Publications
44 results found
Sprangers M, Feldhahn N, Liedtke S, et al., 2006, SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells, Oncogene, Vol: 25, Pages: 5180-5186, ISSN: 0950-9232
Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.
Klein F, Feldhahn N, Herzog S, et al., 2006, BCR-ABL1 induces aberrant splicing of IKAROS and lineage infidelity in pre-B lymphoblastic leukemia cells, Oncogene, Vol: 25, Pages: 1118-1124, ISSN: 0950-9232
Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.
Soh BN, Klein F, Feldhahn N, et al., 2005, B-lymphoid or myeloid lineage identity of cell lines derived from chronic myeloid leukemia blast crisis, Cancer Genet Cytogenet, Vol: 161, Pages: 187-188, ISSN: 0165-4608
Feldhahn N, Rio P, Soh BN, et al., 2005, Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells, Proc Natl Acad Sci U S A, Vol: 102, Pages: 13266-13271, ISSN: 0027-8424
Bruton's tyrosine kinase (BTK) deficiency results in a differentiation block at the pre-B cell stage. Likewise, acute lymphoblastic leukemia cells are typically arrested at early stages of B cell development. We therefore investigated BTK function in B cell precursor leukemia cells carrying a BCR-ABL1, E2A-PBX1, MLL-AF4, TEL-AML1, or TEL-PDGFRB gene rearrangement. Although somatic mutations of the BTK gene are rare in B cell precursor leukemia cells, we identified kinase-deficient splice variants of BTK throughout all leukemia subtypes. Unlike infant leukemia cells carrying an MLL-AF4 gene rearrangement, where expression of full-length BTK was detectable in only four of eight primary cases, in leukemia cells harboring other fusion genes full-length BTK was typically coexpressed with kinase-deficient variants. As shown by overexpression experiments, kinase-deficient splice variants can act as a dominant-negative BTK in that they suppress BTK-dependent differentiation and pre-B cell receptor responsiveness of the leukemia cells. On the other hand, induced expression of full-length BTK rendered the leukemia cells particularly sensitive to apoptosis. Comparing BTK expression in surviving or preapoptotic leukemia cells after 10-Gy gamma radiation, we observed selective survival of leukemia cells that exhibit expression of dominant-negative BTK forms. These findings indicate that lack of BTK expression or expression of dominant-negative splice variants in B cell precursor leukemia cells can (i) inhibit differentiation beyond the pre-B cell stage and (ii) protect from radiation-induced apoptosis.
Klein F, Feldhahn N, Mooster JL, et al., 2005, Tracing the pre-B to immature B cell transition in human leukemia cells reveals a coordinated sequence of primary and secondary IGK gene rearrangement, IGK deletion, and IGL gene rearrangement, J Immunol, Vol: 174, Pages: 367-375, ISSN: 0022-1767
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, Ckappa and Clambda germline transcription, and rearrangement of Ig kappa L chain (IGK) and Ig lambda L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed lambda L, but almost no kappa L chains. This could be explained by STI571-induced rearrangement of the kappa-deleting element (KDE), which can delete productively rearranged Vkappa-Jkappa joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of Vkappa-Jkappa joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of Ckappa and Clambda germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located Jkappa- to downstream Jkappa-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.
Feldhahn N, Klein F, Mooster JL, et al., 2005, Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic leukemia cells, J Exp Med, Vol: 201, Pages: 1837-1852, ISSN: 0022-1007
Pre-B cells undergo apoptosis unless they are rescued by pre-B cell receptor-dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre-B lymphoblastic leukemia bypasses selection for pre-B cell receptor-dependent survival signals. Investigating possible interference of BCR-ABL1 with pre-B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre-B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre-B cell receptor, including activation of PLCgamma1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLX(L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1+ leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1-dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre-B cell receptor.
Klein F, Feldhahn N, Wernet P, et al., 2004, The BCR-ABL1 kinase interferes with pre-B cell receptor signal transduction in B cell precursor leukemia cells., 46th Annual Meeting of the American-Society-of-Hematology, Publisher: AMER SOC HEMATOLOGY, Pages: 524A-525A, ISSN: 0006-4971
Klein F, Feldhahn N, Mooster JL, et al., 2004, Tracing the pre-B to immature B cell transition in human leukemia cells reveals a coordinated sequence of primary and secondary IGK gene rearrangement, <i>IGK</i> deletion and <i>IGL</i> gene rearrangement., 46th Annual Meeting of the American-Society-of-Hematology, Publisher: AMER SOC HEMATOLOGY, Pages: 94A-94A, ISSN: 0006-4971
Klein F, Feldhahn N, Harder L, et al., 2004, The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells, J Exp Med, Vol: 199, Pages: 673-685, ISSN: 0022-1007
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre-B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre-B ALL and normal bone marrow pre-B cells by serial analysis of gene expression, many genes involved in pre-B cell receptor signaling are silenced in the leukemia cells. Although normal pre-B cells are selected for the expression of a functional pre-B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre-B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre-B cell receptor signaling and the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre-) B cell receptor.
Kogler G, Sensken S, Airey JA, et al., 2004, A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential, J Exp Med, Vol: 200, Pages: 123-135, ISSN: 0022-1007
Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.
Su YW, Herzog S, Lotz M, et al., 2004, The molecular requirements for LAT-mediated differentiation and the role of LAT in limiting pre-B cell expansion, Eur J Immunol, Vol: 34, Pages: 3614-3622, ISSN: 0014-2980
Successful recombination of the heavy-chain locus in developing B cells results in the expression of the pre-BCR, which induces the proliferation and expansion of pre-B cells. To avoid uncontrolled proliferation, pre-BCR signals transmitted via the adaptor protein SLP-65 (SH2-domain-containing leukocyte protein of 65 kDa) lead to the down-regulation of pre-BCR expression and to pre-B cell differentiation. Here, we show that, similarly to SLP-65, the adaptor protein LAT (linker for activation of T cells) limits pre-B cell proliferation and reduces the potential of a tumorgenic pre-B cell line to develop leukemia in immune-deficient mice. We further show that the four distal tyrosines are required for LAT activity in pre-B cells. Mutation at Y136 completely abolishes LAT activity, whereas single point-mutations at Y175, Y195 or Y235 impair, but do not block, LAT-induced pre-B cell differentiation. As LAT is also expressed in human pre-B cells, our results suggest that LAT cooperates with SLP-65 to promote the differentiation and control the proliferation of both murine and human pre-B cells.
Klein F, Feldhahn N, Muschen M, 2004, Interference of BCR-ABL1 kinase activity with antigen receptor signaling in B cell precursor leukemia cells, Cell Cycle, Vol: 3, Pages: 858-860, ISSN: 1551-4005
The chromosomal translocation t,(9;22) resulting in the fusion of the BCR and ABL1 genes, represents a recurrent aberration in B cell precursor leukemia cells. Their normal counterparts, B cell precursor cells, are positively selected for survival signals through the antigen receptor, whose expression requires a functional immunoglobulin heavy chain (IGH) gene rearrangement. Unexpectedly, B cell precursor leukemia cells harboring a BCR-ABL1 gene rearrangement do not depend on antigen receptor mediated survival signals. Genes involved in the signaling cascade of the antigen receptor are silenced and in most cases, the dominant tumor clone does not carry a functional IGH gene rearrangement. However, upon inhibition of the BCR-ABL1 kinase activity by STI571, only leukemia cells expressing an antigen receptor are able to survive. Since resistance to STI571 is frequent in the therapy of BCR-ABL1(+) B cell precursor leukemia, antigen receptor signaling may represent a mechanism through which these cells can temporarily evade STI571-induced apoptosis. This may open a time frame, during which leukemia cells acquire secondary transforming events that confer definitive resistance to STI571.
Klein F, Feldhahn N, Lee S, et al., 2003, T lymphoid differentiation in human bone marrow, Proc Natl Acad Sci U S A, Vol: 100, Pages: 6747-6752, ISSN: 0027-8424
The unique role of the thymus in the development of T cells was established >4 decades ago. To elucidate how uncommitted lymphoid progenitor cells are instructed to migrate from bone marrow to the thymus to undergo T lymphoid differentiation, we generated and analyzed a genome-wide gene expression profile of CD7+ CD10+ human bone marrow T cell lineage precursors (TLPs) by using the serial analysis of gene expression technique. Unexpectedly, the serial analysis of gene expression profile identified a high number of (pre-) T cell receptor antigen (TCR)-related transcripts in bone marrow TLPs. To determine the configuration of the TCRbeta locus in these cells at a quantitative level, we sorted and analyzed bone marrow TLPs from five donors by single-cell PCR. Similar proportions of TLPs harbored TCRbeta germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRbeta rearrangement status, suggesting an active recombination machinery that is consistent with the expression of RAG1, RAG2, and TdT in this population. As a hallmark of ongoing TCRbeta V-DJ rearrangement, we could amplify broken-ended recombination-signal sequence DNA intermediates from bone marrow TLPs, but not from mature T cells by ligation-mediated PCR. Approximately half of the TCRbeta rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Talpha on bone marrow TLPs as shown by confocal laser microscopy and flow cytometry. At a frequency <0.5% of mononucleated cells in human bone marrow, this population is rare, yet it exemplifies T lymphoid differentiation in the human already before immigration into the thymus.
Muschen M, Lee S, Zhou G, et al., 2002, Molecular portraits of B cell lineage commitment, Proc Natl Acad Sci U S A, Vol: 99, Pages: 10014-10019, ISSN: 0027-8424
In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in PBCs include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16, and TCL1, the role of which has not been established yet in early B cell development.
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