scholarly journals RNA-sequencing analysis in B-cell acute lymphoblastic leukemia reveals aberrant gene expression and splicing alterations

2017 ◽  
Author(s):  
◽  
Olha Kholod
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Rna Sequencing ◽  
Lymphoblastic Leukemia ◽  
Statistical Testing ◽  
Rna Seq ◽  
Healthy Donors ◽  
Cell Acute Lymphoblastic Leukemia

Background: B-cell acute lymphoblastic leukemia (B-ALL) is a neoplasm of immature lymphoid progenitors and is the leading cause of cancer-related death in children. The majority of B-ALL cases are characterized by recurring structural chromosomal rearrangements that are crucial for triggering leukemogenesis, but do not explain all incidences of disease. Therefore, other molecular mechanisms, such as alternative splicing and epigenetic regulation may alter expression of transcripts that are associated with the development of B-ALL. To determine differentially expressed and spliced RNA transcripts in precursor B-cell acute lymphoblastic leukemia patients a high throughput RNA-seq analysis was performed. Methods: Eight B-ALL patients and eight healthy donors were analyzed by RNA-seq analysis. Statistical testing was performed in edgeR. Each annotated gene was mapped to its corresponding gene object in the Ingenuity KB. Analysis of RNA-seq data for splicing alterations in B-ALL patients and healthy donors was performed with custom Perl script. Results: Using edgeR analysis, 3877 DE genes between B-ALL patients and healthy donors based on TMM (trimmed mean of M-values) normalization method and false discovery rate, FDR less than 0.01, logarithmically transformed fold changes, logFC greater than 2) were identified. IPA revealed abnormal activation of ERBB2, TGFB1 and IL2 transcriptional factors that are crucial for maintaining proliferation and survival potential of leukemic 26 cells. B-ALL specific isoforms were observed for genes with roles in important canonical signaling pathways, such as oxidative phosphorylation and mitochondrial dysfunction. A mechanistic study with the Nalm 6 cell line revealed that some of these gene isoforms significantly change their expression upon 5-Aza treatment, suggesting that they may be epigenetically regulated in B-ALL. Conclusion: Our data provide new insights and perspectives on the regulation of the transcriptome in B-ALL. In addition, we identified transcript isoforms and pathways that may play key roles in the pathogenesis of B-ALL. These results further our understanding of the transcriptional regulation associated with B-ALL development and will contribute to the development of novel strategies aimed towards improving diagnosis and managing patients with B-ALL. Keywords: B-ALL, RNA-sequencing, differential gene expression, alternative splicing.

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (5) ◽  
pp. 2683
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

scholarly journals Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 549-549
Author(s):  
Jason H. Rogers ◽  
Rohit Gupta ◽  
Jaime M. Reyes ◽  
Lorenzo Brunetti ◽  
Michael C. Gundry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Sanger Sequencing ◽  
Lymphoblastic Leukemia ◽  
Clonal Cell ◽  
Ribonucleoprotein Complexes ◽  
Clonal Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models. We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D). We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/-clonal cell lines are distinct from their isogenic IKZF1-/-counterparts. Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/-cells and IKZF1IK6/+cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. Disclosures No relevant conflicts of interest to declare.

scholarly journals ALLSorts: a RNA-Seq classifier for B-Cell Acute Lymphoblastic Leukemia.

2021 ◽  
Author(s):  
Breon M Schmidt ◽  
Lauren M Brown ◽  
Georgina L Ryland ◽  
Andrew Lonsdale ◽  
Hansen J Kosasih ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Learning Algorithm ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Supervised Machine Learning ◽  
World Health ◽  
Rna Seq ◽  
Cancer Subtypes ◽  
Cell Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Subtypes within B-ALL are distinguished by characteristic structural variants and mutations, which in some instances strongly correlate with responses to treatment. The World Health Organisation (WHO) recognises seven distinct classifications, or subtypes, as of 2016. However, recent studies have demonstrated that B-ALL can be segmented into 23 subtypes based on a combination of genomic features and gene expression profiles. A method to identify a patient's subtype would have clear clinical utility. Despite this, no publically available classification methods using RNA-Seq exist for this purpose. Here we present ALLSorts: a publicly available method that uses RNA-Seq data to classify B-ALL samples to 18 known subtypes and five meta-subtypes. ALLSorts is the result of a hierarchical supervised machine learning algorithm applied to a training set of 1223 B-ALL samples aggregated from multiple cohorts. Validation revealed that ALLSorts can accurately attribute samples to subtypes and can attribute multiple subtypes to a sample. Furthermore, when applied to both paediatric and adult cohorts, ALLSorts was able to classify previously undefined samples into subtypes. ALLSorts is available and documented on GitHub (https://github.com/Oshlack/AllSorts/).

Identifying IGH disease clones for MRD monitoring in childhood B-cell acute lymphoblastic leukemia using RNA-Seq

Leukemia ◽  
2020 ◽  
Vol 34 (9) ◽  
pp. 2418-2429 ◽  
Author(s):  
Zhenhua Li ◽  
Nan Jiang ◽  
Evelyn Huizi Lim ◽  
Winnie Hui Ni Chin ◽  
Yi Lu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals Progenitor B-1 B-cell acute lymphoblastic leukemia is associated with collaborative mutations in 3 critical pathways

2017 ◽  
Vol 1 (20) ◽  
pp. 1749-1759 ◽  
Author(s):  
Sheryl M. Gough ◽  
Liat Goldberg ◽  
Marbin Pineda ◽  
Robert L. Walker ◽  
Yuelin J. Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Lymphoblastic Leukemia ◽  
Critical Pathways ◽  
Key Points ◽  
Cell Acute Lymphoblastic Leukemia

Key Points An NUP98-PHF23 fusion collaborates with acquired Bcor and Jak/Stat mutations to produce a pro–B-1 ALL. Gene expression profile of murine pro–B-1 ALL resembles that of a subset of human ALL, suggesting some human ALLs arise from pro–B-1 B cells.

scholarly journals Transcriptional landscape of B cell precursor acute lymphoblastic leukemia based on an international study of 1,223 cases

2018 ◽  
Vol 115 (50) ◽  
pp. E11711-E11720 ◽  
Author(s):  
Jian-Feng Li ◽  
Yu-Ting Dai ◽  
Henrik Lilljebjörn ◽  
Shu-Hong Shen ◽  
Bo-Wen Cui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Rna Sequencing ◽  
Large Scale ◽  
Lymphoblastic Leukemia ◽  
International Study ◽  
Disease Classification ◽  
Cell Precursor

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3–PBX1 fusions, ETV6–RUNX1–positive/ETV6–RUNX1–like, DUX4 fusions, ZNF384 fusions, BCR–ABL1/Ph–like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH–CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4–HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

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