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

Abstract

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, Cκ and Cλ germline transcription, and rearrangement of Ig κ L chain (IGK) and Ig λ L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed λ L, but almost no κ L chains. This could be explained by STI571-induced rearrangement of the κ-deleting element (KDE), which can delete productively rearranged Vκ-Jκ 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 Vκ-Jκ joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of Cκ and Cλ 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 Jκ- to downstream Jκ-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.

Lambda Light Chain Revision in the Human Intestinal IgA Response

“…. The only bias observed was a tendency not to observe J1 in the non-productive rearrangements, consistent with editing of these rearrangements as seen in another study (7,8). In most cells, the somatic hypermutation mechanism was only active on productive rearrangements of VJ (C), presumably due to the activity of the KDE in non-productive rearrangements.…”

“…If IgH rearrangement is successful, rearrangement at the Ig L chain () locus is initiated and if rearrangement of Ig is productive, the B cell matures expressing Ig. If rearrangement fails at both Ig alleles, despite potential editing at the Ig locus, rearrangement at the Ig () locus proceeds, and if this is successful an Ig expressing B cell is generated (6,7). Most nonproductive rearrangements at the loci are inactivated by recombination of the -deleting element (KDE) that excises the constant region and the intronic enhancer from non-functional rearrangements, thus disabling somatic hypermutation, which is dependent on the intronic enhancer (7,8).…”

Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells

“…As shown by us and others (16,19), inhibition of oncogenic kinase activity in BCR-ABL1 ϩ leukemia cells by STI571 induces differentiation in a minor population of cells that survive in the absence of BCR-ABL1 kinase activity. Therefore, STI571-induced differentiation in BCR-ABL1 ϩ pre-B lymphoblastic leukemia cells was used as a model to study the effect of kinase-deficient BTK on differentiation.…”

“…The linker was constructed by annealing the oligonucleotides 5Ј-TTTCTGCTCGAATTCAAGCTTCTAAC-GATGTACGGGGACATG-3Ј and 3Ј amino (C7)-GAC-GAGCTTAAGTTCGAAGATTGCTACATGCCCCT-5Ј, and protruding 3Ј overhangs were removed by 3Ј3 5Ј exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I (Invitrogen). Ligation-mediated PCR (15) was carried out with modifications as described (16). In two seminested rounds of amplification at an annealing temperature of 59°C, recombination signal sequence (RSS) intermediates with a DNA double-strand break at the 5Ј heptamer of J 1 gene segments were amplified (see Fig.…”

“…Among the remaining cells, we sorted annexin V ϩ preapoptotic cells and annexin V Ϫ viable cells and compared BTK splice variant expression between preapoptotic and surviving leukemia cells. In two different PCR approaches, the entire kinase domain (using primers for exons [12][13][14][15][16][17][18][19] and at a higher level of resolution, a hotspot region of aberrant splicing comprising exons 14-17 was amplified. Consistent with a protective effect against ␥ radiation-induced apoptosis, surviving leukemia cells exhibit preferential expression of kinase-deficient dominant-negative BTK splice variants (Fig.…”