinophils in the hepatoprotection against ischemia-reperfusion-induced injury. Together, these data provide insight into a mechanism of eosinophil-mediated liver protection that could serve as a therapeutic target to improve outcomes of patients undergoing liver transplantation.
Assuntos
Eosinófilos , Traumatismo por Reperfusão , Transferência Adotiva , Animais , Humanos , Interleucina-13 , Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Systemic arterial gas embolism (SAGE) is a rare yet serious and underrecognized complication of bronchoscopic procedures. A recent case of presumed SAGE after transbronchial needle aspiration prompted a systematic literature review of SAGE after biopsy procedures during flexible bronchoscopy. METHODS: We performed a systematic database search for case reports and case series pertaining to SAGE after bronchoscopic lung biopsy; reports or series involving only bronchoscopic laser therapy or argon plasma coagulation (APC) were excluded. Patient data were extracted directly from published reports. RESULTS: A total of 29 unique patient reports were assessed for patient demographics, specifics of the procedure, clinical manifestations, diagnostic findings, and clinical outcomes. Cases of SAGE occurred after multiple types of bronchoscopic biopsy and under both positive and negative pressure ventilation. The most common clinical findings were neurologic, followed by cardiac manifestations; temporal patterns included acute onset of cardiac or neurologic emergencies immediately after biopsy, or delayed awakening post-procedure. There was a high mortality rate among cases (28%), with residual neurologic deficits also common (24%). DISCUSSION: SAGE is an underrecognized but severe adverse effect of bronchoscopic lung biopsy, which often presents with acute coronary or cerebral ischemia or delayed awakening from sedation. It is important for all physicians who perform bronchoscopic biopsies to be aware of the clinical manifestations and therapeutic management of SAGE in order to mitigate morbidity and mortality among patients undergoing these procedures.
RESUMO
Defects in the maternal reproductive system that result in early pregnancy loss are important causes of human female infertility. A wide variety of biological processes are involved in implantation and establishment of a successful pregnancy. Although chromatin remodelers have been shown to play an important role in many biological processes, our understanding of the role of chromatin remodelers in female reproduction remains limited. Here, we demonstrate that female mice mutant for chromatin remodeler Cecr2 are subfertile, with defects detected at the peri-implantation stage or early pregnancy. Using both a less severe hypomorphic mutation (Cecr2GT) and a more severe presumptive null mutation (Cecr2Del), we demonstrate a clear difference in the severity of the phenotype depending on the mutation. Although neither strain shows detectable defects in folliculogenesis, both Cecr2GT/GT and Cecr2GT/Del dams show defects in pregnancy. Cecr2GT/GT females have a normal number of implantation sites at embryonic day 5.5 (E5.5), but significant embryo loss by E10.5 accompanied by the presence of vaginal blood. Cecr2GT/Del females show a more severe phenotype, with significantly fewer detectable implantation sites than wild type at E5.5. Some Cecr2GT/Del females also show premature loss of decidual tissue after artificial decidualization. Together, these results suggest a role for Cecr2 in the establishment of a successful pregnancy.
Assuntos
Implantação do Embrião/genética , Perda do Embrião/genética , Infertilidade Feminina/genética , Fatores de Transcrição/genética , Animais , Embrião de Mamíferos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutação , Gravidez , Fatores de Transcrição/fisiologiaRESUMO
The majority of breast cancer specific deaths in women with estrogen receptor positive (ER+) tumors occur due to metastases that are resistant to therapy. There is a critical need for novel therapeutic approaches to achieve tumor regression and/or maintain therapy responsiveness in metastatic ER+ tumors. The objective of this study was to elucidate the role of metabolic pathways that undermine therapy efficacy in ER+ breast cancers. Our previous studies identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression and showed that combining selinexor (SEL), an FDA-approved XPO1 antagonist, synergized with endocrine agents and provided sustained tumor regression. In the current study, using a combination of transcriptomics, metabolomics and metabolic flux experiments, we identified certain mitochondrial pathways to be upregulated during endocrine resistance. When endocrine resistant cells were treated with single agents in media conditions that mimic a nutrient deprived tumor microenvironment, their glutamine dependence for continuation of mitochondrial respiration increased. The effect of glutamine was dependent on conversion of the glutamine to glutamate, and generation of NAD+. PGC1α, a key regulator of metabolism, was the main driver of the rewired metabolic phenotype. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and our findings reveal a critical role that ERα-XPO1 crosstalk plays in reducing cancer recurrences. Combining SEL with current therapies used in clinical management of ER+ metastatic breast cancer shows promise for treating and keeping these cancers responsive to therapies in already metastasized patients.
Assuntos
Cistos/diagnóstico por imagem , Mandíbula/diagnóstico por imagem , Maxila/diagnóstico por imagem , Distrofias Musculares/diagnóstico por imagem , Síndrome de Pierre Robin/diagnóstico por imagem , Esclerose/diagnóstico por imagem , Pré-Escolar , Cistos/complicações , Cistos/cirurgia , Humanos , Mandíbula/cirurgia , Maxila/cirurgia , Distrofias Musculares/complicações , Distrofias Musculares/cirurgia , Síndrome de Pierre Robin/complicações , Síndrome de Pierre Robin/cirurgia , Esclerose/complicações , Esclerose/cirurgiaRESUMO
A majority of breast cancer specific deaths in women with ERα (+) tumors occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and the nuclear transport protein XPO1 in overcoming endocrine resistance. Selinexor (SEL), an XPO1 antagonist, has been evaluated in multiple late stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Our transcriptomics analysis showed that 4-Hydroxytamoxifen (4-OHT), SEL alone or their combination induced differential Akt signaling- and metabolism-associated gene expression profiles. Western blot analysis in endocrine resistant cell lines and xenograft models validated differential Akt phosphorylation. Using the Seahorse metabolic profiler, we showed that ERα-XPO1 targeting changed the metabolic phenotype of TAM-resistant breast cancer cells from an energetic to a quiescent profile. This finding demonstrated that combined targeting of XPO1 and ERα rewired the metabolic pathways and shut down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies to improve therapy response in relapsed ERα (+) tumors, our findings show great promise for uncovering the role that ERα-XPO1 crosstalk plays in reducing cancer recurrences.