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Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia.

Joshi I, Yoshida T, Jena N, Qi X, Zhang J, Van Etten RA, Georgopoulos K - Nat. Immunol. (2014)

Bottom Line: Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL).Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells.Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.

ABSTRACT
Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

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Leukemogenic potential of IkE5Δ/Δ pre-B cellsa, Kaplan-Meier survival curve of NSG mice transplanted with WT or IkE5Δ/Δ pre-B cells. The survival of both cohorts of recipients of IkE5Δ/Δ pre-B cells was significantly shorter than recipients of WT pre-B cells (P = 0.013, Mantel-Cox tests). b, Histopathology of precursor B-cell acute lymphoblastic leukemia/lymphoma derived from IkE5Δ/Δ pre-B cells. (i–iii): Hematoxylin & eosin-stained sections of spleen (i), liver (ii), and BM (iii) from a premorbid NSG recipient (sacrificed day 63 post-transplant) of IkE5Δ/Δ pre-B cells from a CD19-Cre donor. Note the extensive infiltration of all organs with large cells with moderate cytoplasm and prominent nucleoli, and frequent mitotic figures (arrows). Scale bars, 50 μm. (iv) Wright-Giemsa stain of cytospin of BM from this recipient (scale bar, 20 μm). Note predominant population of large lymphoblasts with immature nuclei and basophilic cytoplasm (arrows). c, Integrin expression is elevated in both IkE5Δ/Δ pre-leukemic and leukemic pre-B cells. Percentage of WT, IkE5Δ/Δ pre-leukemic and leukemic pre-B cells expressing integrins α5 (CD49e), α6 (CD49f) and β1 (CD29). d, FAK activation (pFAK) measured by flow cytometry in the presence and absence of FAK inhibitor in WT and mutant pre-B cells. e, FAK inhibition interferes with stromal adhesion of IkE5Δ/Δ preleukemic and leukemic pre-B cells. Inhibitor-treated, closed symbols; vehicle-treated, open symbols. (n=2 each). f, FAK inhibition induces cell death in IkE5Δ/Δ pre-leukemic and leukemic pre-B cells (n=4; *P <10-6, **P <10−7 two-tailed Student's t-test).
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Figure 8: Leukemogenic potential of IkE5Δ/Δ pre-B cellsa, Kaplan-Meier survival curve of NSG mice transplanted with WT or IkE5Δ/Δ pre-B cells. The survival of both cohorts of recipients of IkE5Δ/Δ pre-B cells was significantly shorter than recipients of WT pre-B cells (P = 0.013, Mantel-Cox tests). b, Histopathology of precursor B-cell acute lymphoblastic leukemia/lymphoma derived from IkE5Δ/Δ pre-B cells. (i–iii): Hematoxylin & eosin-stained sections of spleen (i), liver (ii), and BM (iii) from a premorbid NSG recipient (sacrificed day 63 post-transplant) of IkE5Δ/Δ pre-B cells from a CD19-Cre donor. Note the extensive infiltration of all organs with large cells with moderate cytoplasm and prominent nucleoli, and frequent mitotic figures (arrows). Scale bars, 50 μm. (iv) Wright-Giemsa stain of cytospin of BM from this recipient (scale bar, 20 μm). Note predominant population of large lymphoblasts with immature nuclei and basophilic cytoplasm (arrows). c, Integrin expression is elevated in both IkE5Δ/Δ pre-leukemic and leukemic pre-B cells. Percentage of WT, IkE5Δ/Δ pre-leukemic and leukemic pre-B cells expressing integrins α5 (CD49e), α6 (CD49f) and β1 (CD29). d, FAK activation (pFAK) measured by flow cytometry in the presence and absence of FAK inhibitor in WT and mutant pre-B cells. e, FAK inhibition interferes with stromal adhesion of IkE5Δ/Δ preleukemic and leukemic pre-B cells. Inhibitor-treated, closed symbols; vehicle-treated, open symbols. (n=2 each). f, FAK inhibition induces cell death in IkE5Δ/Δ pre-leukemic and leukemic pre-B cells (n=4; *P <10-6, **P <10−7 two-tailed Student's t-test).

Mentions: The rapid development of precursor T-lymphoid neoplasms in IkE5Δ/Δ mice (data not shown and ref. 41) precludes the assessment of B-lymphoid leukemogenesis in these mutant mice. The leukemogenic potential of IkE5Δ/Δ pre-B cells was therefore evaluated by transplantation of this population into immunodeficient NOD-SCID-Il2rg−/− (NSG) recipient mice. Following transplantation with IkE5Δ/Δ pre-B cells isolated from either Cd19-Cre or Cd2-Cre donors, recipient NSG mice uniformly exhibited circulating immature CD19+BP1+CD2− B-lymphoid cells within 7 weeks (data not shown), and developed signs of disseminated leukemia/lymphoma by 3-4 months post-transplant, with weight loss, hyperventilation, and hepatosplenomegaly (mean spleen weight 668 ± 188 mg), whereas recipients of WT pre-B cells remained healthy (Fig. 8a). The disease in recipients of IkE5Δ/ΔCd19-Cre pre-B cells was somewhat more aggressive than in IkE5Δ/ΔCd2-Cre recipients (Fig. 8a; median survival 107d vs. 143d; P = 0.0021, Mantel-Cox test). At necropsy, recipients of IkE5Δ/ΔCd19-Cre pre-B cells had pancytopenia with severe anemia (blood hemoglobin 4.8 ± 0.7 g/dL) that likely contributed to morbidity or death, while IkE5Δ/ΔCd2-Cre recipients tended to develop hind-limb paralysis and malignant pleural effusions.


Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia.

Joshi I, Yoshida T, Jena N, Qi X, Zhang J, Van Etten RA, Georgopoulos K - Nat. Immunol. (2014)

Leukemogenic potential of IkE5Δ/Δ pre-B cellsa, Kaplan-Meier survival curve of NSG mice transplanted with WT or IkE5Δ/Δ pre-B cells. The survival of both cohorts of recipients of IkE5Δ/Δ pre-B cells was significantly shorter than recipients of WT pre-B cells (P = 0.013, Mantel-Cox tests). b, Histopathology of precursor B-cell acute lymphoblastic leukemia/lymphoma derived from IkE5Δ/Δ pre-B cells. (i–iii): Hematoxylin & eosin-stained sections of spleen (i), liver (ii), and BM (iii) from a premorbid NSG recipient (sacrificed day 63 post-transplant) of IkE5Δ/Δ pre-B cells from a CD19-Cre donor. Note the extensive infiltration of all organs with large cells with moderate cytoplasm and prominent nucleoli, and frequent mitotic figures (arrows). Scale bars, 50 μm. (iv) Wright-Giemsa stain of cytospin of BM from this recipient (scale bar, 20 μm). Note predominant population of large lymphoblasts with immature nuclei and basophilic cytoplasm (arrows). c, Integrin expression is elevated in both IkE5Δ/Δ pre-leukemic and leukemic pre-B cells. Percentage of WT, IkE5Δ/Δ pre-leukemic and leukemic pre-B cells expressing integrins α5 (CD49e), α6 (CD49f) and β1 (CD29). d, FAK activation (pFAK) measured by flow cytometry in the presence and absence of FAK inhibitor in WT and mutant pre-B cells. e, FAK inhibition interferes with stromal adhesion of IkE5Δ/Δ preleukemic and leukemic pre-B cells. Inhibitor-treated, closed symbols; vehicle-treated, open symbols. (n=2 each). f, FAK inhibition induces cell death in IkE5Δ/Δ pre-leukemic and leukemic pre-B cells (n=4; *P <10-6, **P <10−7 two-tailed Student's t-test).
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Figure 8: Leukemogenic potential of IkE5Δ/Δ pre-B cellsa, Kaplan-Meier survival curve of NSG mice transplanted with WT or IkE5Δ/Δ pre-B cells. The survival of both cohorts of recipients of IkE5Δ/Δ pre-B cells was significantly shorter than recipients of WT pre-B cells (P = 0.013, Mantel-Cox tests). b, Histopathology of precursor B-cell acute lymphoblastic leukemia/lymphoma derived from IkE5Δ/Δ pre-B cells. (i–iii): Hematoxylin & eosin-stained sections of spleen (i), liver (ii), and BM (iii) from a premorbid NSG recipient (sacrificed day 63 post-transplant) of IkE5Δ/Δ pre-B cells from a CD19-Cre donor. Note the extensive infiltration of all organs with large cells with moderate cytoplasm and prominent nucleoli, and frequent mitotic figures (arrows). Scale bars, 50 μm. (iv) Wright-Giemsa stain of cytospin of BM from this recipient (scale bar, 20 μm). Note predominant population of large lymphoblasts with immature nuclei and basophilic cytoplasm (arrows). c, Integrin expression is elevated in both IkE5Δ/Δ pre-leukemic and leukemic pre-B cells. Percentage of WT, IkE5Δ/Δ pre-leukemic and leukemic pre-B cells expressing integrins α5 (CD49e), α6 (CD49f) and β1 (CD29). d, FAK activation (pFAK) measured by flow cytometry in the presence and absence of FAK inhibitor in WT and mutant pre-B cells. e, FAK inhibition interferes with stromal adhesion of IkE5Δ/Δ preleukemic and leukemic pre-B cells. Inhibitor-treated, closed symbols; vehicle-treated, open symbols. (n=2 each). f, FAK inhibition induces cell death in IkE5Δ/Δ pre-leukemic and leukemic pre-B cells (n=4; *P <10-6, **P <10−7 two-tailed Student's t-test).
Mentions: The rapid development of precursor T-lymphoid neoplasms in IkE5Δ/Δ mice (data not shown and ref. 41) precludes the assessment of B-lymphoid leukemogenesis in these mutant mice. The leukemogenic potential of IkE5Δ/Δ pre-B cells was therefore evaluated by transplantation of this population into immunodeficient NOD-SCID-Il2rg−/− (NSG) recipient mice. Following transplantation with IkE5Δ/Δ pre-B cells isolated from either Cd19-Cre or Cd2-Cre donors, recipient NSG mice uniformly exhibited circulating immature CD19+BP1+CD2− B-lymphoid cells within 7 weeks (data not shown), and developed signs of disseminated leukemia/lymphoma by 3-4 months post-transplant, with weight loss, hyperventilation, and hepatosplenomegaly (mean spleen weight 668 ± 188 mg), whereas recipients of WT pre-B cells remained healthy (Fig. 8a). The disease in recipients of IkE5Δ/ΔCd19-Cre pre-B cells was somewhat more aggressive than in IkE5Δ/ΔCd2-Cre recipients (Fig. 8a; median survival 107d vs. 143d; P = 0.0021, Mantel-Cox test). At necropsy, recipients of IkE5Δ/ΔCd19-Cre pre-B cells had pancytopenia with severe anemia (blood hemoglobin 4.8 ± 0.7 g/dL) that likely contributed to morbidity or death, while IkE5Δ/ΔCd2-Cre recipients tended to develop hind-limb paralysis and malignant pleural effusions.

Bottom Line: Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL).Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells.Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.

ABSTRACT
Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

Show MeSH
Related in: MedlinePlus