Artículos de revistas: "Human B-lineage ALL (B-ALL)"

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Uckun, FM, KJ Gajl-Peczalska, AJ Provisor y NA Heerema. "Immunophenotype-karyotype associations in human acute lymphoblastic leukemia". Blood 73, n.º 1 (1 de enero de 1989): 271–80. http://dx.doi.org/10.1182/blood.v73.1.271.271.

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Abstract The present study is a detailed analysis of the cytogenetic features of leukemic cells from 104 immunologically classified acute lymphoblastic leukemia (ALL) (78 B lineage and 26 T lineage) cases. Clonal chromosomal abnormalities were found in marrow blasts from 77 of 104 (74%) cases. Hyperdiploidy was much more frequent in B-lineage ALL cases, whereas normal diploidy was more common in T-lineage ALL cases. Fifty-nine of 104 cases (46 of 78 B-lineage ALL and 13 of 26 T-lineage ALL cases) had structural chromosomal abnormalities. Structural abnormalities involving 2p11, 7p13, 7p22, proximal q arm of 7 (7q11 or 7q22), 11q23–24, and translocations involving 12p11–13 appeared to be B- lineage specific. By comparison, structural abnormalities involving 7p15, 7q32, and 14q11 displayed T-lineage specificity. Structural abnormalities involving 9p22-p23 or 14q32, del (6)(q21-q23), del (12)(p11-p13), and the Philadelphia chromosome were found in B-lineage as well as T-lineage ALL cases. This study expands the current knowledge about immunophenotype-karyotype associations in ALL.

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Uckun, FM, KJ Gajl-Peczalska, AJ Provisor y NA Heerema. "Immunophenotype-karyotype associations in human acute lymphoblastic leukemia". Blood 73, n.º 1 (1 de enero de 1989): 271–80. http://dx.doi.org/10.1182/blood.v73.1.271.bloodjournal731271.

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The present study is a detailed analysis of the cytogenetic features of leukemic cells from 104 immunologically classified acute lymphoblastic leukemia (ALL) (78 B lineage and 26 T lineage) cases. Clonal chromosomal abnormalities were found in marrow blasts from 77 of 104 (74%) cases. Hyperdiploidy was much more frequent in B-lineage ALL cases, whereas normal diploidy was more common in T-lineage ALL cases. Fifty-nine of 104 cases (46 of 78 B-lineage ALL and 13 of 26 T-lineage ALL cases) had structural chromosomal abnormalities. Structural abnormalities involving 2p11, 7p13, 7p22, proximal q arm of 7 (7q11 or 7q22), 11q23–24, and translocations involving 12p11–13 appeared to be B- lineage specific. By comparison, structural abnormalities involving 7p15, 7q32, and 14q11 displayed T-lineage specificity. Structural abnormalities involving 9p22-p23 or 14q32, del (6)(q21-q23), del (12)(p11-p13), and the Philadelphia chromosome were found in B-lineage as well as T-lineage ALL cases. This study expands the current knowledge about immunophenotype-karyotype associations in ALL.

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3

Uckun, Fatih M., Lei Sun, Hong Ma, Sanjive Qazi, Ilker Dibirdik y Zahide Ozer. "Targeting Human B-Precursor Acute Lymphoblastic Leukemia Cells with Recombinant Human CD19 Ligand". Blood 116, n.º 21 (19 de noviembre de 2010): 599. http://dx.doi.org/10.1182/blood.v116.21.599.599.

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Abstract Abstract 599 CD19 is a 95-kDa B-lineage restricted receptor molecule that functions as a key regulator of transmembrane signals in both B-cells and B-cell precursors. Here we report the cloning and characterization of a novel high-mobility group (HMG)-box protein as the membrane-associated natural ligand of human CD19 receptor (CD19-L) on human immature as well as mature lymphoid cells. We cloned the gene encoding CD19-L from a human thymus cDNA library by yeast two-hybrid screening using the cDNA encoding human CD19 extracellular domain (AA 1 to 273) (CD19ECD) fused to the GAL4 DNA binding domain as the bait plasmid. The cDNA for the surface membrane-associated CD19-L protein is 2290-bp in length encoding a 487-aa protein with a predicted molecular mass of 54-kDa. The comparison of the amino acid sequence of CD19-L protein with reported sequences in Genebank Database revealed that CD19-L is a new member of the HMG-box protein family. CD19-L contains two leucine-rich hydrophobic nuclear export signal (NES) motifs associated with unconventional ER- and Golgi-independent transport of nuclear/cytoplasmic secretory proteins to the surface membrane. Expression of the CD19-L gene expression is limited to the lymphocyte compartment within the human lymphohematopoietic system and particularly abundant in T-lineage cells. CD19-L displays abundant expression on immature double-positive (DP) thymocytes as well as leukemic T-cell precursors from T-lineage ALL patients corresponding to immature double-negative (DN) pro-thymocyte and DP cortico-thymocyte stages of human T-cell ontogeny. CD19-L is also expressed on B-lineage lymphoid cells at all stages of human B-cell ontogeny, including fetal liver derived biphenotypic CD2+CD19+ pro-B/T cells, pro-B cells, pre-B cells and mature B-cells. Soluble recombinant human CD19-L protein produced in a baculovirus expression system exhibited exquisite specificity for the extracellular domain of CD19 and had profound effects on apoptosis-related signaling and gene expression in CD19+ human leukemia cells. Engagement of CD19 co-receptor on B-lineage ALL cells with soluble CD19-L protein perturbed CD19-associated signaling network and triggered tyrosine phosphorylation of CD19 in a time-dependent fashion with peak phosphorylation occurring within 1–5 min. CD19-phosphorylation was associated with rapid and transient activation of SYK tyrosine kinase. Treatment of B-lineage ALL cells with 100 ng/mL CD19-L for 24 h corrupted the regulation of gene expression and altered the expression levels of 13 genes directly involved in regulation of apoptosis. We next examined the ability of CD19-L to induce apoptosis in leukemic B-cell precursors from chemotherapy-resistant CD19-positive human B-lineage ALL cell lines NALM-6 (pre-B ALL), RS4;11 (MLL-AF4+ Pro-B ALL), and ALL-1 (BCR-ABL+ Pre-pre-B ALL). CD19-L (but not control proteins CD19ECD or CD19ICD) caused apoptosis in each of these 3 ALL cell lines. As CD19-L specifically targets CD19ECD, excess CD19ECD protein was able to compete with surface CD19 receptor for CD19-L binding and thereby prevent CD19-L induced apoptosis. Excess CD19 intracellular domain protein (CD19ICD) that was included as a negative control did not affect CD19-L induced apoptosis. CD19-L was also capable of causing apoptosis in chemotherapy-resistant primary leukemic cells from relapsed CD19+ B-lineage ALL patients. This collection of experimental data provides compelling evidence that CD19-L is a potent biotherapeutic new agent candidate against CD19+ lymphoid malignancies. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL as well as other B-lineage lymphoid malignancies by providing an effective alternative to CD19-directed monoclonal antibody-based biotherapeutic agents that have encountered several limitations in clinical settings. Disclosures: No relevant conflicts of interest to declare.

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Dovat, Sinisa, Chunhua Song y Kimberly Payne. "Ikaros directly upregulates transcription of B lineage-specific genes in human B cell leukemia (P4412)". Journal of Immunology 190, n.º 1_Supplement (1 de mayo de 2013): 52.26. http://dx.doi.org/10.4049/jimmunol.190.supp.52.26.

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Abstract Ikaros encodes a DNA-binding protein that functions as a master regulator of hematopoiesis and a tumor suppressor in pre-B acute lymphoblastic leukemia (B-ALL). Loss of Ikaros function is associated with impaired hematopoiesis and high-risk B-ALL in humans. The mechanism of Ikaros tumor suppressor activity is unknown. Using quantitative chromatin immunoprecipitation (qChIP) we demonstrate that Ikaros binds to the promoters of genes required for B cell differentiation in primary leukemia cells. Luciferase reporter assays demonstrated that Ikaros activates transcription of these B lineage-specific genes. Increased expression of Ikaros via retroviral transduction in B-ALL cells resulted in increased transcription of these critical B cell differentiation genes, as well as increased binding of Ikaros to their promoters. The inhibition of Casein Kinase II (CK2) resulted in dephosphorylation of Ikaros and enhanced Ikaros-mediated transcriptional activation of the B lineage-specific genes in B-ALL. Treatment of the pre-B ALL cell line, Nalm6, with a CK2 inhibitor, resulted in increased Ikaros binding to the promoters of B lineage genes, increased transcription of B cell differentiation genes, and cell cycle arrest. Results suggest that Ikaros functions as a positive regulator of B cell differentiation by directly upregulating the transcription of B lineage-specific genes in B-ALL. The presented data suggest that CK2 has a critical role in B cell differentiation and in leukemogenesis.

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5

Sung Min, Woo, Chang W. Song y Fatih M. Uckung. "Thermal sensitivity and thermal tolerance of human b-lineage acute lymphoblastic leukemia (all) cells". International Journal of Radiation Oncology*Biology*Physics 18, n.º 1 (enero de 1990): 147–53. http://dx.doi.org/10.1016/0360-3016(90)90278-r.

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Koss, Brian, Jeffrey Morrison, Rhonda M. Perciavalle, Harpreet Singh, Jerold E. Rehg, Richard T. Williams y Joseph T. Opferman. "Requirement for antiapoptotic MCL-1 in the survival of BCR-ABL B-lineage acute lymphoblastic leukemia". Blood 122, n.º 9 (29 de agosto de 2013): 1587–98. http://dx.doi.org/10.1182/blood-2012-06-440230.

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Key Points Using a mouse genetic mouse model of Ph+ B-lineage ALL, endogenous antiapoptotic MCL-1 is required for leukemia survival. In BCR-ABL+ B-lineage ALL human and mouse cells, combining TKIs with small-molecule inhibitors of BCL-2 can potentiate sensitivity to cell death.

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7

Herfst, Sander, Vicente Mas, Lorena S. Ver, Rutger J. Wierda, Albert D. M. E. Osterhaus, Ron A. M. Fouchier y José A. Melero. "Low-pH-Induced Membrane Fusion Mediated by Human Metapneumovirus F Protein Is a Rare, Strain-Dependent Phenomenon". Journal of Virology 82, n.º 17 (2 de julio de 2008): 8891–95. http://dx.doi.org/10.1128/jvi.00472-08.

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ABSTRACT Membrane fusion promoted by human metapneumovirus (HMPV) fusion (F) protein was suggested to require low pH (R. M. Schowalter, S. E. Smith, and R. E. Dutch, J. Virol. 80:10931-10941, 2006). Using prototype F proteins representing the four HMPV genetic lineages, we detected low-pH-dependent fusion only with some lineage A proteins and not with lineage B proteins. A glycine at position 294 was found responsible for the low-pH requirement in lineage A proteins. Only 6% of all HMPV lineage A F sequences have 294G, and none of the lineage B sequences have 294G. Thus, acidic pH is not a general trigger of HMPV F proteins for activity.

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8

Uckun, FM, K. Gajl-Peczalska, DE Myers, W. Jaszcz, S. Haissig y JA Ledbetter. "Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B- lineage non-Hodgkin's lymphoma cells". Blood 76, n.º 12 (15 de diciembre de 1990): 2449–56. http://dx.doi.org/10.1182/blood.v76.12.2449.bloodjournal76122449.

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Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B- lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence- activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS- sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.

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9

Uckun, Fatih, Ilker Dibirdik, Sanjive Qazi y Seang Yiv. "Therapeutic nanoparticle constructs of a JAK3 tyrosine kinase inhibitor against human B-lineage ALL cells". Arzneimittelforschung 60, n.º 04 (2 de diciembre de 2011): 210–17. http://dx.doi.org/10.1055/s-0031-1296275.

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10

Uckun, FM, K. Gajl-Peczalska, DE Myers, W. Jaszcz, S. Haissig y JA Ledbetter. "Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B- lineage non-Hodgkin's lymphoma cells". Blood 76, n.º 12 (15 de diciembre de 1990): 2449–56. http://dx.doi.org/10.1182/blood.v76.12.2449.2449.

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Abstract Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B- lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence- activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS- sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.

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11

Chen, Z., D. Le Paslier, J. Dausset, L. Degos, G. Flandrin, D. Cohen y F. Sigaux. "Human T cell gamma genes are frequently rearranged in B-lineage acute lymphoblastic leukemias but not in chronic B cell proliferations." Journal of Experimental Medicine 165, n.º 4 (1 de abril de 1987): 1000–1015. http://dx.doi.org/10.1084/jem.165.4.1000.

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T cell rearranging gene gamma (TRG gamma) and T cell antigen receptor beta (TCR beta) chain gene rearrangement and transcription were studied in a series of patients with B-lineage acute lymphoblastic leukemia (ALL), in which the Ig H chain genes are rearranged and the surface phenotype reproduces the stages of normal pre-B maturation. For comparison, polyclonal T cells from peripheral blood of healthy donors and blast cells from 19 cases of T lineage ALL were also studied. In this study we demonstrate the presence of a clonal rearrangement of the TRG gamma in 18 of the 22 B-lineage ALL cases and establish that this rearrangement, which generally involves the J gamma 1 region, is often monoallelic and appears different from the biallelic J gamma 2 rearrangement frequently seen in T-cell ALLs. In 9 of 22 cases, we found rearrangement of the genes of the TCR beta chain, which never involved the J beta 1 region. Conversely, the TRG gamma were seen in germline configuration in all 19 cases of B chronic lymphoid malignancies. In none of the 9 AML cases studied was TRG gamma and TCR beta chain gene rearrangement found. The TCR beta chain genes were rearranged in one B cell chronic lymphocytic leukemia (CLL). We also show that in B-lineage ALL, the cells probably use the same V gamma genes for TRG gamma rearrangements as the malignant cells in T-ALL and the polyclonal T cells. In none of the 13 B-lineage ALL cases investigated by Northern analysis was TCR beta mRNA expression detected, whereas a weak expression of TRG gamma transcripts was found in two of these cases. The correlations between surface phenotype, rearrangement of TRG gamma, TCR beta, and Ig H chain genes were analyzed. The significance of rearrangement of TRG gamma and TCR beta chain genes in B or pre-B cells is also discussed.

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12

Kagoda, Mercy, Yasmin Parrish, Jaqueline Rogerio, Ineavely Baez, Qian-Lin Hao, Lora Barsky, Ewa Zielinska, Monika Smogorzewska y Kimberly Payne. "An All-Human Culture Model Supporting Human B Lymphopoesis". Clinical Immunology 123 (2007): S52. http://dx.doi.org/10.1016/j.clim.2007.03.326.

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Aasheim, Hans-Christian, Leon W. M. M. Terstappen y Ton Logtenberg. "Regulated Expression of the Eph-Related Receptor Tyrosine Kinase Hek11 in Early Human B Lymphopoiesis". Blood 90, n.º 9 (1 de noviembre de 1997): 3613–22. http://dx.doi.org/10.1182/blood.v90.9.3613.

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Abstract Members of the large Eph family of receptor tyrosine kinases (RTKs) display temporally and spatially restricted expression patterns during embryogenesis, suggesting a role in various developmental processes. We have begun to investigate the expression of members of this receptor family during human hematopoiesis, in particular B lymphopoiesis. Expression of Eph RTKs in cells of the B-lymphoid lineage was assessed by using degenerate oligonucleotide primers based on stretches of conserved nucleic acid sequences in members of the Eph family. First, the content of Eph-family RTKs was assessed in freshly sorted fetal bone marrow pro–B cells. This population was found to harbor transcripts of the Hek8 and Hek11 members of this gene family. Subsequent analysis of expression of these genes in B cells representing various differentiation and ontogenic stages showed that the Hek8 transcript is constitutively present in all fetal and adult B-lineage cells, with high levels of expression in peripheral blood B cells. In contrast, the Hek11 transcript was exclusively found in fetal bone marrow pro–B cells and pre–B cells, but not in more mature fetal B-lineage cells. All adult B-lineage cells, from early pro–B cells to end-stage plasma cells, lacked Hek11 transcripts. The developmentally regulated expression of Hek11 during fetal B lymphopoiesis suggests a role for this gene in pre/pro–B cell expansion and/or differentiation and defines a difference in progenitor B cell populations isolated from fetal versus adult human bone marrow.

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Ishii, Eiichi, Yujiro Sadakane, Masafumi Zaitsu, Masanori Nishi, Kanji Sugita, Shuki Mizutani, Akinobu Matsuzaki, Eizaburo Sueoka y Yuhei Hamasaki. "Aberrant Pax5 Production with Deletion of Exon 8 in Childhood B-Lineage Acute Lymphoblastic Leukemia." Blood 108, n.º 11 (16 de noviembre de 2006): 712. http://dx.doi.org/10.1182/blood.v108.11.712.712.

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Abstract Background. B-lineage acute lymphoblastic leukemia (ALL) is a common subtype of childhood leukemia. Among numerous transcription factors that are required for B-cell development, Pax5 regulates the expression of B-cell-specific genes including BLNK, CD19, LEF-1, blk and mb-1 of the Ig genes, and plays a central role in B-cell development and differentiation. Recently, it has been reported that aberrant expression of Pax5 is implicated in the development of a certain type of B-cell lymphomas. These observations suggest that the alteration of Pax5 function might contribute to leukemogenesis by aberrantly regulating the differentiation of early B-cell progenitors. Procedure. Leukemic cells in children with B-lineage ALL, B-lineage ALL cell lines, and normal B lymphocytes from different age groups were used for the study. RNA was purified and cDNA was synthesized for amplification of Pax5, BLNK mRNA. Pax-5 variants were cloned and nucleotide sequences were determined. The production of Pax5 protein was confirmed with anti-human Pax5 antibody specific for the N terminal region of Pax5 to detect eight variant proteins. Results. Twelve of 21 ALL patients (57%) displayed multiple Pax5 mRNA fragments. These fragments corresponded to the full-length human Pax5 mRNA (Pax5 FL) and spliced variants with deletions of exon 7 (ΔE7, exon 8 (ΔE8, exons 7 and 8 (ΔE7/8), exons 8 and 9 (ΔE8/9), exons 6, 7 and 8 (ΔE6/7/8), exons 7, 8 and 9 (ΔE7/8/9), and exons 6, 7, 8 and 9 (ΔE6/7/8/9). Normal B lymphocytes expressed only Pax5 FL and lower level of Pax5 ΔE8. By Western blot analysis, normal B lymphocytes displayed a single band at a molecular weight of 50 kDa corresponding to Pax5 FL, whereas all B-lineage ALL patients except two revealed one protein band at a lower molecular weight of 46 kDa corresponding to Pax5 ΔE8. The production Pax5 and Pax5 ΔE8 proteins was also analyzed in normal B lymphocytes from different age groups. The dominant production of Pax5 ΔE8 was detected in neonates and infants, whereas only Pax5 FL was observed in children and in adults. Since Pax5 regulates the BLNK gene, which endows B lymphocytes with the ability to respond to B-cell-specific signals, BLNK expression and protein production was also analyzed in B-lineage ALL cells. Among 33 ALL samples, 16 (48%) either lacked or weakly expressed BLNK. All ALL samples with multiple Pax5 variants showed low or negative expression of BLNK, regardless of the expression of Pax5 ΔE8. Conclusions. Most of B-lineage ALL displayed multiple Pax5 mRNA transcripts, which were generated through alternative splicing in the 3′ region of the Pax5. On the other hand, all B-lineage ALL patients except two showed only Pax5 ΔE8 protein. The dominant production of Pax5 ΔE8 was also detected in normal B lymphocytes from neonates and infants. These results indicate that Pax5 ΔE8 participates in the early stage of B-cell differentiation predominantly detected in very young children. The altered Pax5 expression might disrupt B-cell hematopoiesis due to the absence or reduction of Pax5-regulated genes and cause the arrest at the stage of early B-cell differentiation and an abnormal expansion of immature B cells in B-lineage ALL in children.

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Linde, Miles H., Christopher G. Dove, Sarah F. Gurev, Paul Phan, Feifei Zhao, Eric J. Gars, Payton L. Marshall, Lindsay P. Miller y Ravindra Majeti. "Reprogramming leukemia cells into antigen presenting cells as a novel cancer vaccination immunotherapy". Journal of Immunology 204, n.º 1_Supplement (1 de mayo de 2020): 91.9. http://dx.doi.org/10.4049/jimmunol.204.supp.91.9.

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Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in malignant transformation. Despite an arrest in B cell maturation, human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage. To test the therapeutic implications of reprogramming, we generated murine models of B-ALL capable of myeloid lineage reprogramming via ectopic expression of CEBPα and PU.1. Once reprogrammed, B-ALL cells acquired an APC phenotype and stimulated antigen-specific T cells. In vivo B-ALL reprogramming in immunodeficient mice led to a modest survival benefit, however, reprogramming in immunocompetent mice led to tumor eradication and protection from subsequent re-challenge, demonstrating successful generation of durable, systemic, and curative immunity. This therapeutic benefit is dependent on the presence of both CD4+ and CD8+ T cells, and characterized by an increased frequency of bone marrow-infiltrating T cells and oligoclonal T cell expansion. Moreover, local myeloid reprogramming of a primary tumor drove systemic immune activation and eradication of distant, non-reprogrammed lesions. Our data suggests that B-ALL cells reprogrammed to the myeloid lineage are potent APCs capable of presenting tumor-associated antigens, and in vivo reprogramming elicits robust, durable, tumor-eradicating, and systemic T cell-mediated immunity. Recent efforts have further identified sarcoma and carcinoma models amenable to myeloid-lineage reprogramming. Thus, reprogramming of malignant cells into APCs represents a novel immunotherapeutic strategy with potential clinical utility in the treatment of a broad array of human malignancies.

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Shojaee, Seyedmehdi, Valeria Cazzaniga, Hilde Schjerven, Maike Buchner, Christian Hurtz, Huimin Geng, Andreas Hochhaus et al. "PTEN Is Essential for Normal Cytokine Signaling and Oncogenic Transformation of Pre-B Cells". Blood 124, n.º 21 (6 de diciembre de 2014): 262. http://dx.doi.org/10.1182/blood.v124.21.262.262.

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Abstract Background: PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor throughout all main types of human cancer. Recent work demonstrated that loss of PTEN in T cell acute lymphoblastic leukemia (ALL) confers glucocorticoid resistance. To develop a model for glucocorticoid resistance in pre-B ALL, we studied Cre-mediated deletion of Pten. Results: Surprisingly, loss of one or both alleles of Pten caused rapid cell death in pre-B ALL cells and was sufficient to cure transplant recipient mice from leukemia. Furthermore, deletion of Pten in pre-B cells disabled oncogenic transformation by BCR-ABL1 and NRASG12D oncogenes. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency across all main subtypes of mature B cell lymphoma, myeloid leukemias and T cell lineage leukemia/lymphoma (11.3% in 1,697 samples). Strikingly, however, lesions of PTEN were not detected in any of 670 pre-B ALL patient samples. Whereas high expression levels of PTEN predict favorable outcomes for patients with T-ALL, pre-B ALL patients with high PTEN expression levels at the time of diagnosis have particularly poor clinical outcomes (P=0.01). Studying the unexpected role of PTEN in B cell lineage ALL, we developed a genetic model for Cre-mediated deletion of Pten in mouse pre-B ALL cells carrying BCR-ABL1 or NRASG12D oncogenes. Deletion of one or both alleles of Pten reduced the colony forming ability (p < 10E-06) and proliferation rate (p < 10E-04) of pre-B ALL cells while strongly increasing cellular senescence (P < 10E-03). Since immediate effects of Pten deletion include hyperactivation of PI3K-AKT signaling, we hypothesized that small molecule inhibitors that limit PI3K-AKT pathway activity may rescue the pre-B ALL cells from cell death when Pten is inducibly deleted. Therefore, we tested the effect of AKT and PI3K inhibitors AZD5363 and BKM120 on Pten deleted cells and confirmed that chemical inhibition of PI3K-AKT pathway indeed rescued cell death of pre-B ALL cells upon Pten deletion. Also, global tyrosine phosphoprofiling by quantitative mass spectrometry revealed that Cre-mediated deletion of Pten strongly induced activation of CBL, a ubiquitin ligase that induces degradation of the IL7 receptor in pre-B cells and oncogenic tyrosine kinases in leukemia. Genetic experiments confirmed that deletion of Pten and hyperactivation of AKT engaged a novel, CBL-dependent pathway of negative feedback inhibition of STAT5 and tyrosine kinase signaling. Consistent with these finding, Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of pre-B ALL revealed a strong negative correlation between STAT5 protein levels and phospho-ATKT308 (P=0.0021) and phospho-AKTS473 (P=0.0005). Unlike pre-B ALL, deletion of Pten in CML like cells showed no toxicity. To further study the lineage specifity of Pten dependency we used a doxycycline-inducible CEBPa construct to differentiate pre-B cell ALL into myeloid lineage leukemia cells. Similar to our finding in CML like cells, unlike the pre-B ALL, myeloid lineage Leukemia showed no dependency on Pten and the deletion of Ptencaused no significant change in the cells viability. Studying the basal level of PTEN, pre-B cells showed ~40-fold higher expression levels than myeloid lineage cells. Consistently, genetic knockdown of PTEN using two different shRNA was only toxic in patient-derived pre-B but not myeloid leukemia cells. Clinical relevance: To find out the clinical potential of our finding, we tested the effects of SF1670 a small molecule inhibitor of PTEN on a panel of pre-B ALL and CML. Interestingly, the pre-B ALL showed more sensitivity to PTEN inhibition compared to CML cells. Conclusion: Our analysis uncovered an unexpected vulnerability of human Pre-B ALL cells toward PTEN inhibition and hyperactivation of the PI3K-AKT pathway, which provides an explanation why activating mutations of this pathway are negatively selected in human pre-B ALL. Since short (few hours) and profound inhibition of Pten is sufficient to commit human pre-B ALL cells to cell death, transient inhibition of this pathway or transient hyperactivation of PI3K-AKT may be a promising strategy to target multi-drug resistant human ALL. Disclosures No relevant conflicts of interest to declare.

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Chen, Xinrong y Paul W. Kincade. "Retinoids accelerate B lineage lymphoid differentiation (83.12)". Journal of Immunology 178, n.º 1_Supplement (1 de abril de 2007): S114. http://dx.doi.org/10.4049/jimmunol.178.supp.83.12.

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Abstract Retinoids are known to have potent effects on hematopoietic cells, and all trans retinoic acid (ATRA) is widely used to treat patients with acute promyelocytic leukemia. Retinoids may normally maintain stem cell integrity, and our main objective was to learn if B lineage lymphoid progenitors express functional receptors. Total CD19+ cells increased substantially in the bone marrow, while early lymphoid progenitors (ELP) and pro-lymphocytes (Pro-L) were reduced in mice treated with time-release ATRA pellets. Culture experiments with highly enriched progenitors demonstrated they were direct drug targets. Overall cell yields at short culture intervals were reduced, but CFSE labeling indicated this did not result from reduced proliferation. ELP are very primitive progenitors and 10 days is normally required for them to generate CD19+ cells in culture. Remarkably, lymphoid cells and IL-7 responding progenitors emerged in just 7 days when ATRA was present. Treatment with a panel of agonists suggested that RAR rather than RXR mediates these responses. ATRA had similar effects on human B cell differentiation. That is, it inhibited the expansion of human CD34+CD38− progenitor cells and accelerated the differentiation of human CD19+ cells. There may be previously unsuspected side effects of ATRA therapy, and retinoids might normally contribute to the lymphopoietic environment in bone marrow.

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18

Xiao, Gang, Chao Hong, Huimin Geng y Markus Muschen. "PON2 Exemplifies a Unique Dependency of B Cell Lineage ALL Cells on Detoxifying Lactonases". Blood 130, Suppl_1 (7 de diciembre de 2017): 882. http://dx.doi.org/10.1182/blood.v130.suppl_1.882.882.

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Abstract Background and significance : Paraoxonase 2 (PON2) is a member of mammalian detoxifying enzymes that are located to the mitochondrial membrane, hydrolyze lactone metabolites and interact with coenzyme Q10 to diminish oxidative stress. While PON2 is highly expressed in the CNS and multiple fetal tissues, expression levels in normal hematopoietic cells are low. We began to study the function of PON2 in B cell lineage ALL, because microarray analyses suggested high mRNA levels of PON2 in B cell lineage ALL cells. In addition, PON2 is used as a diagnostic marker on a 15 gene diagnostic LDA panel (e.g. in AALL1131; NCT02883049) that is used for the identification of Ph-like ALL, a subgroup with particularly poor outcome and specific treatment requirements. Results: Analyzing data from pediatric and adult clinical trials, we found that greater than median PON2 mRNA levels at the time of diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003) with B-lineage ALL. Studying expression levels of PON2 by quantitative RT-PCR and Western blot in normal bone marrow B cell precursors from healthy donors and patient-derived pre-B ALL cells including Ph+ and Ph -like ALL, we found 3-10-fold increased mRNA and protein levels of PON2 throughout multiple pre-B ALL samples. To elucidate potential functions of PON2 in normal B cells and B-lineage ALL, we studied normal B cell development and ALL-models in Pon2-/- mice. While B cell development was unperturbed in Pon2 -deficient mice, deletion of Pon2 had profound effects on both BCR-ABL1- and N RASG12D-driven leukemogenesis. Compared to wildtype, Pon2-/- pre-B ALL cells failed to form colonies in semisolid agar. Pon2-/-ALL cells were arrested in G0/G1 phase and expressed 2-5-fold increased levels of Arf and p21, compared to Pon2+/+ ALL cells. Strikingly, >50% of Pon2-/-ALL cells spontaneously underwent cellular senescence, as shown by b-galactosidase staining in conjunction with increased Arf expression levels. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in transplant experiments based on BCR-ABL1 and N RASG12D ALL models. While Pon2 -deficiency substantially prolonged survival of recipient mice of BCR-ABL1 ALL cells (P=0.0001), mice transplanted with Pon2 -deficient N RASG12D ALL cells survived for indefinite periods of time. Targeting PON2 expression in relapse ALL: Given that PON2 is expressed at very high levels in relapse ALL samples, we tested the concept of targeting PON2 lactonase activity in a prodrug-approach. While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts (Guoping et al., 2016). We therefore tested the therapeutic potential of 3OC12. For genetic validation, we treated wildtype and Pon2-/- ALL cells with 3OC12 and found strong cytotoxic effects in wildtype but not Pon2-/- ALL cells. Likewise, inducible overexpression of Pon2 in patient-derived pre-B ALL cells exacerbated toxicity of 3OC12 compared to empty vector controls. Likewise, CRISPR-Cas9 mediated ablation of PON2 in human ALL PDX reversed sensitivity of the pre-B ALL cells to 3OC12. Conclusion: Here we describe the previously unknown function of the detoxifying PON2 lactonase as an essential prerequisite for pre-B cell transformation and leukemogenesis. PON2 expression is specific for leukemia cells, an outcome predictor for patients with pre-B ALL and a biomarker of Ph-like ALL. While PON2 protects ALL cells and enables malignant growth, we demonstrate that its lactonase-activity can be leveraged for pharmacological targeting as exemplified by the lactone-prodrug 3OC12. Disclosures No relevant conflicts of interest to declare.

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19

Nwabo Kamdje, Armel Hervé, Federico Mosna, Francesco Bifari, Veronica Lisi, Giulio Bassi, Giorgio Malpeli, Mario Ricciardi et al. "Notch-3 and Notch-4 signaling rescue from apoptosis human B-ALL cells in contact with human bone marrow–derived mesenchymal stromal cells". Blood 118, n.º 2 (14 de julio de 2011): 380–89. http://dx.doi.org/10.1182/blood-2010-12-326694.

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Abstract Although many literature data are available on the role of Notch signaling in T-cell acute lymphoblastic leukemia (ALL) biology, the importance of this molecular pathway in the development of B-lineage ALL (B-ALL) cells in the BM microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing Abs and γ-secretase inhibitor (GSI) XII to investigate the role of the Notch signaling pathway in the promotion of human B-ALL cell survival in presence of stromal cell support. The treatment with combinations of anti-Notch molecule neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and B-ALL patients. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. Our data suggest that the stromal cell–mediated antiapoptotic effect on B- ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.

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20

Tedder, T. F., L. T. Clement y M. D. Cooper. "Human lymphocyte differentiation antigens HB-10 and HB-11. I. Ontogeny of antigen expression." Journal of Immunology 134, n.º 5 (1 de mayo de 1985): 2983–88. http://dx.doi.org/10.4049/jimmunol.134.5.2983.

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Abstract T, B, and NK cells appear to represent separate lymphocyte lineages, but indirect evidence suggests that they may be related via a common lymphoid precursor cell. We have produced two monoclonal antibodies, HB-10 (IgM) and HB-11 (IgG1), by fusing spleen cells from mice immunized with the human B cell line SB, and have shown that both antibodies react with lymphocyte-specific cell surface antigens present on T, B, and NK cells, but not on other types of blood cells. The antibodies were reactive with most cell lines and malignancies of B cell origin and with some of T and NK cell lineage. Although the populations of cells expressing these two antigens were virtually identical, the HB-10 and HB-11 antibodies identified separate protease-sensitive determinants on the cell surface. The HB-11 antigenic determinant was also sensitive to neuraminidase and periodate treatments, but the HB-10 determinant was not. Antigen expression by lymphocytes from fetal, newborn, and adult tissues was examined. Within the B cell lineage, these antigens were expressed by most pre-B cells in bone marrow (88% +/- 5) and almost all B cells, but were not expressed by mature plasma cells. Virtually all of the granular lymphocytes in blood marked by the Leu-7 and Leu-11 (anti-Fc receptor) antibodies were HB-10+ and 11+. Among T lineage cells, the HB-10 and 11 antigens were expressed by a subset of relatively mature T3+ thymocytes and by greater than 90% of the T cells in newborn blood. In adults, however, only 65% of blood T cells and 24 to 30% of splenic or tonsillar T cells expressed the HB-10 and HB-11 antigens. The postnatal emergence of T cells which, like plasma cells, do not express these antigens suggests that post-thymic T lymphocyte maturation occurs and may be an activation-dependent process.

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21

Ferrando, Adolfo A., Scott A. Armstrong, Donna S. Neuberg, Stephen E. Sallan, Lewis B. Silverman, Stanley J. Korsmeyer y A. Thomas Look. "Gene expression signatures in MLL-rearranged T-lineage and B-precursor acute leukemias: dominance of HOX dysregulation". Blood 102, n.º 1 (1 de julio de 2003): 262–68. http://dx.doi.org/10.1182/blood-2002-10-3221.

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Abstract Rearrangements of the MLL locus, located on human chromosome 11q23, are frequent in both infant and therapy-related leukemias. Gene expression analysis of MLL-rearranged B-precursor acute lymphoblastic leukemias (MLL B-ALLs) has identified these cases as a unique subtype of leukemia, characterized by the expression of genes associated with both lymphoid and myeloid hematopoietic lineages. Here we show that MLL fusions also generate a distinct genetic subtype of T-lineage ALL (MLL T-ALL), in which leukemic cells are characterized by an early arrest in thymocyte differentiation, with suggestive evidence of commitment to the γδ lineage. Interestingly, multiple genes linked to cell proliferation (eg, PCNA, MYC, CDK2, and POLA) were down-regulated in MLL-fusion samples, relative to those transformed by other T-ALL oncogenes (P < .000 001, Fisher exact test). Overall, MLL T-ALL cases consistently demonstrated increased levels of expression of a subset of major HOX genes—HOXA9, HOXA10, and HOXC6—and the MEIS1 HOX coregulator (P < .008, one-sided Wilcoxon test), a pattern of gene expression that was reiterated in MLL B-ALLs. However, expression of myeloid lineage genes, previously reported in MLL B-ALLs, was not identified in T-lineage cases with this abnormality, suggesting that myeloid gene dysregulation is dispensable in leukemic transformation mediated by MLL fusion proteins. Our findings implicate dysregulation of HOX gene family members as a dominant mechanism of leukemic transformation induced by chimeric MLL oncogenes. (Blood. 2003;102:262-268)

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22

Uckun, F. M., K. J. Gajl-Peczalska, J. H. Kersey, L. L. Houston y D. A. Vallera. "Use of a novel colony assay to evaluate the cytotoxicity of an immunotoxin containing pokeweed antiviral protein against blast progenitor cells freshly obtained from patients with common B-lineage acute lymphoblastic leukemia." Journal of Experimental Medicine 163, n.º 2 (1 de febrero de 1986): 347–68. http://dx.doi.org/10.1084/jem.163.2.347.

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We report a novel colony assay for B-lineage progenitor cells in acute lymphoblastic leukemia (ALL). The primary plating efficiency of blast progenitors freshly obtained from common B-lineage ALL patients varied between 0.09 and 2.63%. Morphological, cytochemical, and immunological analyses of cells from day 7 colonies provided the evidence that they are B-lineage lymphoblasts. Immunological marker analyses of cultured blasts using BA-2 (anti-CD9), BA-3 (anti-CD10), BA-1 (anti-CD24), and B43 mAb have allowed us to define two distinct immunological groups. The first group had BA-2+, BA-3+, BA-1+, B43+ marker profiles, consistent with the phenotype of uncultured bone marrow blasts. The second group differed in that the cells in the blast colonies were BA-3 (anti-CD10)-negative, although many of the cells in the bulk population were BA-3+ before culture. Blasts from both groups were able to proliferate and form secondary colonies when recultured. A pan-B immunotoxin was synthesized by linking B43, a human B cell-specific mAb, to pokeweed antiviral protein (PAP). This study showed that B43-PAP can effectively eradicate leukemic progenitor cells freshly obtained from patients with common B-lineage ALL. B43-PAP eliminated greater than 99.96% of blast progenitors under conditions in which only minimal inhibition of normal bone marrow progenitor cells (CFU-GM, CFU-E, CFU-MK, CFU-GEMM) was observed. Our results establish that the surface determinant recognized by B43 is expressed on B-lineage progenitor cells in ALL, and that these cells are sensitive to PAP at the ribosomal level. To our knowledge, B43-PAP is the first IT to prove effective against common B-lineage ALL cells.

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23

Schiff, C., M. Milili, D. Bossy, A. Tabilio, F. Falzetti, J. Gabert, P. Mannoni y M. Fougereau. "Lambda-like and V pre-B genes expression: an early B-lineage marker of human leukemias". Blood 78, n.º 6 (15 de septiembre de 1991): 1516–25. http://dx.doi.org/10.1182/blood.v78.6.1516.1516.

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Abstract V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these “pre-B-specific” genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu- pseudolight chain complex was actually replaced by the classical mu- kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

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24

Schiff, C., M. Milili, D. Bossy, A. Tabilio, F. Falzetti, J. Gabert, P. Mannoni y M. Fougereau. "Lambda-like and V pre-B genes expression: an early B-lineage marker of human leukemias". Blood 78, n.º 6 (15 de septiembre de 1991): 1516–25. http://dx.doi.org/10.1182/blood.v78.6.1516.bloodjournal7861516.

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V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these “pre-B-specific” genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu- pseudolight chain complex was actually replaced by the classical mu- kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

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25

Guillaume, Nicolas, Celine Alleaume, Jean-Claude Capiod, Brigitte Pautard, Bernard Desablens, Jean-François Claisse, Jean-Jacques Lefrere, Fabrice Gouilleux, Kaiss Lassoued y Valerie Gouilleux-Gruart. "ZAP-70 Tyrosine Kinase Is Constituvely Expressed and Phosphorylated in B-Lineage Acute Lymphoblastic Leukemia Cells." Blood 104, n.º 11 (16 de noviembre de 2004): 1898. http://dx.doi.org/10.1182/blood.v104.11.1898.1898.

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Abstract Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases is normally expressed in T and NK cells. ZAP-70 plays a key role in the signaling pathway of the TCR. It has been recently shown that this protein is also expressed in mouse B lineage cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of chronic lymphocytic leukemia (CLL), with a poor prognosis, that have nonmutated immunoglobulin variable region genes. In this study, we examined the expression and phosphorylation status of ZAP-70 and Syk in B-lineage acute lymphoblastic leukemia (B-ALL) cells. ZAP-70 and Syk were constitutively expressed and phosphorylated (tyr319 for ZAP and tyr352 for Syk) in human precursor B-ALL cell lines as well as in B leukemic cells from all examined B-ALL patients (n=18). The expression of ZAP-70 was specific to these B-ALL cells as it was not detected in the K562 cell line or malignant myeloid cells. This expression was independant of maturation state of leukemic cells (pro-B or pre-B) and caryotypic status. The expression and phosphorylation of the T-cell “specific” ZAP-70 kinase in all B-ALL cells raises important questions concerning the contribution of this protein to the leukemogenesis process resulting in B-ALL.

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26

Chan, Lai N., Zhengshan Chen, Gang Xiao, Jaewoong Lee, Huimin Geng, Hurtz Christian, Valeria Cazzaniga, Giovanni Cazzaniga, Ross A. Dickins y Markus Müschen. "Transcriptional Control of Glucose and Energy Supply Prevents Oncogenic Signaling and B Cell Transformation". Blood 128, n.º 22 (2 de diciembre de 2016): 437. http://dx.doi.org/10.1182/blood.v128.22.437.437.

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Abstract Background and Hypothesis: B-cell identity is determined by a set of B-cell transcription factors including PAX5, IKZF1 and EBF1. However, B-lineage leukemia clones often carry secondary genetic lesions that result in reduced activity or inactivation of these transcription factors. Studying patient samples from clinical trials for B-lineage childhood (P9906; n=187) and adult (MDACC; n=92) leukemia, we found that genetic defects in one or more B-cell transcription factors represent near-obligate lesions in human acute lymphoblastic leukemia (209 of 279 B-lineage ALL cases). While previously of unknown significance, we found that adult ALL cases with known lesions in one or more of these transcription factors had higher activity of lactate dehydrogenase, and phospho-states indicated higher activity of IRS1, PDK1, and AKT, which contribute to glucose uptake. For this reason, we investigated whether B-cell transcription factors set metabolic constraints of oncogenic signaling in leukemia. Results: Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells carrying PAX5 and IKZF1deletions decreased phospho-AKT levels. Furthermore, protein levels of multiple positive regulators involved in glucose uptake and metabolism (including insulin receptor, glucose transporters and hexokinases) were downregulated upon reconstitution of PAX5 and IKZF1. Conversely, protein levels of negative regulators of glucose uptake and metabolism including NR3C1 and TXNIP were upregulated upon reinstatement of PAX5 and IKZF1 function. Analysis of ChIPseq data of human B cells revealed binding of multiple B-cell transcription factors including PAX5, IKZF1 and EBF1 to promoter regions of genes encoding positive regulators of glucose uptake and metabolism. Binding peaks for B-cell transcription factors were also observed at genes that encode negative regulators of glucose uptake and metabolism (NR3C1 and TXNIP). In addition, direct recruitment of PAX5 was confirmed by single-locus quantitative chromatin immunoprecipitation (qChIP). Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells caused depletion from the cell culture in competitive growth assays, in parallel with reduced glucose consumption and depletion of cellular ATP levels. On the contrary, overexpression of dominant-negative PAX5-ETV6 and IK6 in patient-derived B-lineage ALL cells expressing wildtype PAX5 and IKZF1 resulted in a net survival advantage, concomitant with increases in both glycolytic activity and cellular ATP levels. PAX5-mediated impaired survival fitness was significantly rescued by CRISPR-based activation of gene expression of insulin receptor, glucose transporter and hexokinases in patient-derived B-lineage ALL cells. Conversely, CRISPR/Cas9-mediated deletion of NR3C1 and TXNIP largely reversed the effects of PAX5. Finally, reduced survival fitness upon reconstitution of wild-type PAX5 and IKZF1 in patient-derived pre-B ALL cells was mostly rescued by metabolites that can enter the TCA cycle and thus provide ATP. Conclusions: In summary,B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation. Our findings suggest a causative link between impaired glucose uptake and metabolism caused by B-cell transcription factors as well as cell death, and the known tumor suppressive function of PAX5 and IKZF1 in B-lineage ALL. Disclosures No relevant conflicts of interest to declare.

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27

Bertrand, Fred E., Christine Vogtenhuber, Nisha Shah y Tucker W. LeBien. "Pro-B-cell to pre-B-cell development in B-lineage acute lymphoblastic leukemia expressing the MLL/AF4 fusion protein". Blood 98, n.º 12 (1 de diciembre de 2001): 3398–405. http://dx.doi.org/10.1182/blood.v98.12.3398.

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Abstract The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23) that gives rise to the MLL/AF4 fusion gene. Leukemic blasts expressing MLL/AF4 are arrested at an early progenitor stage with lymphoid or monocytoid characteristics. A novel B-lineage ALL cell line termedB-lineage–3 (BLIN-3) requiring human bone marrow (BM) stromal cell contact and interleukin-7 (IL-7) for optimal proliferation has been established. BLIN-3 cells have a CD19+/CD10− phenotype typical of infant ALL, and they harbor the t(4;11)(q21;q23) chromosomal translocation. Reverse transcription–polymerase chain reaction and Western blot analysis confirmed the presence of the MLL/AF4 fusion mRNA and protein in BLIN-3. Initial BLIN-3 cultures had a pro-B cell phenotype and did not express cytoplasmic or surface μ heavy chain. After approximately 5 months in culture on BM stromal cells plus IL-7, BLIN-3 sublines emerged expressing μ heavy chain and VpreB on the cell surfaces (ie, pre-B-cell receptor [BCR]+). BLIN-3 cells expressing pre-BCR had the t(4;11)(q21;q23) translocation and expressed the MLL/AF4 fusion protein. Cross-linking the BLIN-3 pre-BCR led to enhanced cell proliferation, demonstrating that BLIN-3 expressed a functional pre-BCR. Increased acquisition of surface pre-BCR in BLIN-3 sublines was associated with loss of DJ rearrangements and the appearance of VDJ rearrangements. These results indicate that expression of the MLL/AF4 fusion protein is compatible with BM stromal cell and cytokine dependency, functional immunoglobulin gene segment rearrangement, and subsequent expression of a potentially diverse antigen receptor repertoire. Thus, the expression of MLL/AF4 is compatible with the normal developmental program of human B-lineage cells.

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Hao, Qian-Lin, Aswathi A. George, Judy Zhu, Lora Barsky, Ewa Zielinska, Xiuli Wang, Mary Price, Shundi Ge y Gay M. Crooks. "Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7− lympho-myeloid thymic progenitors". Blood 111, n.º 3 (1 de febrero de 2008): 1318–26. http://dx.doi.org/10.1182/blood-2007-08-106294.

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Abstract The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34+lin− thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7−. CD34+lin− thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7hi fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7int (CD10+) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7− fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34+lin−CD7− thymocytes also expressed early T lymphoid genes Tdt, pTα, and IL-7Rα and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7− lympho-myeloid thymic progenitor.

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Rivas, María José, Miguel Alegretti, Leticia Cóppola, Viviana Ramas, Héctor Chiparelli y Natalia Goñi. "Epidemiology and Genetic Variability of Circulating Influenza B Viruses in Uruguay, 2012–2019". Microorganisms 8, n.º 4 (19 de abril de 2020): 591. http://dx.doi.org/10.3390/microorganisms8040591.

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Influenza B viruses (IBV) are an important cause of morbidity and mortality during interpandemic periods in the human population. Two phylogenetically distinct IBV lineages, B/Yamagata and B/Victoria, co-circulate worldwide and they present challenges for vaccine strain selection. Until the present study, there was little information regarding the pattern of the circulating strains of IBV in Uruguay. A subset of positive influenza B samples from influenza-like illness (ILI) outpatients and severe acute respiratory illness (SARI) inpatients detected in sentinel hospitals in Uruguay during 2012–2019 were selected. The sequencing of the hemagglutinin (HA) and neuraminidase (NA) genes showed substitutions at the amino acid level. Phylogenetic analysis reveals the co-circulation of both lineages in almost all seasonal epidemics in Uruguay, and allows recognizing a lineage-level vaccine mismatch in approximately one-third of the seasons studied. The epidemiological results show that the proportion of IBV found in ILI was significantly higher than the observed in SARI cases across different groups of age (9.7% ILI, 3.2% SARI) and patients between 5–14 years constituted the majority (33%) of all influenza B infection (p < 0.05). Interestingly, we found that individuals >25 years were particularly vulnerable to Yamagata lineage infections.

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30

Geng, Huimin, Christian Hurtz, Zhengshan Chen, Wei-Yi Chen, Erica Ballabio, Gang Xiao, Soo-mi Kweon et al. "Targeting Pre-B Cell Receptor and BCL6 In TCF3-PBX1 B-Lineage Acute Lymphoblastic Leukemia". Blood 122, n.º 21 (15 de noviembre de 2013): 349. http://dx.doi.org/10.1182/blood.v122.21.349.349.

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Abstract Background TCF3-PBX1 is one of the most common recurrent translocations which define distinct subtypes of B-lineage Acute Lymphoblastic Leukemia (B-ALL). Patients with TCF3-PBX1 B-ALL have poor clinical outcome, however, the molecular mechanisms underlying poor outcome are poorly understood. It is critical to identify the cellular processes that contribute to the biological and clinical features of this form of ALL and to develop new targeted therapeutic strategies to improve the outcome. Results We first performed ChIP-seq with antibodies against the fusion oncoprotein TCF3-PBX1 in the human B-ALL cell line 697 and found that the pre–B cell receptor (pre-BCR) genes (IGLL1 or λ5, VpreB, CD79A, CD79B) and µ-chain enhancer regions were directly bound by the fusion protein. Gene expression microarray data showed that the pre-BCR signaling genes (IGLL1, VpreB, IGHM, BLK, LCK, SYK, LYN, SRC and BLNK) were overexpressed in TCF3-PBX1, but not other cytogenetic subtypes of B-ALL (n=132 B-ALL patient samples, St. Jude), suggesting a unique high pre-BCR activity in TCF3-PBX1 ALL. Further flow cytometry analysis using µ-chain specific antibodies showed a strong Ca2+ signal in TCF3-PBX1, but not other subtypes of ALL (n=27). The sequencing analysis on IGHM locus in 148 primary B-ALL samples showed that 100% cases of TCF3-PBX1 (n=8) carried functional IGHM VHDJH gene rearrangements, however only 17% for BCR-ABL1 (n=57), 0% for MLL-AF4 (n=7), 31% for ETV6-RUNX1 (n=13), 3.3% for hyperdiploid (n=30), 20% for sporadic (n=20) and 31% for normal karyotype (n=13) ALL. These data demonstrates that TCF3-PBX1 ALL has an unusual spectrum of high activity of pre-BCR signaling. The transcriptional repressor BCL6 has been identified as a critical survival factor in diffuse large B-cell lymphoma. We found that BCL6 was highly expressed in TCF3-PBX1 as compared to other subtypes of ALL by our Western blot (n=15) and also by gene expression microarray data from three B-ALL clinical trials: COG P9906 (n=207), ECOG E2993 (n=191) and St. Jude ALL (n=132). The clinical data showed that high expression of BCL6 correlates with poor clinical outcome. Those data suggested BCL6 might play a critical oncogene role in TCF3-PBX1 ALL. The TCF3-PBX1 ChIPseq data showed no binding enrichment on BCL6 locus. However, forced expression of pre-BCR components (µ-chain and BLNK) resulted in up-regulation of BCL6 (Western blot), suggesting BCL6 upregulation in TCF3-PBX1 ALL was induced by pre-BCR but not the fusion protein binding. To further test this, we applied SYK and BTK inhibitors to treat TCF-PBX1 ALL cells and found that they dramatically decreased BCL6 mRNA and protein levels and also induced cell apoptosis, suggesting BCL6 might be a therapeutic target for TCF3-PBX1 ALL. To test this, we transduced a primary TCF3-PBX1 B-ALL xenograft sample with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell death. When treating primary TCF3-PBX1 ALL cells with the specific BCL6 peptide inhibitor RI-BPI and the small molecule inhibitor PU-H71, they significantly induced cell cycle arrest, compromised colony formation, and prevented leukemia-initiation in transplant recipient mice. Collectively, those data indicates BCL6 is a new therapeutic target for TCF-PBX1 ALL. Dasatinib is a second generation tyrosine kinase inhibitor (TKI) targeting both BCR-ABL1 and Src kinase. We hypothesized that TCF3-PBX1 ALL cells would be more sensitive to Dasatinib than other TKIs because Dasatinib inhibits pre-BCR signaling and hence BCL6. We treated primary TCF3-PBX1 B-ALL cells with Dasatinib and Nilotinib, and found that BCL6 protein expression was abolished with Dasatinib, but no changes with Nilotinib. We also observed a >90% reduction in viability of leukemia cells with 50nM Dasatinib, however only ∼50% cell death at a much higher concentration of Nilotinib (1000nM). These data indicates that Dasatinib can efficiently kill TCF3-PBX1 leukemia cells by inhibiting pre-BCR and BCL6. Conclusions Our study identified TCF3-PBX1 B-ALL as a unique form of ALL which has an unusual high activity of pre-BCR signaling. BCL6 was upregulated by pre-BCR, which we showed was required for proliferation and survival of TCF3-PBX1 ALL cells. Targeting pre-BCR and BCL6 using Dasatinib and specific BCL6 inhibitors (RI-BPI or PU-H71) induced apoptosis of primary TCF3-PBX1 B-ALL cells in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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Cutler, Jevon, Raiha Tahir, Rumen Kostadinov, Raja Sekhar, Tai-Chung Huang, Akhilesh Pandey, Patrick Brown, Mohammad Heydarian y Karen Reddy. "Proteomic/Transcriptomic Signatures of Infant MLL-r Rearranged B-ALL at Diagnosis and Relapse Reveal Lineage Plasticity and Diagnostic Heterogeneity". Blood 128, n.º 22 (2 de diciembre de 2016): 2697. http://dx.doi.org/10.1182/blood.v128.22.2697.2697.

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Abstract Infant leukemia, defined as leukemia before 1 year of age, is an aggressive set of diseases with dramatically poorer outcome when compared to childhood leukemia. A particularly intractable subset of infant leukemia patients, who are prone to relapse, are those with B-cell acute lymphoblastic leukemia (B-ALL) harboring a chromosomal translocations involving the epigenetic modifier gene mixed lineage leukemia (MLL), referred hereafter as MLL rearranged (MLL-r) B-ALL. Rearrangements in MLL occur in 70-80% of all infant B-ALL patients (compared to 5% in pediatric leukemia) and this cohort is characterized by a record setting dearth of cooperating lesions beyond MLL-r. To better understand the mechanism of relapse in this patient cohort we employed RNA sequencing and deep mass spectrometry based proteomics to characterize infant MLL-R B-ALL patient derived xenografts. Five infant MLL-r B-ALL patients with matched diagnosis and relapse primary sample were individually injected into NSG immunocompromised mice. For all patients >95% human engraftment purity was achieved in mouse bone marrow and spleen. Fresh cells were submitted for RNA sequencing and nuclear fractionated as well as whole cell lysates were generated for mass spec protein analysis. Protein and RNA quantitation at relapse and diagnosis across all patients was achieved on molecules that correspond to about 8,000 genes for protein and 17,000 genes for RNA. Hierarchal clustering as well as multi-dimensional scaling analysis of RNA sequencing data revealed a closer intra-patient relationship at the two treatment time points (diagnosis and relapse) than an inter-patient relationship at diagnosis or at relapse. A common relapse signature was simply non-existent according to the data, however this could be attributed to the small sample size. The integration of proteomic and transcriptomic data revealed a strong correlation of relapse/diagnosis fold changes at the level of RNA and protein. Intriguingly, a plasticity at relapse was observed in key developmental and differentiation marker genes associated with the myeloid, T, and B cell lineages. For example, in Patient 1 up-regulation at the level of protein and RNA of myeloid/acute myeloid leukemia (AML) associated genes; RUNX2, MPO, and CD33, and down-regulation of T and B cell associated genes; CD1A, CD2, and BCL6 where observed at relapse. Patient 2 showed an RNA/protein up-regulation of myeloid and AML markers CEBPB and MPO while a concurrent down-regulation of the Ikaros family member Helios, B/T cell attenuator BTLA, and the oncogene ETV6 at relapse. Patient 3 showed an RNA/protein up-regulation of Helios, the oft mutated in B-ALL; ARID5B, and the pre-B-cell leukemia associated transcription factor PBX3 at relapse. Patient 1 and 2 appear to show signs that myeloid programs are being greater utilized at relapse while in Patient 3 seems to intensify its commitment to the B-cell lineage. All patients showed some degree of lineage marker infidelity at relapse and interestingly, this plasticity was not reflected in the traditional flow cytometric markers used to differentiate AML from B-ALL suggesting that this lineage infidelity might be more common than previously thought. Despite the mutational uniformity of infant MLL-R B-ALL genomes it is surprising that such a diversity of RNA expression and subsequent protein levels is observed. This data suggests that MLL fusion proteins have great flexibility to control of the transcriptional regulation/protein expression and could in part help explain why these patients do not rely upon newly acquired mutations to evade treatment protocols. Furthermore, lineage switching in leukemia is rarely observed but as this data suggests that this is more common in MLL-r B-ALL than previously thought. Disclosures No relevant conflicts of interest to declare.

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32

Thomas, Xavier. "Updates in Adult Acute Lymphoblastic Leukemia—Current Status of Antigen-targeted Treatments and Immunotherapy". Oncology & Hematology Review (US) 13, n.º 02 (2017): 92. http://dx.doi.org/10.17925/ohr.2017.13.02.92.

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The outcomes of adult acute lymphoblastic leukemia (ALL) remain inferior to those observed in pediatric populations. Targeted therapy with monoclonal antibodies and immunotherapy represents the most promising new way to fight ALL without significant additive toxicity. Since the use of rituximab in combination chemoimmunotherapy for treatment of B cell lineage ALL, a number of other monoclonal antibodies are under investigation for the treatment of this disease. Deep molecular remissions with anti-CD19- and anti-CD22-directed therapy have been shown in relapsed/refractory (R/R) disease, allowing for the opportunity to transplant. Blinatumomab is the first antigen-directed treatment approved for use in R/R ALL. Adoptive cellular therapy with human T cells genetically engineered to express CD19 redirected chimeric antigen receptors (CARs) has also recently demonstrated efficacy in patients with B cell lineage ALL. In this article, we review the therapeutic implications, primary results, and current status of antigen-targeted treatments and immunotherapy in adult B cell lineage ALL.

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Zhou, L. J., H. M. Smith, T. J. Waldschmidt, R. Schwarting, J. Daley y T. F. Tedder. "Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development". Molecular and Cellular Biology 14, n.º 6 (junio de 1994): 3884–94. http://dx.doi.org/10.1128/mcb.14.6.3884-3894.1994.

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CD19 is a B-cell-specific member of the immunoglobulin superfamily expressed from early pre-B-cell development until plasma cell differentiation. In vitro studies demonstrate that the CD19 signal transduction molecule can serve as a costimulatory molecule for activation through other B-lymphocyte cell surface molecules. However, much remains to be known regarding how CD19 functions in vivo and whether CD19 has different roles at particular stages of B-cell differentiation. Therefore, transgenic mice overexpressing the human CD19 (hCD19) gene were generated to determine whether this transgene would be expressed in a B-lineage-specific fashion and to dissect the in vivo role of CD19 in B-cell development and activation. Expression of the human transgene product was specifically restricted to all B-lineage cells and appeared early in development as occurs with hCD19. In addition, expression of hCD19 severely impaired the development of immature B cells in the bone marrow, with dramatically fewer B cells found in the spleen, peripheral circulation, and peritoneal cavity. The level of hCD19 expressed on the cell surface correlated directly with the severity of the defect in different transgenic lines. These results demonstrate that the hCD19 gene is expressed in a lineage-specific fashion in mice, indicating that the hCD19 gene may be useful for mediating B-lineage-specific expression of other transgene products. In addition, these results indicate an important role for the lineage-specific CD19 molecule during early B-cell development before antigen-dependent activation.

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34

Zhou, L. J., H. M. Smith, T. J. Waldschmidt, R. Schwarting, J. Daley y T. F. Tedder. "Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development." Molecular and Cellular Biology 14, n.º 6 (junio de 1994): 3884–94. http://dx.doi.org/10.1128/mcb.14.6.3884.

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CD19 is a B-cell-specific member of the immunoglobulin superfamily expressed from early pre-B-cell development until plasma cell differentiation. In vitro studies demonstrate that the CD19 signal transduction molecule can serve as a costimulatory molecule for activation through other B-lymphocyte cell surface molecules. However, much remains to be known regarding how CD19 functions in vivo and whether CD19 has different roles at particular stages of B-cell differentiation. Therefore, transgenic mice overexpressing the human CD19 (hCD19) gene were generated to determine whether this transgene would be expressed in a B-lineage-specific fashion and to dissect the in vivo role of CD19 in B-cell development and activation. Expression of the human transgene product was specifically restricted to all B-lineage cells and appeared early in development as occurs with hCD19. In addition, expression of hCD19 severely impaired the development of immature B cells in the bone marrow, with dramatically fewer B cells found in the spleen, peripheral circulation, and peritoneal cavity. The level of hCD19 expressed on the cell surface correlated directly with the severity of the defect in different transgenic lines. These results demonstrate that the hCD19 gene is expressed in a lineage-specific fashion in mice, indicating that the hCD19 gene may be useful for mediating B-lineage-specific expression of other transgene products. In addition, these results indicate an important role for the lineage-specific CD19 molecule during early B-cell development before antigen-dependent activation.

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35

McClellan, James Scott, Christopher Dove, Andrew J. Gentles, Christine E. Ryan y Ravindra Majeti. "Reprogramming of primary human Philadelphia chromosome-positive B cell acute lymphoblastic leukemia cells into nonleukemic macrophages". Proceedings of the National Academy of Sciences 112, n.º 13 (16 de marzo de 2015): 4074–79. http://dx.doi.org/10.1073/pnas.1413383112.

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BCR–ABL1+ precursor B-cell acute lymphoblastic leukemia (BCR–ABL1+ B-ALL) is an aggressive hematopoietic neoplasm characterized by a block in differentiation due in part to the somatic loss of transcription factors required for B-cell development. We hypothesized that overcoming this differentiation block by forcing cells to reprogram to the myeloid lineage would reduce the leukemogenicity of these cells. We found that primary human BCR–ABL1+ B-ALL cells could be induced to reprogram into macrophage-like cells by exposure to myeloid differentiation-promoting cytokines in vitro or by transient expression of the myeloid transcription factor C/EBPα or PU.1. The resultant cells were clonally related to the primary leukemic blasts but resembled normal macrophages in appearance, immunophenotype, gene expression, and function. Most importantly, these macrophage-like cells were unable to establish disease in xenograft hosts, indicating that lineage reprogramming eliminates the leukemogenicity of BCR–ABL1+ B-ALL cells, and suggesting a previously unidentified therapeutic strategy for this disease. Finally, we determined that myeloid reprogramming may occur to some degree in human patients by identifying primary CD14+ monocytes/macrophages in BCR–ABL1+ B-ALL patient samples that possess the BCR–ABL1+ translocation and clonally recombined VDJ regions.

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Koelle, Samson, Chuanfeng Wu, Brian Li, Rong Lu, Robert E. Donahue y Cynthia E. Dunbar. "Long-Term Hematopoietic Clonal Stability Tracked Using Molecular Barcoding in Non-Human Primates". Blood 124, n.º 21 (6 de diciembre de 2014): 2908. http://dx.doi.org/10.1182/blood.v124.21.2908.2908.

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Abstract By transplanting rhesus macaques with autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) labeled with lentivirally delivered high diversity oligonucleotide barcodes, we are able to track complex clonal contributions to hematopoiesis over time and across lineages via low cycle PCR and high-throughput sequencing of the inserted barcode. We reported on patterns of reconstitution for the first 9 months following engraftment in a recent publication (Wu et al, Cell Stem Cell, 2014). For the first 1 to 3 months short lived unilineage progenitors predominated, followed by increased clonal diversity and the emergence of first shared myeloid (My)/B clones, and then My/B/T clones. A major novel finding was a distinct ontogeny for NK cells, with the major CD16+ blood NK population showing non-overlapping origin with My/B and T lineages through 6-9 months. We now report on hematopoietic clonal patterns up to 23 months post-transplant in four animals. In all four animals, a group of high contributing NK clones contributed minimally to other lineages until 9 to 12 months, at which point these unilineage NK clones extinguished and a new generation of NK high contributors appeared. In two animals, these emerging clones continued to be either unilineage or highly biased towards the NK lineage, while in two other animals, they began to contribute highly to all lineages, a discrepancy which possibly derives from differences between animals in barcode marking level. Over the 17 and 23 months since transplant tracked in the two highly marked animals, 5.6±1.4 and 9±0.9 out of the top 10 NK contributing clones, respectively, were highly biased towards NK and away from other lineages, as determined by unsupervised hierarchical clustering of correlations between high contributing clones. Though shared My/B/T progenitors emerged after 6 months in both animals, 3.1±1.3 and 6.4±0.9 out of the T cell top 10 contributing clones were highly biased towards T cells and away from all other lineages up to 17 and 23 months. In contrast to the highly biased NK contributors, these T biased clones did not extinguish in the time surveyed so far, and more frequently were detected at low levels in the My/B lineages. The degree of bias of these high contributing clones towards the NK and T lineages was steady over time, suggesting that strong clonal bias is stable. The top 10 clones in NK contributed a significantly greater fraction of hematopoiesis (20±4% and 38±10%) than the top 10 clones in the T lineage (15±2% and 13±2%) in both animals (p=0.01, p<0.0001). In all lineages at all time points from 1 to 23 months, a small subset (14±3%) of clones detected at each individual time point contributed at least 50% of barcoded hematopoiesis. Polyclonality increased following initial hematopoietic reconstitution but started to fall around 6 months, reflecting the increased contribution of certain high contributing, totipotent clones. These clones did not extinguish after beginning to contribute, and have characteristics of long-term repopulating HSCs. We observed novel instability in the lineage bias of some individual HSPC contributions. For instance, an early totipotent clone which was a top 10 producer of granulocytes halted B cell contribution almost completely at 4.5 months, before regaining it by 5 months later and becoming the highest contributing clone in the entire animal. The high degree of bias towards and away from certain lineages observed in many long term repopulating HSCs is not necessarily in violation of hypothesized progenitor hierarchies, but it indicates the presence of poorly understood nuances in clonal control. In addition to the long-term dominant unbiased HSCs whose hematopoietic contribution increased over time, we noticed a cohort of clones biased towards a combined myeloid/B cell lineage. The varied clonal patterns observed between animals could result from individual differences in transplant progenitor composition or dose, or degree of recipient endogenous HSPC depletion. Understanding the lifespan and population dynamics of repopulating clones in macaques after transplantation is relevant for optimizing human hematopoietic stem cell transplants and also provides an approach for identifying progenitor populations, inferring mechanisms of cell fate control, and calculating rates of differentiation. Disclosures No relevant conflicts of interest to declare.

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37

Aab, A., O. Wirz, W. van de Veen, S. Söllner, B. Stanic, B. Rückert, J. Aniscenko et al. "Human rhinoviruses enter and induce proliferation of B lymphocytes". Allergy 72, n.º 2 (8 de junio de 2016): 232–43. http://dx.doi.org/10.1111/all.12931.

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38

McGinnes, K., M. Letarte y CJ Paige. "B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors". Blood 77, n.º 5 (1 de marzo de 1991): 961–70. http://dx.doi.org/10.1182/blood.v77.5.961.961.

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Abstract We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (Ig) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10+ and CD20+ B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+), plasma cells (PCA-1+), or myeloid cells (CD13+) did not lead to a significant depletion in B-cell colony formation. Pre-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferate into Ig- secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of Ig kappa and Ig lambda from single colonies. These experiments provide evidence that the CD10 antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into Ig-secreting cells.

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39

McGinnes, K., M. Letarte y CJ Paige. "B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors". Blood 77, n.º 5 (1 de marzo de 1991): 961–70. http://dx.doi.org/10.1182/blood.v77.5.961.bloodjournal775961.

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We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (Ig) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10+ and CD20+ B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+), plasma cells (PCA-1+), or myeloid cells (CD13+) did not lead to a significant depletion in B-cell colony formation. Pre-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferate into Ig- secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of Ig kappa and Ig lambda from single colonies. These experiments provide evidence that the CD10 antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into Ig-secreting cells.

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40

Lin, Shan, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy y Michael J. Thirman. "Lymphoid Lineage Preference of MLL-AF4 Is Revealed in a Species-Specific Model". Blood 126, n.º 23 (3 de diciembre de 2015): 2454. http://dx.doi.org/10.1182/blood.v126.23.2454.2454.

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Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes that drive both acute myeloid and acute lymphoid leukemia (AML and ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (also referred to as AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently identified in AML, for example MLL-AF9 caused by t(9;11)(p22;q23), MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It is the most frequent MLL fusion in ALL and accounts for 10-15% of ALL cases. Patients with t(4;11) have a poor prognosis compared with other cytogenetically defined subsets. Although many MLL-fusion leukemia models have successfully been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful human cell based model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Transduced with a modified MLL-AF4 fusion gene, human hematopoietic CD34+ cells successfully initiate ALL in a xenograft system with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells with MLL-AF4 results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNAseq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusions in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to reprogramming in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Strikingly, this decreased BMI1 expression is evident in primary B-ALL patient samples as well, with MLL-AF9 B-ALL samples demonstrating increased BMI1 relative to MLL-AF4. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11) oncogene and highlight the limitations of using murine cells in human disease modeling. The model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. Disclosures Thirman: AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead: Research Funding.

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Hebert, J., JM Cayuela, J. Berkeley y F. Sigaux. "Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias [see comments]". Blood 84, n.º 12 (15 de diciembre de 1994): 4038–44. http://dx.doi.org/10.1182/blood.v84.12.4038.bloodjournal84124038.

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Using a Southern blot approach, deletions of MTS1 (multiple tumor- suppressor gene 1) and MTS2 (multiple tumor-suppressor gene 2) candidate tumor-suppressor genes have been studied in primary neoplastic cells from 55 acute lymphoblastic leukemia (ALL) patients. Homozygous MTS1 deletions were found in 20 of 24 T-ALL cases and in only 2 of 31 B-lineage cases (P < .001). The deletions involved MTS1 and MTS2 in most cases. Homozygous MTS2 deletions were observed in 16 of 24 T-ALL cases and in 1 of 31 B-lineage ALLs (P < .001), all of them displaying homozygous MTS1 deletions. In 5 cases (4 T and 1 B), deletions involved MTS1 but spared the MTS2 gene, showing that one deletion breakpoint was located between the two genes within a 25-kb region. In 1 T-ALL case, an MTS1 gene rearrangement has occurred downstream to exon 2. Possible hemizygous deletions were found in 6 cases, 4 of them of the B-cell lineage. In 7 ALL cases, cells obtained at presentation and at first relapse were studied and identical results were observed in 6 cases. In 1 B-lineage case, a germline pattern was found at presentation and a possible monoallelic MTS1/MTS2 deletion was observed at relapse. The high frequency of MTS1 and MTS2 homozygous deletions in T-ALLs supports the view that inactivation of these genes plays an important role in the pathogenesis of this type of human leukemia.

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Mårtensson, Inga-Lill, Fritz Melchers y Thomas H. Winkler. "A Transgenic Marker for Mouse B Lymphoid Precursors". Journal of Experimental Medicine 185, n.º 4 (17 de febrero de 1997): 653–62. http://dx.doi.org/10.1084/jem.185.4.653.

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Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5′ of the precursor (pre)–B cell–specific λ5 gene. All three strains express human CD25 in parallel to endogenous λ5 on pre–B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19−λ5+ precursor of the B220+CD19+λ5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19− precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre–BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5′ region of the λ5 gene into the mouse genome confers tissue and differentiation stage–specific expression of a transgene.

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Tsuganezawa, Keiko, Nobutaka Kiyokawa, Yoshinobu Matsuo, Fujiko Kitamura, Noriko Toyama-Sorimachi, Keisuke Kuida, Junichiro Fujimoto y Hajime Karasuyama. "Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor". Blood 92, n.º 11 (1 de diciembre de 1998): 4317–24. http://dx.doi.org/10.1182/blood.v92.11.4317.

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Abstract Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.

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44

Tsuganezawa, Keiko, Nobutaka Kiyokawa, Yoshinobu Matsuo, Fujiko Kitamura, Noriko Toyama-Sorimachi, Keisuke Kuida, Junichiro Fujimoto y Hajime Karasuyama. "Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor". Blood 92, n.º 11 (1 de diciembre de 1998): 4317–24. http://dx.doi.org/10.1182/blood.v92.11.4317.423k43_4317_4324.

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Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.

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45

Shah, V. O., C. I. Civin y M. R. Loken. "Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis." Journal of Immunology 140, n.º 6 (15 de marzo de 1988): 1861–67. http://dx.doi.org/10.4049/jimmunol.140.6.1861.

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Abstract We have correlated the intensity of expression of CD45 Ag (T200 common leukocyte Ag) with mAb reactive with various lineages of hemopoietic cells in normal human bone marrow by using two-color immunofluorescence on a flow cytometer. Mature T lymphocytes (CD3+) and NK cells (CD16+ or CD11b+) expressed CD45 at the highest intensity. B lymphoid cells (CD19+) had three distinct levels of CD45 Ag expression. The bright CD45(3+) cells were mature B cells (CD19+, CD20+), whereas the less intense CD45(2+) cells were less mature B lymphoid cells (CD19+, CD10+). The dim CD45+ cells were very early, B lymphoid precursor cells (CD19+, CD10(2+), CD34+). The intensity of CD45 expression increased as cells matured in the monocytic lineage (CD14+, CD11b+). Among marrow granulocytic cells, CD45 intensity did not change on cells during maturation. Within the erythroid lineage, the most immature cells were CD45+ dim, and CD45 expression decreased during erythroid maturation to become undetectable on mature E. Hemopoietic progenitor cells (CD34+) expressed low levels of CD45 Ag. Expression of CD45R on marrow cells also showed intensity differences on different lineages. All NK cells (CD16+) were positive for CD45R, whereas only about one-half of the T lymphocytes (CD3+) were positive for CD45R. Almost all the cells in the erythroid and myelomonocytic lineages were CD45R-. Quantitative differences in expression of CD45R were observed on marrow B lymphoid cells which were correlated with the expression of CD45. The results show that quantitative changes in CD45 Ag expression accompany the differentiation and maturation of cells in the bone marrow. Comparisons with CD45R showed that this Ag was not always correlated with CD45. Since these Ag are the products of the same gene, these data indicate that the regulation of expression of the T200 molecules during normal hemopoietic development must be both quantitative and qualitative.

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46

Rossi, Maria Isabel D., Takafumi Yokota, Kay L. Medina, Karla P. Garrett, Philip C. Comp, Arthur H. Schipul y Paul W. Kincade. "B lymphopoiesis is active throughout human life, but there are developmental age-related changes". Blood 101, n.º 2 (15 de enero de 2003): 576–84. http://dx.doi.org/10.1182/blood-2002-03-0896.

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This study addressed several questions concerning age-related changes in human B lymphopoiesis. The relative abundance of pro-B, pre-B, immature, naive, and mature B cells among the CD19+lymphocyte fraction of human bone marrow was found not to change appreciably over the interval between 24 and 88 years of age. Moreover, proliferation of pro-B and large pre-B cells in adult marrow equaled that observed with fetal marrow specimens. Exceptionally low numbers of lymphocyte precursors were found in some marrow samples, and the values obtained were used to determine parameters that best reflect B lymphopoiesis. Cord blood always contained higher incidences of functional precursors than adult cells. However, sorted CD34+ Lin− CD10+ progenitors from cord blood and adult marrow had equivalent potential for differentiation in culture, and notable age-related changes were found in more primitive subsets. A recently described subset of CD34+CD38−CD7+ cord blood cells had no exact counterpart in adult marrow. That is, all adult CD34+Lin−CD7+CD10−cells expressed CD38, displayed less CD45RA, and had little B-lineage differentiation potential. The CD7+ fractions in either site contained progenitors for erythroid and natural killer (NK) lineages, and ones sorted from marrow expressed high levels of transcripts for the CD122 interleukin 2 (IL-2)/IL-15 receptor required by NK-lineage precursors. Dramatic changes in human B lymphopoiesis occur early in life, and more information is required to construct a probable sequence of differentiation events prior to the acquisition of CD10.

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47

Schjerven, Hilde, Etapong F. Ayongaba, Princess Rodriguez y Seth Frietze. "Ikaros regulates epigenetic and transcriptional programs in progenitor B cell leukemia". Journal of Immunology 200, n.º 1_Supplement (1 de mayo de 2018): 103.26. http://dx.doi.org/10.4049/jimmunol.200.supp.103.26.

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Abstract Hematopoietic cell development is tightly controlled by a network of transcriptional and epigenetic regulators, many of which are mutated or have altered expression in hematological malignancies. Ikaros is a transcription factor that is critical for the proper development of several hematopoietic lineages, and essential for the B-cell lineage. It is recognized as a critical tumor suppressor in precursor B-cell lineage acute lymphoblastic leukemia (pre-B ALL), and is emerging to play roles also in other hematopoietic malignancies. In pre-B ALL, Ikaros mutations are particularly prevalent in the Ph+ (BCR-ABL1+) and ‘Ph-like’ subgroups of leukemia, and Ikaros mutations correlate with poor prognosis. To study the mechanisms of Ikaros tumor suppressor function, we developed a mouse model and a human model system with selective perturbation of Ikaros in Ph+ pre-B ALL cells. This has revealed conserved deregulated genes and pathways, and underscored the role of Ikaros in regulating progenitor-restricted gene programs. Furthermore, our recent studies have highlighted the role of Ikaros in regulation of chromatin structure, and revealed a novel role in epigenetic regulation. It is challenging to therapeutically target mutations in a tumor suppressor factor. It is therefore important to understand the mechanism of action and downstream targets to elucidate targetable vulnerabilities of the Ikaros-mutant leukemic cells. Investigating the tumor suppressor role of Ikaros in developing B cells also sheds light on its role in normal B cell development. We are using our newly established models of Ikaros-mutated pre-B ALL to study the underlying molecular mechanisms and downstream targets, and will present our results to-date at the meeting.

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48

Liang, Xueqing, Jakub Tolar, Jeffery S. Miller, Tucker W. LeBien, Bruce R. Blazar y Wei Chen. "Toll-Like Receptor 7-Targeting of Human B-Lineage Acute Lymphocytic Leukemia Induces Immunogeneicity and Apoptosis of Leukemia Cells." Blood 108, n.º 11 (16 de noviembre de 2006): 706. http://dx.doi.org/10.1182/blood.v108.11.706.706.

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Abstract Acute lymphocytic leukemia (ALL) is the most common childhood leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improved cure rates in both pediatric and adult ALL patients. In this study, we show human B-lineage acute lymphocytic leukemia (B-ALL) cell lines (6/6 tested) and CD19+CD10+ primary B-ALL cells from patients (8/8 tested) express TLR7 mRNA by real-time RT-PCR and TLR7 proteins by Western blot. Triggering TLR7 on B-ALL cells with a TLR7 agonist (imiquimod) significantly increases the cell surface expression of molecules essential for T cell activation (CD40, CD54, CD80, CD86, and HLA-DR), the ligands for NKG2D and ligands for natural cytotoxicity receptors (NKp30, NKp44, and NKp46) which regulate NK-mediate killing. Thus, TLR7 signaling enhances the immunogenicity of B-ALL cells and makes them more suitable targets for T cell and NK cell mediated attack. Most importantly, TLR7 agonists strongly suppress in vitro growth of B-ALL cell lines (RS4;11, BLIN-1) and induces profound apoptosis of primary B-ALL cells from patients in culture in a TLR7 agonist dose-dependent manner. Both t(4;11)-positive RS4;11 cells and t(4;11)-negative BLIN-1 cells proliferate rapidly in culture with a 30–40 fold increases of leukemia cell number in 7 days. The addition of TLR7 agonist at 10 ug/ml fully inhibit the growth of RS4;11 and BLIN-1 cells in culture. Furthermore, TLR7 agonist treatment dramatically induces apoptosis of primary B-ALL cells isolated from the patients (2/2 with t(9;22), 6/6 without t(9;22)) with 0.4%–13.3% leukemia cells left at day 5 of culture. The TLR7 agonist-mediated apoptotic death of B-ALL cells was conformed by viable cell counts, TMRE staining, and, Western blots of the activation and cleavage of caspases. To study the in vivo therapeutic effects of TLR7 agonist against human B-ALL, RS4;11 and BLIN-1 cells were luciferase labeled and injected into NOD/SCID mice. Both RS4;11 and BLIN-1 leukemia cells engrafted in multiple organs (BM, spleen, liver, lymph nodes, kidney) resulting in uniform lethality of RS4;11 mice in 8 weeks and BLIN-1 mice in 12 weeks, respectively. Flow cytometry and tissue staining results confirmed that these organs were massively infiltrated with human CD45+19+ leukemia cells. To determine whether TLR7 preincubation of RS4;11 or BLIN-1 cells would prolong survival due to an apoptotic effect, cohorts of mice were injected with a lethal dose of RS4;11 or BLIN-1 cells with or without pre-incubation with TLR7 agonist. Mice receiving TLR7 agonist pre-pretreated B-ALL cells had a significant increase in long-term survival rate and significant reduction in tumor burden at the time points evaluated. These in vivo results confirm previous in vitro findings and suggest that TLR agonist-treated B-ALL cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established B-ALL is being investigated using these xenograft mouse models. These results form the basis for a clinical trial of systemic TLR7 agonist administration for treating patients with B-ALL. In summary, we have shown that TLR7 targeting increases B-ALL immunogenicity and directly induces B-ALL apoptosis, providing new insights into the biology and therapy of B-ALL.

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49

Wang, Chen, Yoshihiro Hayashi, Xiongwei Cai, Xiaomei Yan, Gang Huang y Yi Zheng. "Inducible Correction of a RUNX1 Mutation of Human AML Causes a Switch of AML to B-ALL". Blood 132, Supplement 1 (29 de noviembre de 2018): 1325. http://dx.doi.org/10.1182/blood-2018-99-116919.

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Abstract Background: Point mutations of RUNX1 gene are frequently seen in myeloid malignancies such as myelodysplasia (MDS) and acute myeloid leukemia (AML), and are associated with unfavorable clinical outcomes. AML patients bearing RUNX1 mutations have a significantly lower complete remission (CR) rate compared with patients with wild-type RUNX1 (30% vs. 73%), whereas in contrast, the CR rate of ALL patients, in which RUNX1 is rarely mutated, are at 92%. It remains unclear whether RUNX1 mutations found in AML patients are causal to AML maintenance, and if RUNX1 mutations serve as a valid therapeutic target in the treatment of such therapy-resistant AML. Experimental Approach: The RUNX1 mutations are found frequently associated with MLL-partial tandem duplication (MLL-PTD) in AML. We have developed two complementary mouse models that express a MLL-PTDknock-in mutation together with a RUNX1 S291fsX300 mutation found in AML patients: in the first model RUNX1 S291fsX300 was introduced into the bone marrow cells of the MLL-PTD knock-in mice by retrovirus transduction, while in the second model the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the Collagen a1 locus and the mice was crossed to MLL-PTD knock-in. In the second model, the mutant RUNX1 protein is only expressed when the mice were fed with doxycycline containing food and the RUNX1 mutant is absent or "corrected" upon doxycycline withdrawn. BM cells of these mice, as well as WT and MLL-PTD controls, were transplanted to syngeneic mice and the recipients were tracked for disease development and progression at bi-monthly intervals. The doxycycline induction or withdraw was carried out after secondary transplant, and the expression of RUNX1 S291fsX300 was monitored by a built-in GFP reporter and verified by RT-PCR.Under tetracycline induction, the RUNX1 S291fsX300 and MLL-PTD double mutation bearing mice developed a spontaneously AML and had a survival time 6-10 months after transplantation, and they showed symptoms of MDS/AML or AML, including thrombocytopenia, anemia, leukocytosis, splenomegaly, abnormal BM and spleen cell morphologies. We transplanted the AML mouse bone marrow to secondary recipients, and observed them with or without continuing doxycycline induction. By using the O-propargyl-puromycin to track the newly synthesized proteins, we found that the RUNX1 mut-on and RUNX1 mut-off mice showed comparable protein synthesis rate at full-blown leukemia stage, indicating that the RUNX1 mutation does not play a restrictive role to protein synthesis in transformed leukemia cells. The c-Kit+ AML cells cultured from the RUNX1 mut-off mice showed reduced viability and significantly reduced CFU-C activities, which are accompanied by a lineage switch in a decrease in expression of Mac1 and an increase in expression of B220. In vivo, the RUNX1 mut-off mice showed a slightly prolonged survival compared with RUNX1 mut-on group (182 vs. 126 days, p=0.0036). An analysis of the lineage distribution in their peripheral blood and bone marrow revealed that the RUNX1 mut-off AML mice underwent a dramatic lineage switch from myelocytes to lymphocytes (Fig. 1): while the Mac1+Gr1+population remained high in the RUNX1 mut-on AML mice, turning off RUNX1 mut expression caused an increased B cell population in expense of the Mac1+Gr1+population. Pathological analysis also showed a drastic decrease of the myeloid marker MPO and increased lymphoid marker B220 in the RUNX1mu-off mice. Consistently, a RT-PCR test of the BM c-Kit+ cells further showed that upon turning off the RUNX1mut, critical B cell regulatory genes including TCF3, EBF1, PAX5, CD79A were significantly up regulated whereas myeloid regulatory genes such as PU.1 were downregulated. In addition, RUNX1 S291fsX300 expression in the AML cells confers a resistance to chemotherapy (Ara-C) treatment compared with that of WT RUNX1 expression, indicating that the RUNX1 mutation is responsible for the AML resistance to therapy. Conclusion:Although RUNX1 mutations found in AML patient may not be essential for the hyperproliferative and growth phenotype of the AML, they play a causal role in maintaining myeloid lineage restriction, expansion and chemo-resistance. Correction or removal of the RUNX1 mutation in AML cells leads to a disease switch from AML to B-ALL, effectively changing the difficult-to-treat drug-resistant AML to another disease with more favorable CR (B-ALL). Disclosures No relevant conflicts of interest to declare.

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50

Wallington-Beddoe, Craig T., Kenneth F. Bradstock y Linda J. Bendall. "Identification of Sphingosine Kinases As Therapeutic Targets in B-Lineage Acute Lymphoblastic Leukemia". Blood 120, n.º 21 (16 de noviembre de 2012): 1499. http://dx.doi.org/10.1182/blood.v120.21.1499.1499.

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Abstract Abstract 1499 Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SphK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SphK1. Little is known about the interaction of SphKs with intracellular signalling pathways. Here we assess the relevance of SphKs in B-lineage acute lymphoblastic leukemia (B-ALL). Gene expression signatures indicative of activation of SphK1 or SphK2 were not publicly available. Therefore we treated ALL cell lines with the SphK1 or SphK2 specific inhibitors SK1-I and ABC294640 respectively, and analysed gene expression by microarray. Although a signature for SphK1 was not obtained, two independent methods of analysis generated SphK2 gene signatures that segregated control and drug treated cell lines. The signatures consisted of 11 and 35 genes and included reduced expression of the NF-κB inhibitor TRIB3 (NF-κB activity is commonly up regulated in ALL), the C/EBP family transcription factor DDIT3, the protein phosphatase 1 regulatory subunit PPP1R15A and the sphingolipid/cholesterol transporter ABCA1, all validated by quantitative RT-PCR. These gene signatures were used to interrogate a large publicly available gene expression dataset (GSE28497) obtained from pediatric ALL patients at the time of diagnosis. SphK2 activity signatures were more highly expressed in ALL samples (p=0.001 and p=0.027 for the smaller and larger signatures respectively) than normal B-cell progenitors. Although SPHK1 or SPHK2 genes were not over expressed in this dataset, SphK1 protein levels were increased in ALL cell lines and in patient samples. The importance of SphK1 and SphK2 in the development of ALL was examined by transducing B-cell progenitors isolated from WT, SphK1−/− or SphK2−/− mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated wild type C57BL/6 mice. Twenty-two of 29 mice receiving cells from WT animals developed ALL, with a mean survival of 52.6 days (95% CI 42.3–62.8 days). Mice receiving cells lacking Sphk1 or Sphk2 had a significantly reduced incidence of ALL development with 14 of 30 (mean survival 71.6 days, 95% CI 60.5–82.8 days) and 16 of 29 animals (mean survival of 68.7 days, 95% CI 57.9–79.5 days) respectively (p=0.001). Lymphoblasts with a B-cell progenitor phenotype (B220+, CD19+, IgM−, CD11b−) were present in blood films and livers from leukemic mice and cells recovered from these animals produced a rapidly fatal ALL in secondary recipients. The presence of BCR/ABL and the expected deletion of Sphk1 or Sphk2 were confirmed by PCR in all murine leukemias examined. We have previously reported that inhibitors of SphK1 and/or SphK2 inhibit proliferation and induce cell death in ALL cell lines, and that these agents can synergize with imatinib in Ph+ ALL cell lines. We have furthered these studies to demonstrate that patient derived ALL cells similarly respond to SphK1 and SphK2 inhibition in vitro. The SphK2 inhibitor ABC294640 (100mg/kg) reduced plasma S1P levels in mice consistent with our previous reports of reduced ALL burden following ABC294640 treatment, and using a NOD/SCID γc−/− xenograft model of human ALL we have now shown that ABC294640 extends survival of ALL-bearing mice (p=0.0012 for Ph− xenograft) and further extends the survival when combined with imatinib treatment in mice engrafted with a human Ph+ ALL patient sample (p=0.044). This is the first report to suggest that sphingosine kinase 2 activity is increased in ALL, providing support for targeting SphK2 as a therapeutic strategy. Loss of SphKs reduces the incidence of ALL in a murine model of BCR/ABL-driven disease and ABC294640 reduces disease and extends survival in a human xenograft model of ALL. This compound synergizes with a number of potential therapeutic agents and further extends survival in a xenograft model of Ph+disease when combined with imatinib. This has potential to translate into a useful anti-leukemic strategy. Disclosures: No relevant conflicts of interest to declare.

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