Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Study of the properties of carotenoids and key carotenoid biosynthesis genes from Deinococcus xibeiensis R13.

To investigate the properties of carotenoids from the extremophile Deinococcus xibeiensis R13, the factors affecting the stability of carotenoids extracted from D. xibeiensis R13, including temperature, illumination, pH, redox chemicals, metal ions, and food additives, were investigated. The results showed that low temperature, neutral pH, reducing agents, Mn2+ , and food additives (xylose and glucose) can effectively improve the stability of Deinococcus carotenoids. The carotenoids of D. xibeiensis R13 exhibited strong antioxidant activity, with the scavenging rate of hydroxyl radicals reaching 71.64%, which was higher than the scavenging efficiency for 1,1-diphenyl-2-picrylhydrazyl free radicals and 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) free radicals (44.55 and 27.65%, respectively). In addition, the total antioxidant capacity reached 0.60 U/ml, which was 2.61-fold that of carotenoids from the model strain Deinococcus radiodurans R1. Finally, we predicted the gene clusters encoding carotenoid biosynthesis pathways in the genome of R13 and identified putative homologous genes. The key enzyme genes (crtE, crtB, crtI, crtLm, cruF, crtD, and crtO) in carotenoid synthesis of D. xibeiensis R13 were cloned to construct the multigene coexpression plasmids pET-EBI and pRSF-LmFDO. The carotenoid biosynthesis pathway was heterologously introduced into engineered Escherichia coli EBILmFDO, which exhibited a higher yield (7.14 mg/L) than the original strain. These analysis results can help us to better understand the metabolic synthesis of carotenoids in extremophiles.

The landscape of coding RNA editing events in pediatric cancer.

BACKGROUND: RNA editing leads to post-transcriptional variation in protein sequences and has important biological implications. We sought to elucidate the landscape of RNA editing events across pediatric cancers. METHODS: Using RNA-Seq data mapped by a pipeline designed to minimize mapping ambiguity, we investigated RNA editing in 711 pediatric cancers from the St. Jude/Washington University Pediatric Cancer Genome Project focusing on coding variants which can potentially increase protein sequence diversity. We combined de novo detection using paired tumor DNA-RNA data with analysis of known RNA editing sites. RESULTS: We identified 722 unique RNA editing sites in coding regions across pediatric cancers, 70% of which were nonsynonymous recoding variants. Nearly all editing sites represented the canonical A-to-I (n = 706) or C-to-U sites (n = 14). RNA editing was enriched in brain tumors compared to other cancers, including editing of glutamate receptors and ion channels involved in neurotransmitter signaling. RNA editing profiles of each pediatric cancer subtype resembled those of the corresponding normal tissue profiled by the Genotype-Tissue Expression (GTEx) project. CONCLUSIONS: In this first comprehensive analysis of RNA editing events in pediatric cancer, we found that the RNA editing profile of each cancer subtype is similar to its normal tissue of origin. Tumor-specific RNA editing events were not identified indicating that successful immunotherapeutic targeting of RNA-edited peptides in pediatric cancer should rely on increased antigen presentation on tumor cells compared to normal but not on tumor-specific RNA editing per se.

Acute depletion of CTCF directly affects MYC regulation through loss of enhancer-promoter looping.

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF's direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF's loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

Pathway engineering of Saccharomyces cerevisiae for efficient lycopene production.

To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100 mg/L of linolenic acid reached 10.12 mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids.

Design and tailoring of an artificial DNA scaffolding system for efficient lycopene synthesis using zinc-finger-guided assembly.

A highly efficient lycopene production system was constructed by assembling enzymes fused to zinc-finger motifs on DNA scaffolds in vitro and in vivo. Three key enzymes of the lycopene synthesis pathway, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, were fused with zinc-finger proteins, expressed and purified. Recombinant plasmids of the pS series containing DNA scaffolds that the zinc-finger proteins can specifically bind to were constructed. In the in vitro system, the production efficiency of lycopene was improved greatly after the addition of the scaffold plasmid pS231. Subsequently, the plasmid pET-AEBI was constructed and introduced into recombinant Escherichia coli BL21 (DE3) for expression, together with plasmids of the pS series. The lycopene production rate and content of the recombinant strain pp231 were higher than that of all strains carrying the DNA scaffold and the control. With the addition of cofactors and substrates in the lycopene biosynthesis pathway, the lycopene yield of pp231 reached 632.49 mg/L at 40 h, representing a 4.7-fold increase compared to the original recombinant strain pA1A3. This DNA scaffold system can be used as a platform for the construction and production of many biochemicals synthesized via multi-enzyme cascade reactions.

Complete genome sequence of Janthinobacterium sp. B9-8, a violacein-producing bacterium isolated from low-temperature sewage.

Janthinobacterium sp. B9-8, isolated from low temperature-sewage in Xinjiang, China, is capable of producing violacein, a promising antibiotic. Here we report the genome sequence of B9-8, which consist of 4,726,850 bp with a G + C content of 48.72%. The violacein biosynthesis gene cluster vioABCDE was identified and analyzed based on the genomic data, which revealed relatively low query coverage (3-44%) and identity (66-87%) with existing strains. Janthinobacterium sp. B9-8 grew fast and reached a high cell density and violacein content within 24 h at 25 °C. The availability of this genome sequence will greatly benefit the industrial production of violacein and facilitate supplementary studies on the mechanism for violacein biosynthesis.

Optimization of fermentation conditions for carotenoid production in the radiation-resistant strain Deinococcus xibeiensis R13.

Deinococcus xibeiensis R13 was isolated from an extreme environment in Xinjiang, China, and can resist gamma-radiation and UV-irradiation. In this study, D. xibeiensis R13 was shown to be capable of efficiently producing carotenoids in culture, and factors influencing its productivity were identified. The maximum carotenoid yield was observed at an initial temperature of 30 °C and pH 7.0 in the presence of fructose, tryptone at a C/N ratio of 1:5, and 10 µM Fe2+. The carotenoid yield under modified culture conditions was 6.64 mg/L after fermentation for 48 h, representing an increase of 84% compared to the original conditions. The biomass reached 7.22 g/L, which was 2.19-fold higher than under non-optimized conditions. The produced carotenoids were extracted from R13 and analyzed by UPLC-MS. This is the first study of carotenoid production by the new strain D. xibeiensis R13, which provides a new source for the microbial fermentation of natural carotenoids, and also provides a good reference for industrial production of other carotenoids and other terpenoid products.

LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma.

BACKGROUND: Oesophageal cancer is one of the most deadly forms of cancer worldwide. Long non-coding RNAs (lncRNAs) are often found to have important regulatory roles. OBJECTIVE: To assess the lncRNA expression profile of oesophageal squamous cell carcinoma (OSCC) and identify prognosis-related lncRNAs. METHOD: LncRNA expression profiles were studied by microarray in paired tumour and normal tissues from 119 patients with OSCC and validated by qRT-PCR. The 119 patients were divided randomly into training (n=60) and test (n=59) groups. A prognostic signature was developed from the training group using a random Forest supervised classification algorithm and a nearest shrunken centroid algorithm, then validated in a test group and further, in an independent cohort (n=60). The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis. RESULTS: LncRNAs showed significantly altered expression in OSCC tissues. From the training group, we identified a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENST00000547963.1) which classified the patients into two groups with significantly different overall survival (median survival 19.2 months vs >60 months, p<0.0001). The signature was applied to the test group (median survival 21.5 months vs >60 months, p=0.0030) and independent cohort (median survival 25.8 months vs >48 months, p=0.0187) and showed similar prognostic values in both. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with OSCC. Stratified analysis suggested that the signature was prognostic within clinical stages. CONCLUSIONS: Our results suggest that the three-lncRNA signature is a new biomarker for the prognosis of patients with OSCC, enabling more accurate prediction of survival.

Protocol for live-cell imaging of immune synapse formation and activation of CAR T cells against cancer cells.

Immune synapse (IS) formation determines T cell antitumor activity. Here, we present a protocol for characterizing the IS formation between chimeric antigen receptor (CAR) T cells and tumor cells by measuring the IS size and calcium flux by live-cell imaging. We describe steps for CAR T cell manufacturing, sample preparation, image acquisition, and data analysis. For complete details on the use and execution of this protocol, please refer to Chockley et al.,1 Ibanez et al.,2 and Zoine et al.3.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with CAR T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons (CSE) present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify CSE targets, we analyzed 1,532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We found 2,933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n=148) or the alternatively spliced (AS) isoform (n=9) level. Expression of selected AS targets, including the EDB domain of FN1 (EDB), and gene targets, such as COL11A1, were validated in pediatric PDX tumors. We generated CAR T cells specific to EDB or COL11A1 and demonstrated that COL11A1-CAR T-cells have potent antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity.

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

MiR-23a in amplified 19p13.13 loci targets metallothionein 2A and promotes growth in gastric cancer cells.

Copy number variation (CNV) and abnormal expression of microRNAs (miRNAs) always lead to deregulation of genes in cancer, including gastric cancer (GC). However, little is known about how CNVs affect the expression of miRNAs. By integrating CNV and miRNA profiles in the same samples, we identified eight miRNAs (miR-1274a, miR-196b, miR-4298, miR-181c, miR-181d, miR-23a, miR-27a and miR-24-2) that were located in the amplified regions and were upregulated in GC. In particular, amplification of miR-23a-27a-24-2 cluster and miR-181c-181d cluster frequently occurred at 19p13.13 and were confirmed by genomic real-time PCR in another 25 paired GC samples. Moreover, in situ hybridization (ISH) experiments represented that mature miR-23a was increased in GCs (75.5%, 40/53) compared with matched normal tissues (28.6%, 14/49, P = 0.001). Knocking down of miR-23a expression inhibited BGC823 cell growth in vitro and in vivo. In addition, the potential target genes of miR-23a were investigated by integration of mRNA profile and miRNA TargetScan predictions, we found that upregulation of miR-23a and downregulation of metallothionein 2A (MT2A) were detected simultaneously in 70% (7/10) of the miRNA and mRNA profiles. Furthermore, an inverse correlation between miR-23a and MT2A expression was detected in GCs and normal tissues. Through combining luciferase assay, we confirmed that MT2A is a potential target of miR-23a. In conclusion, these results suggest that integration of CNV-miRNA-mRNA profiling is a powerful tool for identifying molecular signatures, and that miR-23a might play a role in regulating MT2A expression in GC.

GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.

Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response.

Etiology of oncogenic fusions in 5,190 childhood cancers and its clinical and therapeutic implication.

Oncogenic fusions formed through chromosomal rearrangements are hallmarks of childhood cancer that define cancer subtype, predict outcome, persist through treatment, and can be ideal therapeutic targets. However, mechanistic understanding of the etiology of oncogenic fusions remains elusive. Here we report a comprehensive detection of 272 oncogenic fusion gene pairs by using tumor transcriptome sequencing data from 5190 childhood cancer patients. We identify diverse factors, including translation frame, protein domain, splicing, and gene length, that shape the formation of oncogenic fusions. Our mathematical modeling reveals a strong link between differential selection pressure and clinical outcome in CBFB-MYH11. We discover 4 oncogenic fusions, including RUNX1-RUNX1T1, TCF3-PBX1, CBFA2T3-GLIS2, and KMT2A-AFDN, with promoter-hijacking-like features that may offer alternative strategies for therapeutic targeting. We uncover extensive alternative splicing in oncogenic fusions including KMT2A-MLLT3, KMT2A-MLLT10, C11orf95-RELA, NUP98-NSD1, KMT2A-AFDN and ETV6-RUNX1. We discover neo splice sites in 18 oncogenic fusion gene pairs and demonstrate that such splice sites confer therapeutic vulnerability for etiology-based genome editing. Our study reveals general principles on the etiology of oncogenic fusions in childhood cancer and suggests profound clinical implications including etiology-based risk stratification and genome-editing-based therapeutics.

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. RESULTS: CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. CONCLUSIONS: These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

Improved side-chain modeling by coupling clash-detection guided iterative search with rotamer relaxation.

MOTIVATION: Side-chain modeling has seen wide applications in computational structure biology. Most of the popular side-chain modeling programs explore the conformation space using discrete rigid rotamers for speed and efficiency. However, in the tightly packed environments of protein interiors, these methods will inherently lead to atomic clashes and hinder the prediction accuracy. RESULTS: We present a side-chain modeling method (CIS-RR), which couples a novel clash-detection guided iterative search (CIS) algorithm with continuous torsion space optimization of rotamers (RR). Benchmark testing shows that compared with the existing popular side-chain modeling methods, CIS-RR removes atomic clashes much more effectively and achieves comparable or even better prediction accuracy while having comparable computational cost. We believe that CIS-RR could be a useful method for accurate side-chain modeling. AVAILABILITY: CIS-RR is available to non-commercial users at our website: http://jianglab.ibp.ac.cn/lims/cisrr/cisrr.html.

Decoding tumor microenvironments through artificial tumor transcriptomes.

In this issue of Cancer Cell, Zaitsev et al. (2022) present a machine-learning-based approach, trained from millions of artificial transcriptomes with admixed cell populations, for reconstructing tumor microenvironments (TMEs). The high accuracy of this approach, demonstrated through extensive validation, enables systematic investigation of TMEs in both research and clinical settings.

Safety and efficacy of hemodialysis and peritoneal dialysis in treating end-stage diabetic nephropathy: a meta-analysis of randomized controlled trials.

BACKGROUND: Present investigation aims to elucidate safety and efficacy of hemodialysis as well as peritoneal dialysis in treating end-stage diabetic nephropathy. METHODS: We searched various databases for articles from the database starting date to October 2019. The analysis involved studies that contained outcomes of hemodialysis and peritoneal dialysis in the treatment of end-stage diabetic nephropathy. A total of 12 randomized controlled trials (RCTs) with 932 participants were collected. RESULTS: Meta-analysis results suggested that comparing with peritoneal dialysis group, hemodialysis group had a higher incidence of cardiovascular and cerebrovascular events and bleeding complications. There was no statistically significant difference regarding the infection (P = 0.71) or malnutrition (P = 0.53) incidence between the two forms of dialysis. Hemodialysis could better improve the levels of albumin [mean difference (MD) = 6.80, 95% CI = (4.17-9.44)] and hemoglobin [MD = 3.40, 95% CI = (0.94-5.86)] than peritoneal dialysis after 3 months or more. CONCLUSIONS: In treating end-stage diabetic nephropathy patients, peritoneal dialysis had a lower incidence of cardiovascular and cerebrovascular events, as well as bleeding complication than hemodialysis. However, hemodialysis could better improve albumin and hemoglobin levels than peritoneal dialysis after 3 months.