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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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Reconstitution of Slp65 deficiency in BCR-ABL1 ALL cells suppresses leukemic growth BCR-ABL1–transformed Slp65−/−. ALL cells were reconstituted with Slp65/GFP or a GFP empty vector control (A; single measurements in three independent experiments). For inducible Slp65 reconstitution, we fused Slp65 to the estrogen receptor ligand-binding domain (ERT2). Either ERT2 fused to the N terminus of Slp65 (ERT2-Slp65/GFP) or ERT2 alone (ERT2/GFP) were expressed in the Slp65−/−BCR-ABL1–transformed ALL cells and activated by addition of 1 μmol/l 4-hydroxy-tamoxifen (OHT) using ethanol (EtOH) as vehicle control (B; triplicate measurements, the experiment was repeated once). Primary human leukemia cells from one case of Ph+ ALL cells lacking expression of SLP65 were cultured on OP9 stroma in the presence of IL-7 and transduced with SLP65/GFP or a GFP empty vector control (three independent transductions) and monitored by flow cytometry (C). BCR-ABL1–transformed Slp65−/− ALL cells were labeled by lentiviral firefly luciferase, transduced with retroviral vectors encoding either Slp65/GFP or GFP alone and injected into five sublethally irradiated NOD/SCID mice per group. Engraftment and leukemic growth was monitored by luciferase-bioimaging (D; experiment repeated once). The blue scale bar in (D) corresponds to 1 cm in length. Leukemic infiltration (CD19+ GFP+) of bone marrow, spleen, and peripheral blood (three independent experiments) was documented by flow cytometry (E; representative data from two experiments).
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fig3: Reconstitution of Slp65 deficiency in BCR-ABL1 ALL cells suppresses leukemic growth BCR-ABL1–transformed Slp65−/−. ALL cells were reconstituted with Slp65/GFP or a GFP empty vector control (A; single measurements in three independent experiments). For inducible Slp65 reconstitution, we fused Slp65 to the estrogen receptor ligand-binding domain (ERT2). Either ERT2 fused to the N terminus of Slp65 (ERT2-Slp65/GFP) or ERT2 alone (ERT2/GFP) were expressed in the Slp65−/−BCR-ABL1–transformed ALL cells and activated by addition of 1 μmol/l 4-hydroxy-tamoxifen (OHT) using ethanol (EtOH) as vehicle control (B; triplicate measurements, the experiment was repeated once). Primary human leukemia cells from one case of Ph+ ALL cells lacking expression of SLP65 were cultured on OP9 stroma in the presence of IL-7 and transduced with SLP65/GFP or a GFP empty vector control (three independent transductions) and monitored by flow cytometry (C). BCR-ABL1–transformed Slp65−/− ALL cells were labeled by lentiviral firefly luciferase, transduced with retroviral vectors encoding either Slp65/GFP or GFP alone and injected into five sublethally irradiated NOD/SCID mice per group. Engraftment and leukemic growth was monitored by luciferase-bioimaging (D; experiment repeated once). The blue scale bar in (D) corresponds to 1 cm in length. Leukemic infiltration (CD19+ GFP+) of bone marrow, spleen, and peripheral blood (three independent experiments) was documented by flow cytometry (E; representative data from two experiments).

Mentions: To test this hypothesis in a formal experiment, we reconstituted pre–B cell receptor signaling in BCR-ABL1–transformed bone marrow B cell precursors from mice carrying pre–B cell receptor defects. To this end, we transformed bone marrow B cell precursors from Slp65−/− and Ighm−/− mice with retroviral BCR-ABL1 (Fig. 3). These mice lack expression of the pre–B cell receptor linker molecule Slp65 (Flemming et al., 2003) or fail to express a μ chain because of deletion of the Cμ transmembrane domain. In one set of experiments, we reconstituted Slp65 expression in BCR-ABL1–transformed Slp65−/− pre–B ALL cells either constitutively (Fig. 3 A) or inducibly (Fig. 3 B; Meixlsperger et al., 2007). In both systems, we observed selective depletion of Slp65-reconstituted BCR-ABL1 ALL cells within 9 d after Slp65 reconstitution. We also studied one case of primary human Ph+ ALL with SLP65 deficiency (Jumaa et al., 2003), reconstitution of which had a similar effect (Fig. 3 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)

Reconstitution of Slp65 deficiency in BCR-ABL1 ALL cells suppresses leukemic growth BCR-ABL1–transformed Slp65−/−. ALL cells were reconstituted with Slp65/GFP or a GFP empty vector control (A; single measurements in three independent experiments). For inducible Slp65 reconstitution, we fused Slp65 to the estrogen receptor ligand-binding domain (ERT2). Either ERT2 fused to the N terminus of Slp65 (ERT2-Slp65/GFP) or ERT2 alone (ERT2/GFP) were expressed in the Slp65−/−BCR-ABL1–transformed ALL cells and activated by addition of 1 μmol/l 4-hydroxy-tamoxifen (OHT) using ethanol (EtOH) as vehicle control (B; triplicate measurements, the experiment was repeated once). Primary human leukemia cells from one case of Ph+ ALL cells lacking expression of SLP65 were cultured on OP9 stroma in the presence of IL-7 and transduced with SLP65/GFP or a GFP empty vector control (three independent transductions) and monitored by flow cytometry (C). BCR-ABL1–transformed Slp65−/− ALL cells were labeled by lentiviral firefly luciferase, transduced with retroviral vectors encoding either Slp65/GFP or GFP alone and injected into five sublethally irradiated NOD/SCID mice per group. Engraftment and leukemic growth was monitored by luciferase-bioimaging (D; experiment repeated once). The blue scale bar in (D) corresponds to 1 cm in length. Leukemic infiltration (CD19+ GFP+) of bone marrow, spleen, and peripheral blood (three independent experiments) was documented by flow cytometry (E; representative data from two experiments).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722172&req=5

fig3: Reconstitution of Slp65 deficiency in BCR-ABL1 ALL cells suppresses leukemic growth BCR-ABL1–transformed Slp65−/−. ALL cells were reconstituted with Slp65/GFP or a GFP empty vector control (A; single measurements in three independent experiments). For inducible Slp65 reconstitution, we fused Slp65 to the estrogen receptor ligand-binding domain (ERT2). Either ERT2 fused to the N terminus of Slp65 (ERT2-Slp65/GFP) or ERT2 alone (ERT2/GFP) were expressed in the Slp65−/−BCR-ABL1–transformed ALL cells and activated by addition of 1 μmol/l 4-hydroxy-tamoxifen (OHT) using ethanol (EtOH) as vehicle control (B; triplicate measurements, the experiment was repeated once). Primary human leukemia cells from one case of Ph+ ALL cells lacking expression of SLP65 were cultured on OP9 stroma in the presence of IL-7 and transduced with SLP65/GFP or a GFP empty vector control (three independent transductions) and monitored by flow cytometry (C). BCR-ABL1–transformed Slp65−/− ALL cells were labeled by lentiviral firefly luciferase, transduced with retroviral vectors encoding either Slp65/GFP or GFP alone and injected into five sublethally irradiated NOD/SCID mice per group. Engraftment and leukemic growth was monitored by luciferase-bioimaging (D; experiment repeated once). The blue scale bar in (D) corresponds to 1 cm in length. Leukemic infiltration (CD19+ GFP+) of bone marrow, spleen, and peripheral blood (three independent experiments) was documented by flow cytometry (E; representative data from two experiments).
Mentions: To test this hypothesis in a formal experiment, we reconstituted pre–B cell receptor signaling in BCR-ABL1–transformed bone marrow B cell precursors from mice carrying pre–B cell receptor defects. To this end, we transformed bone marrow B cell precursors from Slp65−/− and Ighm−/− mice with retroviral BCR-ABL1 (Fig. 3). These mice lack expression of the pre–B cell receptor linker molecule Slp65 (Flemming et al., 2003) or fail to express a μ chain because of deletion of the Cμ transmembrane domain. In one set of experiments, we reconstituted Slp65 expression in BCR-ABL1–transformed Slp65−/− pre–B ALL cells either constitutively (Fig. 3 A) or inducibly (Fig. 3 B; Meixlsperger et al., 2007). In both systems, we observed selective depletion of Slp65-reconstituted BCR-ABL1 ALL cells within 9 d after Slp65 reconstitution. We also studied one case of primary human Ph+ ALL with SLP65 deficiency (Jumaa et al., 2003), reconstitution of which had a similar effect (Fig. 3 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