I. Piec, S. Ibata, C. Alleaume, B. Brassard, V. Fuentes, F. Gouilleux, H. Bouhlal and K. Lassoued

1. Pre-B Cell Receptor promotes PI3Kinase/Akt,Ras/MAPKinase and NFkB activation in a Syk-dependent manner and downmodulation of RAG1/2 gene transcription. I. Piec, S. Ibata, C. Alleaume, B. Brassard, V. Fuentes, F. Gouilleux, H. Bouhlal and K. Lassoued INSERM E0351, Laboratory of Immunology, University Picardie Jules Verne, 80000 Amiens, FRANCE Introduction Effect of Pre-BCR stimulation on cell proliferation and cell cycle Analysis of the early steps of the Pre-BCR signaling cascade Pre-B Cell Receptor (pre-BCR) acts as a critical checkpoint guards in pre-B cell selection and expansion. Other functions have been attributed to this receptor including a role in Pre-B cell survival, allelic exclusion, VH repertoire shaping, surrogate light chain (yLC), and RAG1/2 gene regulation. However its functional role is still not fully elucidated and the molecular events that follow its stimulation remain largely unknown. Here, we aimed to extensively analyse the signaling cascade associated to human pre-BCR activation. A C A Methods Pre-BCR aggregation induced (A) cell proliferation, (B) cell cycle progression and (C) the phosphorylation of p21kip1 and the downmodulation of p27waf1 but no modification in c-myc, cyclin D1 and D2 expression and Rb phosphorylation (not shown). For this purpose we used the 697 and Nalm6 human pre-B cell lines and pre-BCR was cross-linked using anti-mu F(ab’)2 antibody (20µg/mL). Pre-BCR stimulation results in a rapid phosphorylation of the Lyn and Blk Src kinases, Syk, ZAP70, BLNK, LAT, Btk and Vav. PI3K/Akt Pathway Ras/MAP Kinase pathway NFkB pathway Pre-BCR regulates RAG1 and 2 gene expression Pre-BCR stimulation results in a downmodulation of RAG1 and RAG2 (as well as ZAP70 and Pax5, not shown) but not of E2a transcripts. EBF, CD79a/b, l5, Vpre-B, Myb, MAZ, LEF1, SP1 transcript expression was also unchanged (not shown). Pre-BCR crosslinking induces phosphorylation of Bcl2 on Ser70 Pre-BCR crosslinking induces phosphorylation of Bcl2 on Ser70 but did not alter Bcl2, Bclx, Bax and Mcl1 expression. Pre-BCR crosslinking induces rapid phosphorylation of Akt and its downstream substrates (GSK3 and FKHRL1), as well as phosphorylation of Ras, p38 MAPKinase and Erk1/2. It also induces a late activation of the canonical NFkB pathway. Effects of Akt inhibition on Pre-BCR functions Syk plays a crucial role in Pre-BCR functions The BAY 6131-06 Syk inhibitor, abrogates BLNK phosphorylation and activation of the PI-3Kinase, MAPKinase and NFkB pathways. LY 294002 or a Dominant Negative (DN) form of Akt inhibits partially cell proliferation and cell cycle progression. NFkB, but not Erk1/2 activation is inhibited by the LY294002 Akt inhibitor. Regulatory aspects of Pre-BCR signaling Conclusion Effects of Src kinase inhibitors on Syk phosphorylation Pre-BCR agregation results in a rapid activation of Syk (ZAP70) Tyrosine Kinases which in turn activates the PI3K/Akt, Ras/MAPK and more lately the NFkB pathways. It also induces cell cycle progression, pre-B cell proliferation and downmodulation of RAG1/2 but not yLC gene expression. c-Cbl and SHIP which are both phosphorylated upon pre-BCR crosslinking are likely involved in regulation of pre-BCR signaling. Further experiments are required to identify new factors implicated in pre-BCR signaling and to better characterize its functions. Experiments are also needed to precise the role of major signaling molecules activated by pre-BCR in human lymphopoïesis and pathology of lymphoid malignancies. The PP2 Src inhibitor, but not PP1, inhibits pre-BCR-induced Syk phospohorylation. Pre-BCR agregation results in a rapid phosphorylation of c-Cbl and its association with Syk and ZAP70. It also induces phosporylation of SHIP1.