RESUMEN
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we use single-cell RNA sequencing (scRNA-seq) to examine immune cells in patient and control cohorts. Postexertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. To detect changes coincident with PEM, we applied scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients display classical monocyte dysregulation suggestive of inappropriate differentiation and migration to tissue. We identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlates with disease severity. Comparing the transcriptome at baseline and postexercise challenge, we discover patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation in platelets.
Asunto(s)
Síndrome de Fatiga Crónica , Humanos , Síndrome de Fatiga Crónica/genética , Síndrome de Fatiga Crónica/diagnóstico , Ejercicio Físico/fisiología , Perfilación de la Expresión Génica , Transcriptoma , MonocitosRESUMEN
The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential roles in translational timing and fertilization in early development, but poly(A) tail dynamics have not been fully explored in post-embryonic systems. Here, we examined the landscape and impact of tail length control during macrophage activation. Upon activation, more than 1500 mRNAs, including proinflammatory genes, underwent distinctive changes in tail lengths. Increases in tail length correlated with mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Strikingly, we found that ZFP36, whose protein product destabilizes target transcripts, undergoes tail extension. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Taken together, this study establishes the complexity, relevance, and widespread nature of poly(A) tail dynamics, and the resulting post-transcriptional regulation during macrophage activation.
Asunto(s)
Activación de Macrófagos , Poli A , Regulación de la Expresión Génica , Activación de Macrófagos/genética , Poli A/genética , Poli A/metabolismo , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Polyadenylation and deadenylation of mRNA are major RNA modifications associated with nucleus-to-cytoplasm translocation, mRNA stability, translation efficiency, and mRNA decay pathways. Our current knowledge of polyadenylation and deadenylation has been expanded due to recent advances in transcriptome-wide poly(A) tail length assays. Whereas these methods measure poly(A) length by quantifying the adenine (A) base stretch at the 3' end of mRNA, we developed a more cost-efficient technique that does not rely on A-base counting, called tail-end-displacement sequencing (TED-seq). Through sequencing highly size-selected 3' RNA fragments including the poly(A) tail pieces, TED-seq provides accurate measure of transcriptome-wide poly(A)-tail lengths in high resolution, economically suitable for larger scale analysis under various biologically transitional contexts.
Asunto(s)
Poliadenilación , Genoma , Poli A/genética , Poli A/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARNRESUMEN
Post-transcriptional RNA processing is a core mechanism of gene expression control in cell stress response. The poly(A) tail influences mRNA translation and stability, but it is unclear whether there are global roles of poly(A)-tail lengths in cell stress. To address this, we developed tail-end displacement sequencing (TED-seq) for an efficient transcriptome-wide profiling of poly(A) lengths and applied it to endoplasmic reticulum (ER) stress in human cells. ER stress induced increases in the poly(A) lengths of certain mRNAs, including known ER stress regulators, XBP1, DDIT3, and HSPA5. Importantly, the mRNAs with increased poly(A) lengths are both translationally de-repressed and stabilized. Furthermore, mRNAs in stress-induced RNA granules have shorter poly(A) tails than in the cytoplasm, supporting the view that RNA processing is compartmentalized. In conclusion, TED-seq reveals that poly(A) length is dynamically regulated upon ER stress, with potential consequences for both translation and mRNA turnover.
Asunto(s)
Estrés del Retículo Endoplásmico , Poli A/metabolismo , Poliadenilación , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Poli A/química , Análisis de Secuencia de ARN/métodos , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Transcriptoma , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
A novel series of arylurea and arylamide derivatives 1a-z, 2a-d having aminoquinazoline scaffold was designed and synthesized. Their in vitro antiproliferative activities against RT112 bladder cancer cell line and inhibitory activities against FGFR3 kinase were tested. Most compounds showed good antiproliferative activities against RT112 bladder cancer cell line, and arylurea compounds 1a-z were more potent than arylamide compounds 2a-d. Among them, eight compounds 1a, 1d-g, 1l, 1y, and 1z showed potent activities with GI50 values below submicromolar range. Especially, arylurea compounds 1d and 1g possessing 2,3-dimethyl and 3,4-dimethyl moieties exhibited superior or similar antiproliferative activity (GI50=8.8nM and 30.2nM, respectively) to AZD4547 (GI50=29.2nM) as a reference standard.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Quinazolinas/síntesis química , Quinazolinas/farmacología , Urea/síntesis química , Urea/farmacología , Neoplasias de la Vejiga Urinaria/patología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Quinazolinas/química , Urea/químicaRESUMEN
Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, 6i rationally designed. Compound 6i inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in 6i is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, 6i not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/genética , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Fosforilación , Piperidinas/síntesis química , Piperidinas/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Relación Estructura-Actividad , Especificidad por Sustrato , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genéticaRESUMEN
Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.
Asunto(s)
Neoplasias Endometriales/tratamiento farmacológico , Imidazoles/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridazinas/administración & dosificación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Humanos , Ratones , Mutación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Uncontrolled activation of FGFRs induces the progression of various cancers. It was recently reported that FGFR2-activating mutants are implicated in about 12% of endometrial carcinomas. AZD4547, a potent pan-FGFR inhibitor, is currently being evaluated in clinical trials for several FGFR-driven cancers. However, AZD4547 has not been examined yet against FGFR2 mutant-driven endometrial cancers. Thus, we evaluated the activity of AZD4547 against four different endometrial cancer cells, including AN3-CA, MFE296, MFE280, and HEC1A, where all but HEC1A cells express distinctive FGFR2 mutations. We found that AZD4547 exhibits potent antiproliferative activity (EC50 = 31 nmol/L) against AN3-CA cells harboring FGFR2-K310R/N550K mutant. Analysis using a phospho-kinase array revealed that AZD4547 blocks FGFR2 downstream signaling, such as p38, ERK1/2, JNK, p70S6K, and PLCγ. Moreover, oral administration of AZD4547 (30 mg/kg, every day) remarkably delayed tumor growth in a mouse xenograft model of AN3-CA cells. Unbiased reporter gene assay showed that AZD4547 antagonizes the aFGF-induced activation of several transcription factors, including EGR1, ELK-1/SRF, AP-1, and NFκB. Genome-wide transcriptome analysis revealed that AZD4547 perturbs a number of transcriptions, and EGR1 was identified as one of the major targets of AZD4547. The significance of the FGFR2-EGR1 axis in endometrial cancer progression has not been reported. In addition, using kinome-wide inhibition profiling analysis, we first identified potential new target kinases of AZD4547, including MAP4K3, MAP4K5, IRR, RET, and FLT3. Our study demonstrated that AZD4547 exhibits its therapeutic activity against endometrial cancer cells by perturbing various regulatory mechanisms related to FGFR signaling.