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

Trageser D, Iacobucci I, Nahar R, Duy C, von Levetzow G, Klemm L, Park E, Schuh W, Gruber T, Herzog S, Kim YM, Hofmann WK, Li A, Storlazzi CT, Jäck HM, Groffen J, Martinelli G, Heisterkamp N, Jumaa H, Müschen M - J. Exp. Med. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, USA.

ABSTRACT
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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Pre-B cell receptor–mediated suppression of leukemic growth requires IKAROS function. mRNA levels of Ikaros after μ chain induction were measured by quantitative RT-PCR (A; three experiments). BCR-ABL1–transformed ALL cells were retrovirally transduced with the dominant-negative IKAROS splice variant IK6/GFP or a GFP empty vector control (B–D). Leukemia cells transduced with IK6/GFP (+IK6) or an empty vector control (-IK6) were studied in the presence or absence of pre–B cell receptor activation (+/− μ chain). IK6/GFP- and GFP-transduced leukemia cells were subjected to cell cycle analysis in the presence or absence of inducible pre–B cell receptor signaling (B–D). The percentages of cells in G0/G1, S and G2/M phase are indicated and means of three experiments are indicated in C. In D, the relative increase or decrease of IK6/GFP+ versus GFP+ leukemia cells in the presence or absence of pre–B cell receptor activation (± μ chain) was measured. The experiment was repeated once.
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fig6: Pre-B cell receptor–mediated suppression of leukemic growth requires IKAROS function. mRNA levels of Ikaros after μ chain induction were measured by quantitative RT-PCR (A; three experiments). BCR-ABL1–transformed ALL cells were retrovirally transduced with the dominant-negative IKAROS splice variant IK6/GFP or a GFP empty vector control (B–D). Leukemia cells transduced with IK6/GFP (+IK6) or an empty vector control (-IK6) were studied in the presence or absence of pre–B cell receptor activation (+/− μ chain). IK6/GFP- and GFP-transduced leukemia cells were subjected to cell cycle analysis in the presence or absence of inducible pre–B cell receptor signaling (B–D). The percentages of cells in G0/G1, S and G2/M phase are indicated and means of three experiments are indicated in C. In D, the relative increase or decrease of IK6/GFP+ versus GFP+ leukemia cells in the presence or absence of pre–B cell receptor activation (± μ chain) was measured. The experiment was repeated once.

Mentions: We then focused our investigation of the mechanism of pre–B cell receptor–induced leukemia-suppression on the IKAROS transcription factor based on the following rationale. Two recent studies showed that pre–B cell receptor signaling in normal B cell precursors results in up-regulation of IKAROS, which subsequently induces cell cycle arrest (Thompson et al., 2007; Ma et al., 2008). In addition, a recent landmark study demonstrated that deletions of the IKZF1 (IKAROS) gene represents a defining feature of Ph+ ALL (deletions in >80% of the cases; Mullighan et al., 2008). Validation by quantitative RT-PCR confirmed previous findings (Thompson et al., 2007; Ma et al., 2008) that inducible pre–B cell receptor activation resulted in a 6–8-fold up-regulation of full-length IKAROS mRNA levels within 24 h (Fig. 6 A). Within 42 h after induction of pre–B cell receptor expression, close to 70% of viable BCR-ABL1 ALL cells were arrested in G0/G1 (Fig. 5, B and C, and Fig. 6, B and C). To investigate whether IKAROS function is required for pre–B cell receptor–induced cell cycle arrest, we transduced BCR-ABL1 ALL cells with IK6, a dominant-negative splice variant of IKAROS (Iacobucci et al., 2008; Klein et al., 2006; Reynaud et al., 2008). The IK6 splice variant neutralizes IKAROS function by forming nonfunctional heterodimers with full-length IKAROS (Reynaud et al., 2008) and is abundantly expressed in most if not all cases of Ph+ ALL (Klein et al., 2006; Iacobucci et al., 2008; Mullighan et al., 2008). To test this hypothesis, pre–B cell receptor signaling was induced in BCR-ABL1–transformed Rag2−/− tTA/μ chain transgenic ALL cells that were transduced with the dominant-negative IK6 splice variant (IK6/GFP) or an empty vector control (GFP; Fig. 6, B–D). Whereas IK6/GFP-overexpression did not confer a significant growth advantage over GFP background levels in pre–B cell receptor–negative BCR-ABL1–transformed ALL cells, pre–B cell receptor induction resulted in an up to 25-fold increase of IK6/GFP-transduced leukemia cells (Fig. 6 D; a 5-fold increase was measured in one repeat experiment). Previous studies demonstrated that IKAROS negatively regulates the G1–S phase transition of the cell cycle in pre–B cells (Gómez-del Arco et al., 2004; Kathrein et al., 2005), mainly through histone H3 acetylation and up-regulation of negative cell cycle regulators, including CDKN1B (Kathrein et al., 2005; Fig. S1). Consistent with these findings, we observed that pre–B cell receptor/IKAROS-induced cell cycle arrest can be reversed by dominant-negative IK6 (Fig. 6, B and C).


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

Trageser D, Iacobucci I, Nahar R, Duy C, von Levetzow G, Klemm L, Park E, Schuh W, Gruber T, Herzog S, Kim YM, Hofmann WK, Li A, Storlazzi CT, Jäck HM, Groffen J, Martinelli G, Heisterkamp N, Jumaa H, Müschen M - J. Exp. Med. (2009)

Pre-B cell receptor–mediated suppression of leukemic growth requires IKAROS function. mRNA levels of Ikaros after μ chain induction were measured by quantitative RT-PCR (A; three experiments). BCR-ABL1–transformed ALL cells were retrovirally transduced with the dominant-negative IKAROS splice variant IK6/GFP or a GFP empty vector control (B–D). Leukemia cells transduced with IK6/GFP (+IK6) or an empty vector control (-IK6) were studied in the presence or absence of pre–B cell receptor activation (+/− μ chain). IK6/GFP- and GFP-transduced leukemia cells were subjected to cell cycle analysis in the presence or absence of inducible pre–B cell receptor signaling (B–D). The percentages of cells in G0/G1, S and G2/M phase are indicated and means of three experiments are indicated in C. In D, the relative increase or decrease of IK6/GFP+ versus GFP+ leukemia cells in the presence or absence of pre–B cell receptor activation (± μ chain) was measured. The experiment was repeated once.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2722172&req=5

fig6: Pre-B cell receptor–mediated suppression of leukemic growth requires IKAROS function. mRNA levels of Ikaros after μ chain induction were measured by quantitative RT-PCR (A; three experiments). BCR-ABL1–transformed ALL cells were retrovirally transduced with the dominant-negative IKAROS splice variant IK6/GFP or a GFP empty vector control (B–D). Leukemia cells transduced with IK6/GFP (+IK6) or an empty vector control (-IK6) were studied in the presence or absence of pre–B cell receptor activation (+/− μ chain). IK6/GFP- and GFP-transduced leukemia cells were subjected to cell cycle analysis in the presence or absence of inducible pre–B cell receptor signaling (B–D). The percentages of cells in G0/G1, S and G2/M phase are indicated and means of three experiments are indicated in C. In D, the relative increase or decrease of IK6/GFP+ versus GFP+ leukemia cells in the presence or absence of pre–B cell receptor activation (± μ chain) was measured. The experiment was repeated once.
Mentions: We then focused our investigation of the mechanism of pre–B cell receptor–induced leukemia-suppression on the IKAROS transcription factor based on the following rationale. Two recent studies showed that pre–B cell receptor signaling in normal B cell precursors results in up-regulation of IKAROS, which subsequently induces cell cycle arrest (Thompson et al., 2007; Ma et al., 2008). In addition, a recent landmark study demonstrated that deletions of the IKZF1 (IKAROS) gene represents a defining feature of Ph+ ALL (deletions in >80% of the cases; Mullighan et al., 2008). Validation by quantitative RT-PCR confirmed previous findings (Thompson et al., 2007; Ma et al., 2008) that inducible pre–B cell receptor activation resulted in a 6–8-fold up-regulation of full-length IKAROS mRNA levels within 24 h (Fig. 6 A). Within 42 h after induction of pre–B cell receptor expression, close to 70% of viable BCR-ABL1 ALL cells were arrested in G0/G1 (Fig. 5, B and C, and Fig. 6, B and C). To investigate whether IKAROS function is required for pre–B cell receptor–induced cell cycle arrest, we transduced BCR-ABL1 ALL cells with IK6, a dominant-negative splice variant of IKAROS (Iacobucci et al., 2008; Klein et al., 2006; Reynaud et al., 2008). The IK6 splice variant neutralizes IKAROS function by forming nonfunctional heterodimers with full-length IKAROS (Reynaud et al., 2008) and is abundantly expressed in most if not all cases of Ph+ ALL (Klein et al., 2006; Iacobucci et al., 2008; Mullighan et al., 2008). To test this hypothesis, pre–B cell receptor signaling was induced in BCR-ABL1–transformed Rag2−/− tTA/μ chain transgenic ALL cells that were transduced with the dominant-negative IK6 splice variant (IK6/GFP) or an empty vector control (GFP; Fig. 6, B–D). Whereas IK6/GFP-overexpression did not confer a significant growth advantage over GFP background levels in pre–B cell receptor–negative BCR-ABL1–transformed ALL cells, pre–B cell receptor induction resulted in an up to 25-fold increase of IK6/GFP-transduced leukemia cells (Fig. 6 D; a 5-fold increase was measured in one repeat experiment). Previous studies demonstrated that IKAROS negatively regulates the G1–S phase transition of the cell cycle in pre–B cells (Gómez-del Arco et al., 2004; Kathrein et al., 2005), mainly through histone H3 acetylation and up-regulation of negative cell cycle regulators, including CDKN1B (Kathrein et al., 2005; Fig. S1). Consistent with these findings, we observed that pre–B cell receptor/IKAROS-induced cell cycle arrest can be reversed by dominant-negative IK6 (Fig. 6, B and C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus