CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

DKC1 aggravates gastric cancer cell migration and invasion through up-regulating the expression of TNFAIP6.

Gastric cancer (GC) is one hackneyed malignancy tumor accompanied by high death rate. DKC1 has been discovered to serve as a facilitator in several cancers. Additionally, it was discovered from one study that DKC1 displayed higher expression in GC tissues than in the normal tissues. Nevertheless, its role and regulatory mechanism in GC is yet to be illustrated. In this study, it was proved that DKC1 expression was upregulated in GC tissues through GEPIA and UALCAN databases. Moreover, we discovered that DKC1 exhibited higher expression in GC cells. Functional experiments testified that DKC1 accelerated cell proliferation, migration, and invasion in GC. Further investigation disclosed that the weakened cell proliferation, migration, and invasion stimulated by DKC1 knockdown can be reversed after TNFAIP6 overexpression. Lastly, through in vivo experiments, it was demonstrated that DKC1 strengthened tumor growth. In conclusion, our work uncovered that DKC1 aggravated GC cell migration and invasion through upregulating the expression of TNFAIP6. This discovery might highlight the function of DKC1 in GC treatment.

G3BP2 promotes tumor progression and gemcitabine resistance in PDAC via regulating PDIA3-DKC1-hENT in a stress granules-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is distinguished by its aggressive malignancy, limited treatment avenues and a tendency towards chemotherapy resistance, underscoring the critical need for advanced research to uncover new therapeutic approaches. Stress granules (SGs) that is implicated in cellular self-protection mechanism, along with its associated family molecules have shown pro-cancer effects and are closely related to tumor chemotherapy resistance. In this study we investigated the relationship between Ras GTPase-activating protein-binding proteins 2 (G3BP2), a core component of SGs, and the malignancy of PDAC as well as its resistance to the chemotherapy drug gemcitabine. Analyzing TCGA dataset revealed that the expression of G3BP1 and G3BP2 was significantly upregulated in PDAC compared with adjacent normal pancreatic tissues, and the high expression of G3BP2 rather than G3BP1 was significantly associated with poorer overall survival (OS) in PDAC patients. We demonstrated that knockdown of G3BP2 inhibited the proliferation and invasion of PANC-1 and CFPAC-1 cells in vitro and in vivo. By analyzing the differentially expressed genes in G3BP2 knockdown and overexpressed PANC-1 cells, we identified DKC1 that was associated with RNA stability and regulation as the target of G3BP2. We demonstrated that G3BP2 bound to PDIA3 mRNA and recruited them into SGs, increasing the stability of PDIA3 mRNA and attenuating its translation efficiency, thereby promoting DKC1 expression. Furthermore, DKC1 could bind to hENT mRNA and inhibited its expression, which enhanced gemcitabine resistance of PDAC. Therefore, we propose a novel mechanism wherein G3BP2 facilitates PDAC's resistance to chemotherapy by modulating PDIA3-DKC1-hENT in a SGs-dependent way, suggesting G3BP2 SGs a protentional therapeutic target for the treatment in PDAC.

Dyskerin: an essential pseudouridine synthase with multifaceted roles in ribosome biogenesis, splicing, and telomere maintenance.

Dyskerin and its homologs are ancient and conserved enzymes that catalyze the most common post-transcriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently-it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlights the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations in DKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural, and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency, and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential, and cancer.

Evidence from a meta-analysis for the prognostic and clinicopathological importance of DKC1 in malignancies.

Aim: We conducted a meta-analysis to evaluate the prognostic and clinicopathological relevance of DKC1 in various cancers. Methods: We searched Web of Science, Embase, PubMed, Wanfang and CNKI. Stata SE15.1 was used to calculate the hazard ratio and relative risk with 95% CIs to assess the possible correlations between DKC1 expression levels and overall and disease-free survival, as well as with clinicopathological parameters. Results: We included nine studies, with a total of 2574 patients. There was a meaningful link between elevated DKC1 and poorer disease-free (p < 0.001) and overall survival (p < 0.001). Also, it was linked to advanced tumor node metastasis stage (p = 0.005). Conclusion: High DKC1 expression was predictive of worse prognosis and poorer clinicopathological parameters.

What is this summary about? This brief summary reports the effects of high or low levels of a gene expression product called DKC1 on survival time and clinicopathological parameters in a small group of people with cancer. The DKC1 gene encodes a protein, DKC1, that is important for cancer cell proliferation. We systematically reviewed nine studies involving 2574 patients. What was the result? In this research, we revealed that people with cancer who had poor DKC1 expression had considerably longer survival without disease and better overall survival. In addition, the increased expression of DKC1 was linked to late-stage cancers. What do these results mean? The study has shown encouraging results, suggesting that DKC1 is a promising target for cancer therapy, as targeting it may hinder its ability to impair ribosome production and normal telomerase complex function to prolong patient life and prevent progression to a later disease stage. These studies demonstrate the need for more studies involving more people to definitively confirm how effective targeting DKC1 may be in treating cancer patients.

A pan-cancer analysis of Dyskeratosis congenita 1 (DKC1) as a prognostic biomarker.

BACKGROUND: Dyskeratosis congenita 1 (DKC1), a critical component of telomerase complex, is highly expressed in a variety of human cancers. However, the association of DKC1 with cancer occurrence and development stages is not clear, making a pan-cancer analysis crucial. METHODS: We conducted a study using various bioinformatic databases such as TIMER, GEPIA, UALCAN, and KM plotter Analysis to examine the different expressions of DKC1 in multiple tissues and its correlation with pathological stages. Through KEGG analysis, GO enrichment analysis and Venn analysis, we were able to reveal DKC1-associated genes and signaling pathways. In addition, we performed several tests including the CCK, wound healing assay, cell cycle arrest assay, transwell assay and Sa-ß-gal staining on DKC1-deleted MDA-231 cells. RESULTS: Our study demonstrates that DKC1 has relatively low expression specificity in different tissues. Furthermore, we found that in ACC, KICH, KIRP and LIHC, the expression level of DKC1 is positively correlated with pathological stages. Conversely, in NHSC, KIRP, LGG, LIHC, MESO and SARC, we observed a negative influence of DKC1 expression level on the overall survival rate. We also found a significant positive correlation between DKC1 expression and Tumor Mutational Burden in 14 tumors. Additionally, we observed a significantly negative impact of DKC1 DNA methylation on gene expression at the promoter region in BRCA. We also identified numerous phosphorylation sites concentrated at the C-terminus of the DKC1 protein. Our GO analysis revealed a correlation between DKC1 and ribosomal biosynthesis pathways, and the common element UTP14A was identified. We also observed decreased rates of cell proliferation, migration and invasion abilities in DKC1-knockout MDA-MB-231 cell lines. Furthermore, DKC1-knockout induced cell cycle arrest and caused cell senescence. CONCLUSIONS: Our findings suggest that the precise expression of DKC1 is closely associated with the occurrence and developmental stages of cancer in multiple tissues. Depletion of DKC1 can inhibit the abilities of cancer cells to proliferate, migrate, and invade by arresting the cell cycle and inducing cell senescence. Therefore, DKC1 may be a valuable prognostic biomarker for the diagnosis and treatment of cancer in various tissues.

Inhibition of DKC1 induces telomere-related senescence and apoptosis in lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the most widely spread cancers in the world and half of the non-small cell lung cancers are lung adenocarcinoma (LUAD). Although there were several drugs been approved for LUAD therapy, a large portion of LUAD still cannot be effectively treated due to lack of available therapeutic targets. Here, we investigated the oncogenic roles of DKC1 in LUAD and its potential mechanism and explored the possibility of targeting DKC1 for LUAD therapy. METHODS: The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas Program (TCGA) databases were used to examine the DKC1 transcript levels. Gene expression with clinical information from tissue microarray of LUAD were analyzed for associations between DKC1 expression and LUAD prognosis. In addition, loss- and gain-of-function assays were used for oncogenic function of DKC1 both in vitro and in vivo. RESULTS: DKC1 is overexpressed in LUAD compared with adjacent normal tissues. High expression of DKC1 predicts the poor overall survival. DKC1 knockdown in LUAD cell lines induced G1 phase arrest and inhibited cell proliferation. Ectopic expression of DKC1 could rescue the growth of LUAD cell lines. In addition, the abundance of DKC1 is positively correlated with telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) levels in LUAD. DKC1 downregulation resulted in decreased TERC expression, reduced telomerase activity and shorten telomere, and thus eventually led to cell senescence and apoptosis. CONCLUSIONS: Our results show that high DKC1 expression indicates poor prognosis of LUAD and DKC1 downregulation could induce telomere-related cell senescence and apoptosis. This study suggests that DKC1 could serve as a candidate diagnostic biomarker and therapeutic target for LUAD.

Successful liver transplantation in short telomere syndromes without bone marrow failure due to DKC1 mutation.

Short telomere syndromes are a heterogenous spectrum of disorders leading to premature cellular aging. These may involve bone marrow failure, adult-onset idiopathic pulmonary fibrosis, and liver disease, and classical entities such as dyskeratosis congenita. We report a patient who presented with common variable immunodeficiency at 3 years of age and autoimmune cytopenias at 8 years of age. He was found to have short telomeres, and genetic testing confirmed a hemizygous mutation NM_001363.4: c.-142C > G in DKC1 gene. He subsequently developed cirrhosis with severe portal hypertension and hepatopulmonary syndrome, prompting liver transplantation at 11 years of age. He remains well 10 years after transplant with no progression of bone marrow failure or progressive lung disease. In conclusion, short telomere syndromes should be considered as a potential cause of pediatric liver disease of unknown etiology, and in severe cases, isolated liver transplantation may be both appropriate and successful.

CD8+ T-cell senescence and skewed lymphocyte subsets in young Dyskeratosis Congenita patients with PARN and DKC1 mutations.

BACKGROUND: Dyskeratosis congenita (DC) is a syndrome resulting from defective telomere maintenance. Immunodeficiency associated with DC can cause significant morbidity and lead to premature mortality, but the immunological characteristics and molecular hallmark of DC patients, especially young patients, have not been described in detail. METHODS: We summarize the clinical data of two juvenile patients with DC. Gene mutations were identified by whole-exome and direct sequencing. Swiss-PdbViewer was used to predict the pathogenicity of identified mutations. The relative telomere length was determined by QPCR, and a comprehensive analysis of lymphocyte subsets and CD57 expression was performed by flow cytometry. RESULTS: Both patients showed typical features of DC without severe infection. In addition, patient 1 (P1) was diagnosed with Hoyeraal-Hreidarsson syndrome due to cerebellar hypoplasia. Gene sequencing showed P1 had a compound heterozygous mutation (c.204G > T and c.178-245del) in PARN and P2 had a novel hemizygous mutation in DKC1 (c.1051A > G). Lymphocyte subset analysis showed B and NK cytopenia, an inverted CD4:CD8 ratio, and decreased naïve CD4 and CD8 cells. A significant increase in CD21low B cells and skewed numbers of helper T cells (Th), regulatory T cells (Treg), follicular regulatory T cells (Tfr), and follicular helper T cells (Tfh) were also detected. Short telomere lengths, increased CD57 expression, and an expansion of CD8 effector memory T cells re-expressing CD45RA (TEMRA) were also found in both patients. CONCLUSION: Unique immunologic abnormalities, CD8 T-cell senescence, and shortened telomere together as a hallmark occur in young DC patients before progression to severe disease.

Increased DKC1 expression in glioma and its significance in tumor cell proliferation, migration and invasion.

The dyskeratosis congenita 1 (DKC1) gene is located on the X chromosome at Xq28. Dyskerin encoded by the DKC1 gene is associated with the formation of certain small RNAs and the telomerase activity. Inherited mutations in DKC1 inactivate the dyskerin and causes dyskeratosis congenital, which is characterized by skin defects, hematopoiesis failure, and increased susceptibility to cancer. DKC1 reportedly up-regulates in several human cancers, including renal cell carcinoma and prostate cancer. Dyskerin is deregulated in B-chronic lymphocytic leukemia and breast carcinomas, but its expression and function in glioma have hardly been investigated. Hence, we were prompted to collect tissue samples and implement cell experiments. Our study reveals that DKC1 expression is significantly increased in the pathological tissues of glioma compared with that in normal tissues. The increased staining of DKC1 is related to the World Health Organization stages of tumors. DKC1 knockdown also significantly inhibits glioma cell growth by altering the expression of cell cycle-relative molecules to arrest at the G1 phase. In the transwell chamber, DKC1 knockdown glioma cells exhibit low motility. Consistent with classic oncogenic pathways, N-cadherin, HIF-1α, and MMP2 expression levels are lower compared with those of the control group. Therefore, DKC1 up-regulation in gliomas is common and necessary for extensive tumor growth. The phenotype of glioma cell lines after DKC1 down-regulation suggests its use as a valuable clinical treatment strategy.

Dyskeratosis congenita with a novel genetic variant in the DKC1 gene: a case report.

BACKGROUND: Dyskeratosis congenita (DC) is a rare genetic disorder of bone marrow failure inherited in an X-linked, autosomal dominant or autosomal recessive pattern. It has a wide array of clinical features and patients may be cared for by many medical sub specialties. The typical clinical features consist of lacy reticular skin pigmentation, nail dystrophy and oral leukoplakia. As the disease advances, patients may develop progressive bone marrow failure, pulmonary fibrosis, oesophageal stenosis, urethral stenosis, liver cirrhosis as well as haematological and solid malignancies. Several genes have been implicated in the pathogenesis of dyskeratosis congenita, with the dyskerin pseudouridine synthase 1 (DKC1) gene mutations being the X-linked recessive gene. CASE PRESENTATION: Herein, we report a 31-year-old male with history of recurrent febrile episodes who was found to have reticulate skin pigmentation interspersed with hypopigmented macules involving the face, neck and extremities, hyperkeratosis of palms and soles, nail dystrophy, leukoplakia of the tongue, premature graying of hair, watery eyes and dental caries. Several of his male relatives, including two maternal uncles and three maternal cousins were affected with a similar type of disease condition. Pedigree analysis suggested a possible X-linked pattern of inheritance. Genetic testing in the proband showed a novel hemizygous, non-synonymous likely pathogenic variant [NM_001363.4: c.1054A > G: p.Thr352Ala] in the PUA domain of the DKC1 gene. Quantitative polymerase chain reaction for relative telomere length measurements performed in the proband showed that he had very short telomeres [0.38, compared to a control median of 0.71 (range 0.44-1.19)], which is consistent with the DC diagnosis. Co-segregation analysis of the novel mutation and telomere length measurements in the extended family members could not be performed as they were unwilling to provide consent for testing. CONCLUSIONS: The novel variant detected in the DKC1 gene adds further to the existing scientific literature on the genotype-phenotype correlation of DC, and has important implications for the clinical and molecular characterization of the disease.

Homology Model and Docking-Based Virtual Screening for Ligands of Human Dyskerin as New Inhibitors of Telomerase for Cancer Treatment.

Immortality is one of the main features of cancer cells. Tumor cells have an unlimited replicative potential, principally due to the holoenzyme telomerase. Telomerase is composed mainly by dyskerin (DKC1), a catalytic retrotranscriptase (hTERT) and an RNA template (hTR). The aim of this work is to develop new inhibitors of telomerase, selecting the interaction between hTR⁻DKC1 as a target. We designed two models of the human protein DKC1: homology and ab initio. These models were evaluated by different procedures, revealing that the homology model parameters were the most accurate. We selected two hydrophobic pockets contained in the PUA (pseudouridine synthase and archaeosine transglycosylase) domain, using structural and stability analysis. We carried out a docking-based virtual screen on these pockets, using the reported mutation K314 as the center of the docking. The hDKC1 model was tested against a library of 450,000 drug-like molecules. We selected the first 10 molecules that showed the highest affinity values to test their inhibitory activity on the cell line MDA MB 231 (Monroe Dunaway Anderson Metastasis Breast cancer 231), obtaining three compounds that showed inhibitory effect. These results allowed us to validate our design and set the basis to continue with the study of telomerase inhibitors for cancer treatment.

Identifying and validating the roles of the cuproptosis-related gene DKC1 in cancer with a focus on esophageal carcinoma.

BACKGROUND: Esophageal cancer is a common malignancy of the digestive tract. Despite remarkable advancements in its treatment, the overall prognosis for patients remains poor. Cuproptosis is a form of programmed cell death that affects the malignant progression of tumors. This study aimed to examine the impact of the cuproptosis-associated gene DKC1 on the malignant progression of esophageal cancer. METHODS: Clinical and RNA sequencing data of patients with esophageal cancer were extracted from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis was used to identify the differentially expressed genes related to cuproptosis that are associated with prognosis. We then validated the difference in the expression of DKC1 between tumor and normal tissues via three-dimensional multiomics difference analysis. Subsequently, we investigated the association between DKC1 expression and the tumor microenvironment by employing the TIMER2.0 algorithm, which was further validated in 96 single-cell datasets obtained from the TISCH database. Additionally, the functional role of DKC1 in pancarcinoma was assessed through GSEA. Furthermore, a comprehensive pancancer survival map was constructed, and the expression of DKC1 was verified in various molecular subtypes. By utilizing the CellMiner, GDSC, and CTRP databases, we successfully established a connection between DKC1 and drug sensitivity. Finally, the involvement of DKC1 in the progression of esophageal cancer was investigated through in vivo and in vitro experiments. RESULTS: In this study, we identified a copper death-related gene, DKC1, in esophageal cancer. Furthermore, we observed varying levels of DKC1 expression across different tumor types. Additionally, we conducted an analysis to determine the correlation between DKC1 expression and clinical features, revealing its association with common cell cycle pathways and multiple metabolic pathways. Notably, high DKC1 expression was found to indicate poor prognosis in patients with various tumors and to influence drug sensitivity. Moreover, our investigation revealed significant associations between DKC1 expression and the expression of molecules involved in immune regulation and infiltration of lymphocyte subtypes. Ultimately, the increased expression of DKC1 in esophageal cancer tissues was verified using clinical tissue samples. Furthermore, DKC1-mediated promotion of esophageal cancer cell proliferation and migration was confirmed through both in vitro and in vivo experiments. Additionally, it is plausible that DKC1 may play a role in the regulation of cuproptosis. CONCLUSION: In this study, we conducted a systematic analysis of DKC1 and its regulatory factors and experimentally validated its excellent diagnostic and prognostic abilities in various cancers. Further research indicated that DKC1 may reshape the tumor microenvironment (TME), highlighting the potential of DKC1-based cancer treatment and its usefulness in predicting the response to chemotherapy.

Presumptive Cytomegalovirus Retinitis as a Complication of Dyskeratosis Congenita: A Case Report.

Introduction: Dyskeratosis congenita is a rare genetic disorder characterized by abnormalities of the skin, nails, and oral mucosa. Retinal involvement in this condition is uncommon. Here, we present a case of a young male patient diagnosed with presumptive cytomegalovirus retinitis, ultimately found to be concomitant with dyskeratosis congenita. Case Presentation: A non-HIV-infected young male with recurrent infections, including aspergillus pneumonia and pneumocystis pneumonia, presented with presumptive cytomegalovirus retinitis in both eyes. Systemic manifestations included cutaneous hyperpigmentation, nail dystrophy, and oral mucosal leukoplakia. Genetic testing revealed a mutation in the DKC1 gene. The final diagnosis was dyskeratosis congenita complicated by presumptive cytomegalovirus retinitis. Conclusion: Cytomegalovirus retinitis can serve as an ocular complication of dyskeratosis congenita. When a patient presents with cytomegalovirus retinitis, a comprehensive systematic examination should be conducted as it indicates severe immunodeficiency.

Telomere phenotypes in females with heterozygous mutations in the dyskeratosis congenita 1 (DKC1) gene.

Dyskeratosis congenita (DC) is a telomere-mediated syndrome defined by mucocutaneous features. The X-linked mode of inheritance accounts for half the cases, and is thought to predominantly manifest in childhood as bone marrow failure. We identified two male probands who presented in the fifth decade with idiopathic pulmonary fibrosis and cancer. Their pedigrees displayed consecutively affected generations. Five of six females (83%) manifested mucocutaneous features of DC, and two had wound-healing complications. No mutations in autosomal dominant telomere genes were present, but exome sequencing revealed novel variants in the X-chromosome DKC1 gene that predicted missense mutations in conserved residues, p.Thr49Ser and p.Pro409Arg. Variants segregated with the telomere phenotype, and affected females were heterozygotes, showing skewed X-inactivation. Telomerase RNA levels were compromised in cells from DKC1 mutation carriers, consistent with their pathogenic role. These findings indicate that females with heterozygous DKC1 mutations may be at increased risk for developing penetrant telomere phenotypes that, at times, may be associated with clinical morbidity.

Severity of X-linked dyskeratosis congenita (DKCX) cellular defects is not directly related to dyskerin (DKC1) activity in ribosomal RNA biogenesis or mRNA translation.

Dyskerin (encoded by the DKC1 locus) is the pseudouridine synthase responsible for the modification of noncoding RNA. Dyskerin is also an obligate member of the telomerase enzyme, and participates in the biogenesis of telomerase. Genetic lesions at the DKC1 locus are associated with X-linked dyskeratosis congenita (X-DC) and the Hoyeraal-Hreidarsson Syndrome (HHS). Both syndromes have been linked to deficient telomere maintenance, but little is known about the RNA modification activities of dyskerin in X-DC and HHS cells. To evaluate whether X-DC-associated dyskerin mutations affect the modification or function of ribosomal RNA, we studied five telomerase-rescued X-DC cells (X-DC(T) ). Our data revealed a small reproducible loss of pseudouridines in mature rRNA in two X-DC variants. However, we found no difference in protein synthesis between telomerized wild-type (WT(T) ) and X-DC(T) cells, with an internal ribosomal entry site translation assay, or by measuring total protein synthesis in live cells. X-DC(T) cells and WT(T) cells also exhibited similar tolerances to ionizing radiation and endoplasmic reticulum stress. Despite the loss in rRNA pseudouridine modification, functional perturbations from these changes are secondary to the telomere maintenance defects of X-DC. Our data show that telomere dysfunction is the primary and unifying etiology of X-DC.

Dyskerin and telomerase RNA component are sex-differentially associated with outcomes and Sunitinib response in patients with clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) displays sex-biased incidence, outcomes, molecular alterations and treatment efficacy; however, clinical managements are largely identical in male and female patients. Moreover, many biomarkers have been identified as predictors for ccRCC outcomes and response to therapeutic drugs, such as multitargeted tyrosine-kinase receptor (TKR) inhibitors, but little is known about their sex-specificity. Dyskerin (DKC1), encoded by the DKC1 gene within Xq28, is a telomerase co-factor stabilizing telomerase RNA component (TERC) and overexpressed in various cancers. Here, we determined whether DKC1 and/or TERC affect ccRCC sex-differentially. METHODS: DKC1 and TERC expression in primary ccRCC tumors was assessed using RNA sequencing and qPCR. DKC1 association with molecular alterations and overall or progression-free survival (OS or PFS) was analyzed in the TCGA cohort of ccRCC. The IMmotion 151 and 150 ccRCC cohorts were analyzed to evaluate impacts of DKC1 and TERC on Sunitinib response and PFS. RESULTS: DKC1 and TERC expression was significantly upregulated in ccRCC tumors. High DKC1 expression predicts shorter PFS independently in female but not male patients. Tumors in the female DKC1-high group exhibited more frequent alterations in PIK3CA, MYC and TP53 genes. Analyses of the IMmotion 151 ccRCC cohort treated with the TKR inhibitor Sunitinib showed that female patients in the DKC1-high group was significantly associated with lower response rates (P = 0.021) accompanied by markedly shortened PFS (6.1 vs 14.2 months, P = 0.004). DKC1 and TERC expression correlated positively with each other, and higher TERC expression predicted poor Sunitinib response (P = 0.031) and shorter PFS (P = 0.004), too. However, DKC1 rather than TERC acted as an independent predictor (P < 0.001, HR = 2.0, 95% CI 1.480-2.704). In male patients, DKC1 expression was associated with neither Sunitinib response (P = 0.131) nor PFS (P = 0.184), while higher TERC levels did not predict response rates. Similar results were obtained from the analysis of the Sunitinib-treated IMmotion 150 ccRCC patients. CONCLUSIONS: DKC1 serves as an independent female-specific predictor for survival and Sunitinib efficacy in ccRCC, which contribute to better understanding of the sex-biased ccRCC pathogenesis and improve personalized interventions of ccRCC.

Many types of cancer including clear cell renal cell carcinoma (ccRCC) are known to display sex-biased survival, genomic alterations and treatment efficacy; however, clinical managements are largely identical in male and female ccRCC patients. Many molecules have been identified as predictors for ccRCC survival and response to therapeutic drugs, such as multitargeted tyrosine-kinase receptor inhibitor Sunitinib, but little is known about their sex-specificity. Dyskerin (DKC1), encoded by the DKC1 gene on X chromosome, is a telomerase co-factor stabilizing telomerase RNA component (TERC), whereas telomerase plays key roles in cancer development and progression. In this study, we observed increased DKC1 expression in ccRCC tumors. High DKC1 expression predicts shorter disease progression-free survival (PFS) in female but not male patients. Oncogene activation and tumor suppressor inactivation are more frequent in the female DKC1-high tumors. By analyzing two cohorts of ccRCC patients treated with Sunitinib, we showed that female patients in the DKC1-high group was significantly associated with lower response rates accompanied by markedly shortened PFS. DKC1 and TERC expression correlated positively with each other, and higher TERC expression predicted poor Sunitinib response and shorter PFS, too. However, DKC1 rather than TERC acted as an independent predictor. In male patients, DKC1 expression was associated with neither Sunitinib response nor PFS. Thus, DKC1 serves as a female-specific predictor for survival and Sunitinib response in ccRCC. Our findings are expected to improve personalized management of ccRCC.

AC125611.3 promotes the progression of colon cancer by recruiting DKC1 to stabilize CTNNB1.

BACKGROUND AND STUDY AIMS: Previous studies have suggested that lncRNAs impact cancer progression. The lncRNA AC125611.3 (also referred to as RP11-161H23.5) is highly expressed in colon cancer but rarely studied; understanding its regulation may provide novel insights on treating colon cancer. MATERIALS AND METHODS: qRT-PCR was performed to quantify RNAs. CCK-8 and EdU assays were performed to assess cell proliferation. Western blot analysis was used to detect levels of proteins related to cell apoptosis and EMT. Wound healing assay and Transwell invasion assay were conducted to evaluate cell migratory and invasive capabilities, respectively. Luciferase reporter assay, RIP assay, and pull-down assay were used to verify RNA-RNA and RNA-protein interactions. RESULTS: AC125611.3 was highly overexpressed in colon cancer cells. AC125611.3 depletion curbed cell proliferative, invasive, migratory, and EMT processes while enhancing apoptosis. Furthermore, AC125611.3 activated the Wnt signaling pathway in colon cancer cells by regulating catenin beta-1 (CTNNB1). Moreover, AC125611.3 recruited dyskeratosis congenita 1 (DKC1) to stabilize CTNNB1. CONCLUSION: AC125611.3 recruits DKC1 to stabilize CTNNB1 and activate Wnt signaling, thereby promoting the progression of colon cancer.

Patient-Induced Pluripotent Stem Cell-Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies.

BACKGROUND & AIMS: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. METHODS: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. RESULTS: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. CONCLUSIONS: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

New Insights into Dyskerin-CypA Interaction: Implications for X-Linked Dyskeratosis Congenita and Beyond.

The highly conserved family of cyclophilins comprises multifunctional chaperones that interact with proteins and RNAs, facilitating the dynamic assembly of multimolecular complexes involved in various cellular processes. Cyclophilin A (CypA), the predominant member of this family, exhibits peptidyl-prolyl cis-trans isomerase activity. This enzymatic function aids with the folding and activation of protein structures and often serves as a molecular regulatory switch for large multimolecular complexes, ensuring appropriate inter- and intra-molecular interactions. Here, we investigated the involvement of CypA in the nucleus, where it plays a crucial role in supporting the assembly and trafficking of heterogeneous ribonucleoproteins (RNPs). We reveal that CypA is enriched in the nucleolus, where it colocalizes with the pseudouridine synthase dyskerin, the catalytic component of the multifunctional H/ACA RNPs involved in the modification of cellular RNAs and telomere stability. We show that dyskerin, whose mutations cause the X-linked dyskeratosis (X-DC) and the Hoyeraal-Hreidarsson congenital ribosomopathies, can directly interact with CypA. These findings, together with the remark that substitution of four dyskerin prolines are known to cause X-DC pathogenic mutations, lead us to indicate this protein as a CypA client. The data presented here suggest that this chaperone can modulate dyskerin activity influencing all its partecipated RNPs.