IKAROS and BCL6 Limit Pre-B Cell Expansion and Prevent Leukemogenesis Downstream of the Pre-B Cell Receptor

The pre-B cell receptor promotes differentiation of normal pre-B cells and induces cell cycle arrest at the transition from large cycling pre-B cells (Fraction C') to small resting pre-B cells (Fraction D). While pre-B cell receptor-induced cell cycle arrest represents a critical safeguard against pre-B cell leukemogenesis, the mechanism of pre-B cell receptor-dependent tumor suppression is only poorly understood. We recently established that pre-B cell receptor signaling leads to upregulation of Ikaros (Trageser et al., J Exp Med, 2009). Ikaros functions as a tumor suppressor in BCR-ABL1 pre-B ALL and is deleted in >80% of the cases. In addition, we recently reported that BCL6 is upregulated during pre-B cell receptor-induced cell cycle arrest (Duy et al., J Exp Med 2010).

To elucidate the mechanism of pre-B cell receptor-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of Stat5-phosphorylation as a central mediator of survival and proliferation downstream of the BCR-ABL1 kinase. Forced expression of the pre-B cell receptor resulted in rapid dephosphorylation of Stat5 Y694 and concomitant upregulation of BCL6. Pre-B cell receptor-mediated upregulation of BCL6 was sensitive to expression of a constitutively active mutant of Stat5. Therefore, upregulation of BCL6 likely occurs indirectly through dephosphorylation of Stat5 downstream of the pre-B cell receptor. Upregulation of BCL6 is indeed causally linked to pre-B cell receptor-induced cell cycle arrest: By genome-wide ChIP-on-chip analysis and single-locus qChIP verification, we observed direct recruitment of the BCL6 transcriptional repressor to the promoter regions of CCND2 and MYC, which represent central mediators of cell cycle progression in BCR-ABL1 ALL. The negative effect of BCL6 on cell cycle progression was confirmed by retroviral overexpression, which induced cell cycle arrest in the vast majority of BCR-ABL1 ALL cells. In addition, overexpression of Myc could rescue BCL6-dependent cell cycle arrest downstream of the pre-B cell receptor. To verify the role of BCL6 in negative cell cycle regulation in a genetic experiment, we tested the function of the pre-B cell receptor in BCL6+/+ and BCL6-Null BCR-ABL1-transformed pre-B ALL cells. Forced expression of the pre-B cell receptor rapidly induced cell cycle arrest in BCL6+/+ but not BCL6-Null pre-B ALL cells. We conclude that upregulation of BCL6 leads to transcriptional repression of Myc/CCND2 and is required for pre-B cell receptor-mediated cell cycle arrest. Since our experiments established that BCL6 upregulation required Stat5-dephosphorylation, we next studied how pre-B cell receptor signaling leads to dephosphorylation of Stat5 and, hence, transcriptional activation of BCL6 as key effector to induce cell cycle arrest. Surprisingly, this analysis identified Ikaros as the key-mediator of Stat5-dephosphorylation is direct cooperation with the pre-B cell receptor signaling cascade. Reconstitution of Ikaros expression resulted in dramatic Stat5-dephosphorylation, which was comparable to the effect of Imatinib. Ikaros-dependent Stat5-dephosphorylation directly intersects with the pre-B cell receptor signaling pathway, because the pre-B cell receptor-associated linker molecule BLNK (SLP65) is required for Ikaros-mediated dephosphorylation. In BLNK-Null BCR-ABL1 pre-B ALL cells, Ikaros expression did neither affect Stat5-phosphorylation nor proliferation and survival of leukemia cells. As an indirect consequence of Stat5-dephosphorylation, Ikaros/BLNK signaling resulted in upregulation of BCL6 and subsequent cell cycle arrest.

The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of BCR-ABL1-driven pre-B ALL, however, the mechanisms of Ikaros-dependent tumor suppression remained elusive. Here we describe for the first time that Ikaros functions as tumor suppressor via dephosphorylation of Stat5. Thereby, the Ikaros tumor suppressor requires direct interaction with the pre-B cell receptor signaling pathway including BLNK. Ikaros/BLNK inactivate Stat5 and, hence, a critical survival and proliferation signal. In addition, Ikaros/BLNK signaling leads to activation of BCL6, which functions as negative regulator of Myc/CCND2-dependent proliferation.

No relevant conflicts of interest to declare.