Regeneration pattern of precursor-B-cells in bone marrow of acute lymphoblastic leukemia patients depends on the type of preceding chemotherapy

Abstract Abstract 1466 Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures: No relevant conflicts of interest to declare.

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Pediatric Acute Lymphoblastic Leukemia Outcome Predictor Genes OPAL1 and RANTES Modulate Transformation by the V-Abl Oncogene

Blood ◽

10.1182/blood.v112.11.5318.5318 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5318-5318

Author(s):

Jason H. Rogers ◽

Kristin S. Owens ◽

Jake M. Vargas ◽

Cheryl L. Willman ◽

Robert Hromas ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Lymph Nodes ◽

B Cell ◽

Drug Targets ◽

Lymphoblastic Leukemia ◽

Novel Drug ◽

Novel Drug Targets

Abstract B-precursor acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. While it represents a highly curable malignancy, a significant number of children still relapse or present with disease that is resistant to therapeutic intensification. With the increasing intensity of curative treatment, there is also an increased incidence of late effects that adversely affect the quality of life of survivors. Previously, a large scale microarray gene expression analysis was undertaken in order to identify genes predictive of outcome that could enhance risk classification, thereby identifying children who might be cured with less intensive therapy or those who fail current regimens and require novel therapies for cure. From analysis of 254 pediatric ALL samples registered to various COG clinical trials, gene expression classifiers were identified that are highly predictive of poor and favorable outcome in precursor B-cell ALL. The goal of this project is to study the biologic function and role in hematopoiesis and leukemogenesis of the genes predictive of outcome and to determine whether any of these genes may serve as novel drug targets. To this end, we have developed a system to examine the effect of these genes on the cancer-promoting activity of the v-Abl oncogene. We have established pre-B cell lines by infecting primary mouse bone marrow cells with a virus expressing v-Abl. These lines were then engineered to express two of the genes we identified that were associated with poor or good treatment outcomes, RANTES (CCL5) and OPAL1 respectively. Consistent with the association of RANTES expression with poor outcome, v-Abl/RANTES pre-B cells are more proliferative than v-Abl cells, and form larger colonies in methylcellulose cultures without added cytokines. OPAL1 had the opposite effect on pre-B cell growth. V-Abl pre-B cells expressing OPAL1 were less proliferative and generated fewer colonies in methylcellulose cultures. To determine whether RANTES made the v-Abl pre-B cells more leukemic, we transplanted v-Abl or v-Abl/RANTES pre-B cells into syngeneic mice. Mice were noticeably ill 30 days post-transplant. Mice injected with v-Abl/RANTES cells had enlarged lymph nodes and increased numbers of white cells in their peripheral blood. The percentage of pre-B cells in the bone marrow, lymph nodes and spleen was higher in v-Abl/RANTES transplanted mice compared to v-Abl transplanted mice. Currently our data demonstrates that the outcome genes are playing a mechanistic role in the susceptibility of leukemic cells to therapy, and are not merely epiphenomenon. This suggests that the products of these genes and their associated pathways may be novel drug targets. Such targeted therapy has the potential of being less toxic than current nonspecific treatment regimens.

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Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein

Blood ◽

10.1182/blood.v81.4.1025.bloodjournal8141025 ◽

1993 ◽

Vol 81 (4) ◽

pp. 1025-1031 ◽

Cited By ~ 1

Author(s):

D Campana ◽

E Coustan-Smith ◽

A Manabe ◽

M Buschle ◽

SC Raimondi ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Prolonged Survival ◽

Allogeneic Bone ◽

Immature B Cells ◽

B Lineage

Overexpression of bcl-2 delays the onset of apoptosis in lymphohematopoietic cells. We measured levels of bcl-2 protein in normal and leukemic human B-cell progenitors with a specific monoclonal antibody and flow cytometry. Normal immature B cells had low levels of bcl-2 protein; the intensity of fluorescence, expressed as molecules of soluble fluorochrome per cell, within CD10+ cells was 3,460 +/- 1,050 (mean +/- SD; 5 samples). In 16 cases of B-lineage acute lymphoblastic leukemia (ALL), cells had levels of bcl-2 that were strikingly higher than those of their normal counterparts (33,560 +/- 14,570; P < .001 by t-test analysis). We next investigated whether the intensity of bcl-2 expression correlated with the resistance of immature B cells to in vitro culture. In 12 cases of B-lineage ALL, the cells recovered after 7 days of culture on allogeneic bone marrow stromal layers were 69% to 178% (median, 95.5%) of those originally seeded. Prolonged survival of leukemic cells in vitro was observed even in the absence of stromal layers in 6 of these 12 cases; the intensity of bcl-2 protein expression in these cases was 45,000 +/- 13,270, compared with 21,500 +/- 7,260 in the 6 cases in which greater than 99.5% of cells rapidly died by apoptosis under the same culture conditions (P = .003). Five immature B-cell lines, continuously growing in the absence of stroma, had the highest bcl-2 expression (79,400 +/- 20,330). By contrast, most normal CD19+, sIg-immature B cells died despite the presence of bone marrow stromal layers; 9.7% to 28.2% were recovered after 7 days of culture in three experiments. We conclude that abnormal bcl-2 gene expression influences the survival ability of B-cell progenitors. This may contribute to leukemogenesis and explain the aptitude of leukemic lymphoblasts to expand outside the bone marrow microenvironment.

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Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2012-05-429811 ◽

2012 ◽

Vol 120 (22) ◽

pp. 4407-4417 ◽

Cited By ~ 81

Author(s):

Charles Gawad ◽

Francois Pepin ◽

Victoria E. H. Carlton ◽

Mark Klinger ◽

Aaron C. Logan ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Heavy Chain ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Immunoglobulin Heavy Chain ◽

Igh Locus ◽

Precursor B Cells

Abstract The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

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Haploinsufficiency of Ebf1 Collaborates with BCR-ABL1 to Decrease the Latency of Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v112.11.3363.3363 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3363-3363

Author(s):

Salil Goorha ◽

Noel T. Lenny ◽

Christopher B Miller ◽

S. Scott Perry ◽

Xiaoping Su ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

B Cell Development ◽

B Lineage ◽

B Lymphoid

Abstract In previously published genome-wide copy number analysis of leukemic samples from 242 pediatric acute lymphoblastic leukemia (ALL) patients, we reported that mutations in genes regulating B lymphoid development are the most common lesion in B-progenitor ALL, and these include PAX5, IKZF1, and EBF1. Mono-allelic deletion of EBF1 was observed in 8/200 B-progenitor leukemia samples, including a BCR-ABL1 ALL. EBF1 encodes a transcription factor that is required for the development of B cells, and with E2A regulates the expression of B-lineage specific genes. Mice null for Ebf1 arrest B cell development at the pro-B cell stage, whereas Ebf1+/− mice have a 50% reduction in the number of immature and mature B cells but a normal number of pro-B cells. Importantly, neither haploinsufficiency nor the complete loss of Ebf1 results in the development of leukemia in mice. To examine the role of genetic alterations targeting B-lymphoid differentiation in the pathogenesis in BCR-ABL1 ALL, we transduced Ebf1+/+ and Ebf1+/− bone marrow cells with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus and transplanted the resultant cells into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 Ebf1+/− cells developed B lineage ALLs at a shorter latency than observed with BCR-ABL1 Ebf1+/+ cells (median overall survival of 17 days in Ebf1+/− vs 42 days in Ebf1+/+, P<0.0001). All leukemias had a B220+Cd19+Bp1+ pre-B cell immunophenotype; however, the leukemias that developed from the Ebf1+/− cells aberrantly expressed high levels of the stem cell marker Sca1 (mean fluorescence level for Sca1 of 69.6 in Ebf1+/− (n=22) vs 16.8 in Ebf1+/+ (n=14), p<0.0001). To begin to understand how a decrease in the copy number of Ebf1 may contribute to leukemogenesis, we examined early B cell development in bone marrow (BM) cells from two week-old C57BL6 Ebf1+/− and Ebf1+/+ mice. Our analysis confirmed previous reports indicating a 2-fold reduction of B220+CD43− B cells in Ebf1+/− compared to Ebf1+/+ mice. Interestingly, however, we also detected an approximately 6-fold increase in a transitional population of B220loIL-7R+cKitlo Pre-pro B cells that also expressed Sca1 (2194 mean number of Ebf1+/− cells per 100,000 BM cells (n=10) vs 372 mean number of Ebf1+/+ cells per 100,000 BM cells (n=8), p<0.0001), an observation that raises the possibility that Ebf1 haploinsufficiency expands the pool of cells that are susceptible to transformation by BCR-ABL expression. It will be important to examine whether the accelerated tumorigenesis resulting from Ebf1 haploinsufficiency is a consequence of a subtle shift in differentiation, or some alternative mechanism of oncogenic cooperativity. Studies are underway to directly assess the role of B220loIL-7R+cKitlo Sca1+ cells in BCR-ABL1 driven ALL.

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C/EBPβ expressed By Bone Marrow Mesenchymal Stromal Cells Is Indispensable For Precursor B-Cell Lymphopoiesis

Blood ◽

10.1182/blood.v122.21.1213.1213 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1213-1213

Author(s):

Satoshi Yoshioka ◽

Yasuo Miura ◽

Hisayuki Yao ◽

Masaki Iwasa ◽

Atsushi Sato ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factors ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Stromal Cells ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Transcriptional Factor ◽

Precursor B Cells

Abstract B-cell lymphopoiesis is critically dependent on the bone marrow microenvironment. Early B-cell lymphopoiesis is regulated by direct interaction with bone marrow stromal cells (BM-MSCs) and by soluble factors produced by BM-MSCs. The roles of transcription factors expressed by hematopoietic cells that involve early B-cell lymphopoiesis have been well investigated; those include PU.1, Ikaros, E2A, early B-cell factor, and paired box protein 5. By contrast, transcriptional factors expressed by BM-MSCs that are important for early B-cell lymphopoiesis remain unknown. We show that CCAAT-enhancer binding protein (C/EBP) β expressed by BM-MSCs contributes to the early B-cell lymphopoiesis. In addition, the proliferation of precursor B-cell acute lymphoblastic leukemia (B-ALL) cells is also associated with BM-MSCs, in which C/EBPβ is one of the regulatory transcription factors. When c-kit+ Sca-1+ lineage- (KSL) HSCs from Wild-type (WT) mice were co-cultured with C/EBPβ-deficient BM-MSCs in the presence of stem cell factor (SCF), Flt3-L, and interleukin (IL)-7 (Figure A), the generation of hematopoietic cells from KSL cells was significantly lower than when they were co-cultured with WT BM-MSCs. In addition, the generation of B220+ B-cells from KSL cells was also significantly lower when they were co-cultured with C/EBPβ-deficient BM-MSCs than when co-cultured with WT BM-MSCs. Detailed analysis of the generated B-cell subsets showed that differentiation of KSL cells into precursor B-cells was reduced and differentiation from pre-pro-B-cells to pro-B-cells/pre-BI cells was suppressed when cells were co-cultured with C/EBPβ-deficient BM-MSCs (Figure B). Therefore, C/EBPβ was an indispensable transcriptional factor expressed by BM-MSCs for supporting the differentiation of HSCs into precursor B-cells. We next examined the expression of B-cell lymphopoiesis-associated molecules in C/EBPβ-deficient BM-MSCs by quantitative real-time PCR analysis. Levels of IL-7 and SCF mRNA tended to be lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs, although the difference was not statistically significant in our analysis. Levels of CXCL12/SDF-1 and Flt3-L mRNA were significantly lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs. In addition, the protein concentration of CXCL12/SDF-1 was significantly lower in the culture supernatant of C/EBPβ-deficient BM-MSCs than that of WT BM-MSCs. The concentration of CXCL12/SDF-1 in the supernatant of BM-MSC co-cultures strongly correlated with the number of B220+ B-cells that differentiated from KSL cells. Thus, the impaired differentiation of HSCs into B-cells is associated, at least in part, with reduced production of CXCL12/SDF-1 by C/EBPβ-deficient BM-MSCs. Precursor B-ALL is a hematological disease characterized by malignant transformation of precursor B-cells. We examined whether C/EBPβ expressed by BM-MSCs has effects on the precursor B-ALL cells. When the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with WT BM-MSCs, the number of BaF3/Bcr-Abl cells was significantly increased than when they were cultured alone. Interestingly, when the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with C/EBPβ-deficient BM-MSCs, the number of BaF3/Bcr-Abl cells was reduced to the level similar to when they were cultured alone. This was true in co-cultures with BM-MSCs that were pharmacologically treated to down-regulate the C/EBPβ expression. As well as the correlation of SDF-1 concentration in co-cultures with precursor B-cell differentiation, the proliferation of BaF3/Bcr-Abl cells was associated with the correlation of SDF-1 concentration in co-cultures. In conclusion, this work demonstrates that C/EBPβ expressed by BM-MSCs is a critical transcriptional factor for both the differentiation of physiological precursor B-cells and pathological proliferation of leukemic precursor B-cells. Disclosures: No relevant conflicts of interest to declare.

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CD58 Expression Decreases as Nonmalignant B Cells Mature in Bone Marrow and Is Frequently Overexpressed in Adult and Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

American Journal of Clinical Pathology ◽

10.1309/x5vv6fkjq6mublpx ◽

2005 ◽

Vol 123 (1) ◽

pp. 119-124 ◽

Cited By ~ 15

Author(s):

Ronald V. Lee ◽

Raul C. Braylan ◽

Lisa M. Rimsza

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Acute Lymphoblastic Leukemia

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Infectious stimuli promote malignant B-cell acute lymphoblastic leukemia in the absence of AID

Nature Communications ◽

10.1038/s41467-019-13570-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Guillermo Rodríguez-Hernández ◽

Friederike V. Opitz ◽

Pilar Delgado ◽

Carolin Walter ◽

Ángel F. Álvarez-Prado ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Ex Vivo ◽

Mutagenic Activity ◽

Lymphoblastic Leukemia ◽

Natural Infection ◽

Current Model ◽

Cell Acute Lymphoblastic Leukemia ◽

Precursor B Cells

AbstractThe prerequisite to prevent childhood B-cell acute lymphoblastic leukemia (B-ALL) is to decipher its etiology. The current model suggests that infection triggers B-ALL development through induction of activation-induced cytidine deaminase (AID; also known as AICDA) in precursor B-cells. This evidence has been largely acquired through the use of ex vivo functional studies. However, whether this mechanism governs native non-transplant B-ALL development is unknown. Here we show that, surprisingly, AID genetic deletion does not affect B-ALL development in Pax5-haploinsufficient mice prone to B-ALL upon natural infection exposure. We next test the effect of premature AID expression from earliest pro-B-cell stages in B-cell transformation. The generation of AID off-target mutagenic activity in precursor B-cells does not promote B-ALL. Likewise, known drivers of human B-ALL are not preferentially targeted by AID. Overall these results suggest that infections promote B-ALL through AID-independent mechanisms, providing evidence for a new model of childhood B-ALL development.

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Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein

Blood ◽

10.1182/blood.v81.4.1025.1025 ◽

1993 ◽

Vol 81 (4) ◽

pp. 1025-1031 ◽

Cited By ~ 103

Author(s):

D Campana ◽

E Coustan-Smith ◽

A Manabe ◽

M Buschle ◽

SC Raimondi ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Prolonged Survival ◽

Allogeneic Bone ◽

Immature B Cells ◽

B Lineage

Abstract Overexpression of bcl-2 delays the onset of apoptosis in lymphohematopoietic cells. We measured levels of bcl-2 protein in normal and leukemic human B-cell progenitors with a specific monoclonal antibody and flow cytometry. Normal immature B cells had low levels of bcl-2 protein; the intensity of fluorescence, expressed as molecules of soluble fluorochrome per cell, within CD10+ cells was 3,460 +/- 1,050 (mean +/- SD; 5 samples). In 16 cases of B-lineage acute lymphoblastic leukemia (ALL), cells had levels of bcl-2 that were strikingly higher than those of their normal counterparts (33,560 +/- 14,570; P < .001 by t-test analysis). We next investigated whether the intensity of bcl-2 expression correlated with the resistance of immature B cells to in vitro culture. In 12 cases of B-lineage ALL, the cells recovered after 7 days of culture on allogeneic bone marrow stromal layers were 69% to 178% (median, 95.5%) of those originally seeded. Prolonged survival of leukemic cells in vitro was observed even in the absence of stromal layers in 6 of these 12 cases; the intensity of bcl-2 protein expression in these cases was 45,000 +/- 13,270, compared with 21,500 +/- 7,260 in the 6 cases in which greater than 99.5% of cells rapidly died by apoptosis under the same culture conditions (P = .003). Five immature B-cell lines, continuously growing in the absence of stroma, had the highest bcl-2 expression (79,400 +/- 20,330). By contrast, most normal CD19+, sIg-immature B cells died despite the presence of bone marrow stromal layers; 9.7% to 28.2% were recovered after 7 days of culture in three experiments. We conclude that abnormal bcl-2 gene expression influences the survival ability of B-cell progenitors. This may contribute to leukemogenesis and explain the aptitude of leukemic lymphoblasts to expand outside the bone marrow microenvironment.

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Global DNA methylation and gene expression analysis in pre-B cell acute lymphoblastic leukemia

10.32469/10355/47112 ◽

2015 ◽

Author(s):

◽

Md Almamun

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Expression Analysis ◽

Lymphoblastic Leukemia ◽

Regulatory Elements ◽

Differentially Methylated Regions ◽

Precursor B Cells

Acute lymphoblastic leukemia (ALL) is a hematological cancer associated with precursor B-cells and is the most common cancer diagnosed in children under the age of 15. Our complete understanding of all mechanisms responsible for ALL induction is inadequate. DNA methylation is an epigenetic modification and is accountable for regulating gene expression and plays a significant role in hematopoiesis. In addition to genomic rearrangements and hyperdiploidy, other mechanisms like alteration of DNA methylation may be involved in ALL pathogenesis. Therefore, the identification of altered DNA methylation on key regulatory regions of the genome is critical to gaining a better understanding of ALL pathogenesis. This dissertation identified the dynamic establishment of DNA methylation during normal B-cell development, and alterations of DNA methylation in the pathogenesis of ALL. First, a protocol to isolate the subsets of precursor B-cells from human umbilical cord blood (HCB) was developed. Using this protocol, we were able to isolate sufficient numbers of pro-B, pre-BI, pre-BII and naive B-cells from a single HCB unit. This method can be adapted for any type of cell present in HCB at any stage of differentiation. Next, genome-wide DNA methylation profiles using the methylated CpG island recovery assay followed by next generation sequencing (MIRA-seq) were generated for each of the four subsets of precursor B-cells. We report for the first time that gaining of DNA methylation in certain regions of genome were associated with the transition of pro-B to pre-BI cells. Differentially methylated regions were identified and the majority of the regions were present within intronic and intergenic regions that harbor putative regulatory elements. The development of methylation profiles in normal precursor B-cells will aid in revealing the role of altered DNA methylation in the pathogenesis of precursor B-cell related disorders including ALL. In order to identify epigenetic alterations in ALL, we next determined the differentially methylated regions (DMRs) between ALL and healthy precursor B-cells. Further, whole genome genes expression analysis was performed to determine the regulatory potential of the DMRs. Our studies identified ALL specific epigenetically deregulated genes, novel putative enhancers, and biological pathways that showed concurrent DNA methylation and changes in gene expression during malignant transformation. These altered epigenetic marks could be used as prospective biomarkers for ALL and/or as potential targets to reset the altered DNA methylation in order to modify gene expression which may enhance the present treatment protocol for ALL and eventually improve patient outcome."

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