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.

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells in humans 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 light chain-associated transcription factors IRF4 and SPIB. Upregulation of RAG1 and RAG2, Cκ and Cλ germline transcription and rearrangement of IGK and IGL genes were also observed in cell culture experiments. However, STI571-treated pre-B ALL cells expressed λ- but almost no κ-light chains. This could be explained by STI571-induced rearrangement of the κ-deleting element (KDE), which can delete productively rearranged Vκ-Jκ joints. Amplifying short-lived DNA intermediates from 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 is already initiated within one hour after STI571-treatment, followed by KDE-mediated deletion of Vκ-Jκ-joints six hours later and, ultimately, IGL gene rearrangement after 12 hours. Consistently, upregulation of Cκ and Cλ germline transcripts, indicating opening of IGK and IGL loci, was detected after one and six hours 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 i.) recapitulates early B cell development, ii.) directly shows that IGK, KDE and IGL genes are rearranged in sequential order and iii.) provides a model for Ig light chain gene regulation in the human.

Figure Legend:BCR-ABL1⁺ BV173, NALM1 and SUP-B15 cells were were cultured in the presence or absence of 10 μmol/l STI571 for the times indicated. Genomic DNA was isolated from 5 x 10⁶ cells. Broken-ended DNA strand breaks were ligated to linker molecules and subjected to two rounds of PCR-amplification using primers specific for breaks at Jκ-RSS, KDE-RSS and Jλ-RSS sites.

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