PU.1 Opposes IL-7–Dependent Proliferation of Developing B Cells with Involvement of the Direct Target Gene Bruton Tyrosine Kinase

Abstract

Deletion of genes encoding the E26 transformation-specific transcription factors PU.1 and Spi-B in B cells (CD19-CreΔPB mice) leads to impaired B cell development, followed by B cell acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 wk. However, little is known about the target genes that explain leukemogenesis in these mice. In this study we found that immature B cells were altered in frequency in the bone marrow of preleukemic CD19-CreΔPB mice. Enriched pro–B cells from CD19-CreΔPB mice induced disease upon transplantation, suggesting that these were leukemia-initiating cells. Bone marrow cells from preleukemic CD19-CreΔPB mice had increased responsiveness to IL-7 and could proliferate indefinitely in response to this cytokine. Bruton tyrosine kinase (BTK), a negative regulator of IL-7 signaling, was reduced in preleukemic and leukemic CD19-CreΔPB cells compared with controls. Induction of PU.1 expression in cultured CD19-CreΔPB pro–B cell lines induced Btk expression, followed by reduced STAT5 phosphorylation and early apoptosis. PU.1 and Spi-B regulated Btk directly as shown by chromatin immunoprecipitation analysis. Ectopic expression of BTK was sufficient to induce apoptosis in cultured pro–B cells. In summary, these results suggest that PU.1 and Spi-B activate Btk to oppose IL-7 responsiveness in developing B cells.

PU.1 Regulates Ig Light Chain Transcription and Rearrangement in Pre-B Cells during B Cell Development

“…The 660BM and i660BM cell lines used in this study were described previously (19). Cells were cultured in IMDM (Wisent, QC, Canada) containing 5% IL-7-conditioned medium from the J558L-IL-7 cell line (21), 10% FBS (Wisent), 13 penicillin/streptomycin/L-glutamine (Lonza, Shawinigan, QC, Canada), and 5 3 10 25 M 2-ME (Sigma-Aldrich, St. Louis, MO).…”

“…We previously generated mice that delete Spi1 encoding PU.1 under control of CD19-Cre on a Spib 2/2 background (18). These mice had impaired development of follicular B cells and perturbation of BM B cell development, demonstrating a complementary role for PU.1 and Spi-B in B cell development and function (18,19). However, CD19-Cre does not delete alleles efficiently in BM (20), precluding an examination of PU.1 and Spi-B function in early B cell development.…”

Deletion of genes encoding PU.1 and Spi-B leads to B cell acute lymphoblastic leukemia associated with driver mutations in Janus Kinases

“…However, PU.1 and Spi-B are involved in attenuating IL-7R signaling in developing pro- and pre-B cells. Target genes of PU.1 and Spi-B involved in regulating IL-7-induced proliferation include Btk encoding the tumour suppressor Bruton tyrosine kinase 15 , and Blnk encoding B Cell Linker Protein 45 . Btk and Blnk work together to attenuate IL-7R signaling in developing B cells 46 .…”

“…These two proteins share a conserved DNA binding domain and interact with an overlapping set of DNA binding sites within the genome 12 . PU.1 and Spi-B complement one anothers function, and activate multiple genes involved in B cell receptor signaling 12–15 . Lack of these factors in developing B cells results in a block to B development at the small pre-B cell stage associated with impaired Ig light chain rearrangement 15,16 .…”

“…PU.1 and Spi-B complement one anothers function, and activate multiple genes involved in B cell receptor signaling 12–15 . Lack of these factors in developing B cells results in a block to B development at the small pre-B cell stage associated with impaired Ig light chain rearrangement 15,16 . Importantly, conditional deletion of Spi-B and PU.1 in developing B-cells leads to high incidence of B-ALL in mice, but the mechanisms of leukemogenesis in the absence of these transcription factors are still undetermined 17 .…”