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Prognostic markers and DNA methylation profiling in lymphoid malignanciesBhoi, Sujata January 2017 (has links)
In recent years, great progress has been achieved towards identifying novel biomarkers in lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), at the genomic, transcriptomic and epigenomic level for accurate risk-stratification and prediction of treatment response. In paper I, we validated the prognostic relevance of a recently proposed RNA-based marker in CLL, UGT2B17, and analyzed its expression levels in 253 early-stage patients. Besides confirming its prognostic impact in multivariate analysis, we could identify 30% of IGHV-mutated CLL (M-CLL) cases with high expression and poor outcome, which otherwise lacked any other poor-prognostic marker. In paper II, we investigated the prognostic impact of a previously reported 5 CpG signature that divides CLL patients into three clinico-biological subgroups, namely naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL) and intermediate CLL (i-CLL), in 135 CLL patients using pyrosequencing. We validated the signature as an independent marker in multivariate analysis and further reported that subset #2 cases were predominantly classified as i-CLL, although displaying a similar outcome as n-CLL. In paper III, we investigated the methylation status and expression level of miR26A1 in both CLL (n=70) and MCL (n=65) cohorts. High miR26A1 methylation was associated with IGHV-unmutated (U-CLL) and shorter overall survival (OS) in CLL, while it was uniformly hypermethylated in MCL. Furthermore, overexpression of miR26A1 resulted in significant downregulation of EZH2 that in turn led to increased apoptosis. In paper IV, we performed DNA methylation profiling in 176 CLL cases assigned to one of 8 major stereotyped subsets (#1-8) in relation to non-subset CLL (n=325) and different normal B-cell subpopulations. Principal component analysis of subset vs. non-subset CLL revealed that U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL, respectively, indicating common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL subgroup, which in turn alludes to a distinct cellular origin for subset #2 and i-CLL patients. Altogether, this thesis confirms the prognostic significance of RNA and epigenetic-based markers in CLL, provides insight into the mechanism of miRNA deregulation in lymphoid malignancies and further unravels the DNA methylation landscape in stereotyped subsets of CLL.
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Recurrent Genetic Mutations in Lymphoid MalignanciesYoung, Emma January 2017 (has links)
In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.
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Molecular Insights into Lymphoid Malignancy : Role of Transcription Factor BCL11B in T-cell Leukemia Genesis and Biochemical Characterization of DNA Binding Domain of RAG1Deepthi, R January 2017 (has links) (PDF)
The lymphoid tissues consist of distinct cell subpopulations of B and T cell lineages and possess complex signaling pathways that are controlled by a myriad of molecular interactions. During the fine-tuned developmental process of the lymphoid system, inappropriate activation of oncogenes and loss of tumor suppressor gene activity can push lymphocytes into uncontrolled clonal expansion, causing several lymphoid malignancies. V(D)J recombination is one such essential process, important for the proper development of the mammalian immune system. However, mistakes in normal V(D)J recombination can lead to deletion of tumor suppressor genes or activation of proto-oncogenes. In the first part of the study, the physiological and pathological roles of DNA binding domain of RAG1 have been characterized.
RAG (Recombination Activating Gene) complex consisting of RAG1 and RAG2, is a site specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves a specific DNA sequence termed as recombination signal sequence (RSS), comprising of a conserved heptamer and nonamer. Recent studies have shown that RAGs can also act as a structure-specific nuclease by cleaving flaps, heterologous loops, bubbles, hairpins etc. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS during its sequence specific activity. To investigate its DNA binding properties, NBD of murine RAG1 was cloned, overexpressed and purified from E. coli. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS. However, it did not bind to heteroduplex DNA, irrespective of the sequence of the single-stranded region. Interestingly, when a nonamer was present next to a heteroduplex DNA, NBD exhibited robust binding. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G and T were used. Biolayer interferometry studies showed that the observed poly T binding to NBD was robust with a binding constant of 0.45±0.16 µM. >23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C stretches or random sequences. Although NBD is indispensable for sequence-specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore the sequence specific activity, suggesting that the overall domain architecture of RAG1 is important for maintaining its properties. Therefore, we define the sequence requirements of NBD binding to double- and single-stranded DNA, which will have implications in generation of chromosomal rearrangement and genomic instability in lymphoid cells.
Genetic alterations are one of the hallmarks of lymphoid malignancies. Many genes involved in chromosomal abnormalities are known to play central roles in the development of normal lymphocytes. In the second part of the study, molecular mechanism associated with fragility of the transcription factor, B cell leukemia 11B (BCL11B) that drives malignant transformation of T-cells has been studied. BCL11B is a zinc finger protein transcription factor with multiple functions. It plays a key role in both development and subsequent maintenance of T-cells. BCL11B gene alterations are implicated in a number of diseases including T-cell malignancies. It acts as a haplo-insufficient tumor suppressor and loss of BCL11B allele leads to susceptibility to mouse thymic lymphoma and human T-ALL. Recent studies reveal heterozygous BCL11B mutations and deletions across each of the major molecular subtypes of T-ALL (15% of patients). Most of the BCL11B missense mutations identified so far affected the residues within BCL11B zinc finger domains of the exon 4. However, mechanism of generation of such specific mutations leading to altered functions of BCL11B remains to be explored.
In the present study, we address the potential mechanism of fragility of BCL11B gene during leukemia genesis. Firstly, we have evaluated different regions of BCL11B gene for presence of non-B DNA sequence motifs. Studies using non-B DB database reveal clustering of several non-B DNA forming motifs at the region spanning exon 4 of BCL11B gene. In order to biochemically evaluate the potential of non-B DNA structure formation, two different regions of exon 4 were PCR amplified and cloned. Using bisulfite modification assay we demonstrate that, single strandedness exists at both region I and II of BCL11B exon 4, when the region is present on a plasmid DNA. Bisulfite reactivity on chromosomal DNA confirmed existence of such altered DNA structures in the context of human genome. In vitro gel shift assays showed formation of both intra and intermolecular G-quadruplexes. Primer extension studies revealed that non-B DNA structures could block polymerization during replication on a plasmid, leading to DNA replication arrest. Extrachromosomal assays showed that non-B DNA structure motifs, in contrast to its mutants, blocked transcription leading to reduced expression of green fluorescent protein (GFP) within cells.
Many non-B DNA-forming sequences have been mapped to regions of common chromosomal breakpoints in human tumors, known as “hotspots”, which are associated with leukemia, lymphomas and genomic disorders. Thus, alternative DNA conformations are believed to contribute to mutations, deletions and other genetic instability, leading to the deregulation of cancer-related genes in malignant diseases such as leukemia and lymphoma. Activation induced cytidine deaminase (AID), is an essential enzyme involved in antibody diversification of immunoglobulin genes. However, aberrant AID expression in B- cell and non-B cell background is reported in various cancers including leukemia and lymphoma. AID activity requires single stranded DNA (ssDNA) as a substrate. Since activation induced cytidine deaminase (AID) deaminates cytosines when present on a single stranded DNA and its expression is deregulated in many cancers, we investigated the role of AID in BCL11B gene mutagenesis. We observed substantial AID expression in many T-cell leukemic cell lines. Thus, we hypothesize that AID might be targeted to single stranded DNA present at BCL11B exon 4 due to formation of non-B DNA structures such as G-quadruplexes causing AID mediated deamination, further leading to nucleotide alterations and the mutational signature observed at BCL11B exon 4 resulting in T-ALL. Based on our findings, we propose that single strandedness resulted due to formation of non-B DNA structures such as G-quadruplex DNA, triplex DNA or cruciform DNA during physiological processes like DNA replication and transcription at exon 4 of BCL11B, can act as the target for AID. Thus, our findings uncover a new possible link between non-B DNA structure motifs and AID expression in causing mutations at BCL11B exon 4 which could lead to T cell leukemia genesis.
BCL11B is a bifunctional transcriptional regulator that can act as a repressor and transactivator, and is known to differentially control the expression of specific genes in a context-dependent manner. In order to understand the transcriptional network involving BCL11B, it was cloned, overexpressed and purified from E. coli. To investigate the DNA binding properties of BCL11B protein, electrophoretic mobility shift assays were performed. Our results lead to identification of a specific sequence motif that is responsible for DNA binding. Competition experiments in presence of specific and nonspecific oligomers further confirmed the binding specificity.
Thus, in the present study, we have characterized the binding properties of nonamer binding domain of RAG1, emphasizing its pathological relevance in causing genomic instability in lymphoid cells. The study may help in better understanding of RAG induced genomic instability in lymphoid tissues and role of aberrant AID expression in inducing mutations at BCL11B Zinc finger domain, leading to its deregulation and culminating into T-cell leukemia
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