RESUMEN
The molecular pathogenesis of extranodal NK/T-cell lymphoma (NKTCL) remains obscured despite the next-generation sequencing (NGS) studies explored on ever larger cohorts in the last decade. We addressed the highly variable mutation frequencies reported among previous studies with comprehensive amplicon coverage and enhanced sequencing depth to achieve higher genomic resolution for novel genetic discovery and comparative mutational profiling of the oncogenesis of NKTCL. Targeted exome sequencing was conducted to interrogate 415 cancer-related genes in a cohort of 36 patients with NKTCL, and a total of 548 single nucleotide variants (SNVs) and 600 Copy number variances (CNVs) were identified. Recurrent amplification of the MCL1 (67%) and PIM1 (56%) genes was detected in a dominant majority of patients in our cohort. Functional mapping of genetic aberrations revealed that an enrichment of mutations in the JAK-STAT signaling pathway, including the cytokine receptor LIFR (copy number loss) upstream of JAK3, STAT3 (activating SNVs), and downstream effectors of MYC, PIM1 and MCL1 (copy number gains). RNA in situ hybridization showed the significant consistence of MCL1 RNA level and copy number of MCL1 gene. We further correlated molecular and clinical parameters with overall survival (OS) of these patients. When correlations were analyzed by univariate followed by multivariate modelling, only copy number loss of LIFR gene and stage (III-IV) were independent prognostic factors of reduced OS. Our findings identified that novel loss of LIFR gene significantly correlated with the adverse clinical outcome of NKTCL patients and provided therapeutic opportunities for this disease through manipulating LIFR.
Asunto(s)
Linfoma Extranodal de Células NK-T , Mutación , Humanos , Linfoma Extranodal de Células NK-T/genética , Linfoma Extranodal de Células NK-T/terapia , Linfoma Extranodal de Células NK-T/mortalidad , Masculino , Femenino , Persona de Mediana Edad , Adulto , Anciano , Pronóstico , Variaciones en el Número de Copia de ADNRESUMEN
The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.
Asunto(s)
Antígeno B7-H1/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Interferón gamma/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Factor de Transcripción STAT1/genética , Afatinib/farmacología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Biomarcadores , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Humanos , Inmunofenotipificación , Masculino , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT1/metabolismoRESUMEN
BACKGROUND: Cancer stem cells are capable of undergoing cell division after surviving cancer therapies, leading to tumor progression and recurrence. Inhibitory agents against cancer stem cells may be therapeutically used for efficiently eradicating tumors. Therefore, the aim of this study was to identify the relevant driver genes that maintain cancer stemness in epidermal growth factor receptor (EGFR)-positive colorectal cancer (CRC) cells and to discover effective therapeutic agents against these genes. METHODS: In this study, EGFR-positive cancer stem-like cells (CSLCs) derived from HCT116 and HT29 cells were used as study models for in vitro inductions. To identify the differential genes that maintain CSLCs, RNAseq analysis was conducted followed by bioinformatics analysis. Moreover, a panel containing 172 therapeutic agents targeting the various pathways of stem cells was used to identify effective therapeutics against CSLCs. RESULTS: RNAseq analysis revealed that 654 and 840 genes were significantly upregulated and downregulated, respectively, in the HCT116 CSLCs. Among these genes, notably, platelet-derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3) were relevant according to the cancer pathway analyzed using NetworkAnalyst. Furthermore, therapeutic screening revealed that the agents targeting STAT3 and Wnt signaling pathways were efficient in reducing the cell viabilities of both HCT116 and HT29 cells. Consequently, we discovered that STAT3 inhibition using homoharringtonine and STAT3 knockdown significantly reduced the formation and survival of HT29-derived tumorspheres. We also observed that STAT3 phosphorylation was regulated by epidermal growth factor (EGF) to induce PDGFA and Wnt signaling cascades. CONCLUSIONS: We identified the potential genes involved in tumorsphere formation and survival in selective EGFR-positive CRCs. The results reveal that the EGF-STAT3 signaling pathway promotes and maintains CRC stemness. In addition, a crosstalk between STAT3 and Wnt activates the Wnt/ß-catenin signaling pathway, which is also responsible for cancer stemness. Thus, STAT3 is a putative therapeutic target for CRC treatment.
Asunto(s)
Neoplasias Colorrectales/genética , Receptores ErbB/genética , Células Madre Neoplásicas/patología , Factor de Transcripción STAT3/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Detección Precoz del Cáncer , Factor de Crecimiento Epidérmico/genética , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/genética , Análisis de Secuencia de ARN , Vía de Señalización WntRESUMEN
The unsatisfactory real-world efficacy of the hypomethylating agent azacitidine in treating myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has prompted us to investigate the hematological adverse events and host variables that may compromise the use of this epigenetic drug. Using the zebrafish, we found that azacitidine destroyed their myeloid precursors and impaired myeloid function by inhibiting antigen processing, allogeneic response and phagocytic activity, resulting in increased susceptibility to infection even by the normal flora E. coli. In addition, iron overload, a MDS-associated condition following repeated transfusions, exacerbated bacterial infection especially by V. vulnificus with known iron dependence. Furthermore, we show that the tp53M214K mutant zebrafish survived longer than the wild-type (WT) when challenged with bacteria following azacitidine treatment. This was attributed to the mutant's hematopoietic cells rather than its general genetic background, since the WT animals reconstituted with the tp53M214K mutant kidney marrow became more resistant to bacterial infection following treatment with azacitidine. The clinical relevance of our findings was indicated by a MDS case with severe azacitidine-induced bone marrow suppression and by the association of hyperferritinemia with bacteremia in azacitidine-treated patients, while tp53M214K-mediated resistance to azacitidine-induced myelosuppression may explain the survival advantage of malignant MDS and AML clones over their normal counterparts under azacitidine treatment. Together, we propose that myelosuppression, iron overload and TP53 mutations may represent the host variables that compromise the azacitidine efficacy.
RESUMEN
OBJECTIVES: YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. MATERIALS AND METHODS: The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. RESULTS: We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. CONCLUSION: This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Imidazoles/farmacología , Neoplasias Pulmonares/metabolismo , Naftoquinonas/farmacología , Células Madre Neoplásicas/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Células A549 , Afatinib/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Humanos , Imidazoles/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Naftoquinonas/administración & dosificación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas del Factor Nuclear 90/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1-5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.
Asunto(s)
ADN de Neoplasias/genética , Mutación , Trastornos Mieloproliferativos/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Calreticulina/genética , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/sangre , Femenino , Mutación de Línea Germinal , Humanos , Janus Quinasa 2/genética , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/sangre , Receptores de Trombopoyetina/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6×10-19, 1.9×10-19 and 3.1×10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6×10-21, 4.4×10-21 and 8.6×10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population.
RESUMEN
Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm. We have reported that increased activated B cells can facilitate platelet production mediated by cytokines regardless JAK2 mutational status in ET. Recently, calreticulin (CALR) mutations were discovered in ~30% JAK2/MPL-unmutated ET and primary myelofibrosis. Here we sought to screen for CALR mutations and to evaluate B cell immune profiles in a cohort of adult Taiwanese ET patients. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) levels, B cells toll-like receptor 4 (TLR4) expression and intracellular levels of interleukin (IL)-1ß/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF concentration was measured by ELISA. 48 healthy adults were used for comparison. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Compared to JAK2V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. JAK2- and CALR-mutated ET have significantly higher fraction of B cells with TLR4 expression when compared to triple-negative ET (p=0.019 and 0.02, respectively). CALR-mutated ET had significantly higher number of CD69-positive activated B cells when compared to triple-negative ET (p=0.035). In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.
Asunto(s)
Linfocitos B/inmunología , Calreticulina/genética , Calreticulina/inmunología , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Trombocitemia Esencial/genética , Trombocitemia Esencial/inmunología , Adulto , Anciano , Linfocitos B/patología , Calreticulina/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Janus Quinasa 2/metabolismo , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Mutación , Trombocitemia Esencial/sangre , Trombocitemia Esencial/patología , Adulto JovenRESUMEN
BACKGROUND: Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations. METHODS: 32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden. RESULTS: The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative. CONCLUSION: The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.