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

Abstract Abstract 146 Background: 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). Result: 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. Conclusion: 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. Disclosures: No relevant conflicts of interest to declare.

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Pre–B cell receptor–mediated cell cycle arrest in Philadelphia chromosome–positive acute lymphoblastic leukemia requires IKAROS function

Journal of Experimental Medicine ◽

10.1084/jem.20090004 ◽

2009 ◽

Vol 206 (8) ◽

pp. 1739-1753 ◽

Cited By ~ 92

Author(s):

Daniel Trageser ◽

Ilaria Iacobucci ◽

Rahul Nahar ◽

Cihangir Duy ◽

Gregor von Levetzow ◽

...

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

Tumor Suppressor ◽

B Cell ◽

Cell Cycle Arrest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Cell Receptor ◽

B Cell Receptor ◽

Cycle Arrest

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph+) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph+ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph+ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph+ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression.

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Mechanisms of Ikaros-Mediated Tumor Suppression

Blood ◽

10.1182/blood.v118.21.408.408 ◽

2011 ◽

Vol 118 (21) ◽

pp. 408-408

Author(s):

Rahul Nahar ◽

Parham Ramezani-Rad ◽

Sinisa Dovat ◽

Maike Buchner ◽

Thomas G Graeber ◽

...

Keyword(s):

Tumor Suppressor ◽

B Cell ◽

Tumor Suppression ◽

Cell Receptor ◽

B Cell Receptor ◽

Leukemia Cells ◽

Receptor Signaling ◽

B Cell Receptor Signaling ◽

Adapter Molecule ◽

Cell Receptor Signaling

Abstract Abstract 408 Background: The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of Ph+ ALL. While Ikaros cooperates with pre-B cell receptor signaling to induce cell cycle exit in Ph+ ALL (Trageser et al., J Exp Med, 2009), the mechanism of Ikaros-mediated tumor suppression is poorly understood. Here we report on a series of genetic experiments that show that Ikaros (i) interferes with key survival pathways downstream of the BCR-ABL1 kinase, (ii) inhibits leukemia cell proliferation through interaction with the pre-B cell receptor signaling pathway and (iii) activates the tumor suppressors p53, p21 and p27. Results: To elucidate the mechanism of Ikaros-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of critical phosphorylation events downstream of the BCR-ABL1 kinase as a central mediators of survival and proliferation. Reconstitution of Ikaros expression in BCR-ABL1-transformed pre-B ALL cells resulted in rapid and global dephosphorylation comparable to the effect of Imatinib. A detailed analysis showed that Ikaros-induced dephosphorylation events affect activation of Stat5 (Y694), AKT (S473), ERK1/2 (T202 and Y204) and SRC (Y416). Interestingly, both Imatinib-treatment and reconstitution of pre-B cell receptor signaling using retroviral vectors for expression of the m heavy chain or the BLNK adapter molecule have the same effects as reconstitution of Ikaros. In fact, a comprehensive gene expression analysis demonstrated that Ikaros reconstitution resulted in similar gene expression changes as reconstitution of pre-B cell receptor signaling (m heavy chain or BLNK), reconstitution of PAX5, Cre-mediated deletion of Stat5 or Myc, or treatment with Imatinib. The signature of common gene expression changes shared between reconstitution of Ikaros, Pax5, m heavy chain, BLNK and inducible deletion of Stat5 or Myc and Imatinib-treatment involves known tumor suppressors including SPIB, BTG1, and BTG2. These findings suggest that reconstitution of tumor suppressive transcription factor (Ikaros, Pax5) converges with pre-B cell receptor-mediated tumor suppression. To better understand how pre-B cell receptor signaling and Ikaros intersect, we combined reconstitution of Ikaros with genetic deletion of either the (more proximal) SYK kinase or the (more distal) BLNK adapter molecule. While inducible Cre-mediated deletion of Syk had no effect on Ikaros-mediated tumor suppression, deletion of the BLNK adapter compromised the ability of Ikaros to function as tumor suppressor. These findings were confirmed in an in vivo transplantation experiment. While mice transplanted with Ikaros+ BLNK+ leukemia cells survived indefinitely, mice transplanted with Ikaros- BLNK+, Ikaros+ BLNK- or Ikaros- BLNK- leukemia cells died after 24 to 31 days post transplantation. While these findings provide genetic evidence for collaboration between the Ikaros and pre-B cell receptor tumor suppressor pathways, Ikaros and pre-B cell receptor signaling differ with respect to activation of classical tumor suppressor pathways. While reconstitution of pre-B cell receptor signaling failed to activate Arf, p53 or p27, protein levels of all these molecules were strongly upregulated by Ikaros. In agreement with these findings, reconstitution of pre-B cell receptor signaling had the same tumor suppressive effect in wildtype leukemia cells as in Arf−/−, p53−/− as well as p27−/− leukemia cells. Conversely, deletion of Arf and p53 significantly diminished the ability of Ikaros to function as tumor suppressor. Conclusion: Ikaros deletion represents a near-obligatory lesion in the pathogenesis of Ph+ ALL. Here we provide genetic evidence for three novel pathways of Ikaros-mediated tumor suppression. Like PAX5, Ikaros reconstitution results in multiple dephosphorylation events (Stat5, AKT, ERK1/2 and SRC are affected). In collaboration with the pre-B cell receptor and its downstream adapter molecule BLNK, Ikaros suppressed MYC and inhibits cell cycle progression. Induction of the Arf/p53 pathway represents a distinct function of Ikaros, which is not shared with the pre-B cell receptor signaling pathway. Disclosures: No relevant conflicts of interest to declare.

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Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC

Blood ◽

10.1182/blood.v118.21.1406.1406 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1406-1406

Author(s):

Rahul Nahar ◽

Parham Ramezani-Rad ◽

Maximilian Mossner ◽

Cihangir Duy ◽

Leandro Cerchietti ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Transcriptional Repression ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

Cell Cycle Exit ◽

B Cell Receptor Signaling ◽

Cell Receptor Signaling ◽

Inducible Activation

Abstract Abstract 1406 Background: Pre-B cell receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. Results: This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell receptor/BCL6-induced cell cycle exit. Conclusion: Hence, pre-B cell receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2. Disclosures: No relevant conflicts of interest to declare.

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