RNA Interferenz-Untersuchungen zur Entstehung von B-, T- und myeloischen Vorläuferzellen im Knochenmark
Final Report Abstract
This project focused on the regulation of lymphoid lineage commitment in human bone marrow. The studies identified a yet unrecognized T lymphoid progenitor cell population in human bone marrow, which could give rise to “extrathymic” T cell development and also highlighted the role of IKAROS in normal lymphoid vs myeloid lineage commitment. In the absence of IKAROS function, which is a frequent feature in acute lymphoblastic feature, lineage commitment is compromised and the leukemia cells exhibit “lineage infidelity”. 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 TCRß 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 TCRß germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRß 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 TCR? 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 TCRß rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Tß 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. 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.
Publications
- T lymphoid differentiation in human bone marrow. Proc Natl Acad Sci USA 100:6747-6752, 2003
Barath VS, Kronke M, Wernet P, Rowley JD, Müschen M
- Interference of BCR-ABL1 kinase activity with antigen receptor signaling in B cell precursor leukemia cells. Cell Cycle 3:858-860, 2004
Klein F, Feldhahn N, Müschen M
- 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 199:673-685, 2004
Klein F, Feldhahn N, Harder L, Wang H, Wartenberg M, Hofmann WK, Wernet P, Siebert R, Müschen M
- Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells. Proc Natl Acad Sci USA 102:13266-13271, 2005
Feldhahn N, Rio P, Soh BN, Liedtke S, Sprangers M, Klein F, Wernet P, Jumaa H, Hofmann WK, Hanenberg H, Rowley JD, Müschen M
- Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic leukemia cells. J Exp Med 201:1837-1852, 2005
Feldhahn N, Klein F, Mooster JL, Hadweh P, Sprangers M, Wartenberg M, Bekhite MM, Hofmann WK, Herzog S, Jumaa H, Rowley JD, Müschen M
- 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 174:367-375, 2005
Klein F, Feldhahn N, Mooster JL, Sprangers M, Hofmann WK, Wernet P, Wartenberg M, Müschen M
- BCR-ABL1 induces aberrant splicing of IKAROS and lineage infidelity in pre-B lymphoblastic leukemia cells. Oncogene 25:1118-1124, 2006
Klein F, Feldhahn N, Herzog S, Sprangers M, Mooster JL, Jumaa H, Müschen M
- SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells. Oncogene 25:5180-5186, 2006
Sprangers M, Feldhahn N, Liedtke S, Jumaa H, Siebert R, Müschen M
- The SRC family kinase LYN redirects B cell receptor signaling in human SLP65-deficient B cell lymphoma cells. Oncogene 25:5056-5062, 2006
Sprangers M, Feldhahn N, Herzog S, Hansmann ML, Reppel M, Hescheler J, Jumaa H, Siebert R, Müschen M
