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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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Pre-B cell differentiation is dependent on the Ikaros gene familya, Strategy to generate a conditional Ikzf1 dominant-negative allele. Non-coding (black) and coding (white) exons, with exon 5 flanked by loxP sites (black arrowheads) for deletion are shown at the Ikzf1 locus. Stars mark zinc fingers involved in DNA binding (E4-E6) or protein dimerization (E8). b, Immunoblot analysis of Ikaros isoforms (Ik-1 and Ik-2) in WT and IkE5Δ/Δ pre-B cells. Shift in size indicates exon 5 deletion. c, Flow cytometric analysis of wild-type (WT) and IkE5fl/fl CD2-Cre bone marrow (BM) cells. Expression of stage-specific markers (as in Supplementary Fig. 1a) identify large pre-B cells (CD19+CD43+BP1+), small pre-B cells (CD19+CD2+IgM−), and immature B cells (CD19+IgM+) in the BM. d, Absolute number of cells/(femur + tibia)×2 in various B cell subsets in WT and IkE5Δ/Δ BM are shown as a graph of means ± standard deviation (s.d.). Asterisks indicate a statistically significant change between WT and mutant B cell subsets (n=10 for WT and mutant; *P < 0.01, **P < 0.0001, two-tailed Student's t-test). e, Representative cell cycle analysis of ex-vivo isolated large pre-B cells from WT and IkE5fl/fl CD2-Cre mice. Gates show relative number of cells in G0/G1 and S/G2/M phase. f, Igh and Igk rearrangements in Ikaros-deficient pre-B cells. Diagram of Igh and Igk loci depicting proximal and distal V, D and J clusters tested for recombination with primers and probes used for detection. Recombination products were amplified by PCR with decreasing amounts of pre-B cell DNA (depicted as black triangles) and with amplification of Ikzf1 non-deleted genomic fragment as loading control. g, Igk recombination fails to rescue the IkE5Δ/Δ large pre-B cell block. Analysis as described in Fig. 1c with intracellular staining for Igκ chain performed on IkE5Δ/Δ and IkE5Δ/Δ: D23 large pre-B cells (CD19+CD43+BP1+).
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Figure 1: Pre-B cell differentiation is dependent on the Ikaros gene familya, Strategy to generate a conditional Ikzf1 dominant-negative allele. Non-coding (black) and coding (white) exons, with exon 5 flanked by loxP sites (black arrowheads) for deletion are shown at the Ikzf1 locus. Stars mark zinc fingers involved in DNA binding (E4-E6) or protein dimerization (E8). b, Immunoblot analysis of Ikaros isoforms (Ik-1 and Ik-2) in WT and IkE5Δ/Δ pre-B cells. Shift in size indicates exon 5 deletion. c, Flow cytometric analysis of wild-type (WT) and IkE5fl/fl CD2-Cre bone marrow (BM) cells. Expression of stage-specific markers (as in Supplementary Fig. 1a) identify large pre-B cells (CD19+CD43+BP1+), small pre-B cells (CD19+CD2+IgM−), and immature B cells (CD19+IgM+) in the BM. d, Absolute number of cells/(femur + tibia)×2 in various B cell subsets in WT and IkE5Δ/Δ BM are shown as a graph of means ± standard deviation (s.d.). Asterisks indicate a statistically significant change between WT and mutant B cell subsets (n=10 for WT and mutant; *P < 0.01, **P < 0.0001, two-tailed Student's t-test). e, Representative cell cycle analysis of ex-vivo isolated large pre-B cells from WT and IkE5fl/fl CD2-Cre mice. Gates show relative number of cells in G0/G1 and S/G2/M phase. f, Igh and Igk rearrangements in Ikaros-deficient pre-B cells. Diagram of Igh and Igk loci depicting proximal and distal V, D and J clusters tested for recombination with primers and probes used for detection. Recombination products were amplified by PCR with decreasing amounts of pre-B cell DNA (depicted as black triangles) and with amplification of Ikzf1 non-deleted genomic fragment as loading control. g, Igk recombination fails to rescue the IkE5Δ/Δ large pre-B cell block. Analysis as described in Fig. 1c with intracellular staining for Igκ chain performed on IkE5Δ/Δ and IkE5Δ/Δ: D23 large pre-B cells (CD19+CD43+BP1+).

Mentions: To determine the role of the Ikaros family during B cell differentiation, exon 5 of the Ikzf1 gene (defined hereafter as IkE5), encoding two Ikaros DNA binding zinc fingers, was floxed (IkE5fl/fl; Fig. 1a) and deleted from either the common lymphoid progenitor or the downstream definitive pro-B cell precursor using CD2-Cre or CD19-Cre transgenes, respectively (Supplementary Fig. 1a). Deletion of IkE5 generates Ikaros protein isoforms that lack DNA binding activity and are structurally similar to those encountered in human B-ALL (Ik6)24 (Fig. 1b, pre-B IkE5Δ/Δ. These mutant Ikaros isoforms act in a dominant-negative fashion by dimerizing with co-expressed family members, including Aiolos, and interfering with their DNA binding activity30,32. We confirmed the dominant-negative phenotype by combining the Ikzf3 (Aiolos gene) homozygous and the Ikzf1 heterozygous mutations (Ikzf3−/−Ikzf1+/−). Deletion of IkE5 or the combined Ikzf3−/−Ikzf1+/− mutations caused a similar block and expansion of large pre-B cells (CD19+CD43+BP1+; Fig. 1c, d and Supplementary Fig. 1b, c). These normally represent a minor population but were now found in numbers that were similar to those of all bone marrow (BM) B cells in wild type (WT) mice (Fig. 1d). As in WT, the majority of mutant large pre-B cells were in cell cycle (Fig. 1e). The few immature B (CD19+IgM+) cells detected in the IkE5fl/flCD2-Cre mice (Fig. 1d) had not deleted IkE5fl/fl (Supplementary Fig. 1d), indicating that transition from the large to the small pre-B cell is absolutely dependent on the DNA binding activities of Ikaros gene family members expressed at this stage of differentiation.


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)

Pre-B cell differentiation is dependent on the Ikaros gene familya, Strategy to generate a conditional Ikzf1 dominant-negative allele. Non-coding (black) and coding (white) exons, with exon 5 flanked by loxP sites (black arrowheads) for deletion are shown at the Ikzf1 locus. Stars mark zinc fingers involved in DNA binding (E4-E6) or protein dimerization (E8). b, Immunoblot analysis of Ikaros isoforms (Ik-1 and Ik-2) in WT and IkE5Δ/Δ pre-B cells. Shift in size indicates exon 5 deletion. c, Flow cytometric analysis of wild-type (WT) and IkE5fl/fl CD2-Cre bone marrow (BM) cells. Expression of stage-specific markers (as in Supplementary Fig. 1a) identify large pre-B cells (CD19+CD43+BP1+), small pre-B cells (CD19+CD2+IgM−), and immature B cells (CD19+IgM+) in the BM. d, Absolute number of cells/(femur + tibia)×2 in various B cell subsets in WT and IkE5Δ/Δ BM are shown as a graph of means ± standard deviation (s.d.). Asterisks indicate a statistically significant change between WT and mutant B cell subsets (n=10 for WT and mutant; *P < 0.01, **P < 0.0001, two-tailed Student's t-test). e, Representative cell cycle analysis of ex-vivo isolated large pre-B cells from WT and IkE5fl/fl CD2-Cre mice. Gates show relative number of cells in G0/G1 and S/G2/M phase. f, Igh and Igk rearrangements in Ikaros-deficient pre-B cells. Diagram of Igh and Igk loci depicting proximal and distal V, D and J clusters tested for recombination with primers and probes used for detection. Recombination products were amplified by PCR with decreasing amounts of pre-B cell DNA (depicted as black triangles) and with amplification of Ikzf1 non-deleted genomic fragment as loading control. g, Igk recombination fails to rescue the IkE5Δ/Δ large pre-B cell block. Analysis as described in Fig. 1c with intracellular staining for Igκ chain performed on IkE5Δ/Δ and IkE5Δ/Δ: D23 large pre-B cells (CD19+CD43+BP1+).
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Related In: Results  -  Collection

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Figure 1: Pre-B cell differentiation is dependent on the Ikaros gene familya, Strategy to generate a conditional Ikzf1 dominant-negative allele. Non-coding (black) and coding (white) exons, with exon 5 flanked by loxP sites (black arrowheads) for deletion are shown at the Ikzf1 locus. Stars mark zinc fingers involved in DNA binding (E4-E6) or protein dimerization (E8). b, Immunoblot analysis of Ikaros isoforms (Ik-1 and Ik-2) in WT and IkE5Δ/Δ pre-B cells. Shift in size indicates exon 5 deletion. c, Flow cytometric analysis of wild-type (WT) and IkE5fl/fl CD2-Cre bone marrow (BM) cells. Expression of stage-specific markers (as in Supplementary Fig. 1a) identify large pre-B cells (CD19+CD43+BP1+), small pre-B cells (CD19+CD2+IgM−), and immature B cells (CD19+IgM+) in the BM. d, Absolute number of cells/(femur + tibia)×2 in various B cell subsets in WT and IkE5Δ/Δ BM are shown as a graph of means ± standard deviation (s.d.). Asterisks indicate a statistically significant change between WT and mutant B cell subsets (n=10 for WT and mutant; *P < 0.01, **P < 0.0001, two-tailed Student's t-test). e, Representative cell cycle analysis of ex-vivo isolated large pre-B cells from WT and IkE5fl/fl CD2-Cre mice. Gates show relative number of cells in G0/G1 and S/G2/M phase. f, Igh and Igk rearrangements in Ikaros-deficient pre-B cells. Diagram of Igh and Igk loci depicting proximal and distal V, D and J clusters tested for recombination with primers and probes used for detection. Recombination products were amplified by PCR with decreasing amounts of pre-B cell DNA (depicted as black triangles) and with amplification of Ikzf1 non-deleted genomic fragment as loading control. g, Igk recombination fails to rescue the IkE5Δ/Δ large pre-B cell block. Analysis as described in Fig. 1c with intracellular staining for Igκ chain performed on IkE5Δ/Δ and IkE5Δ/Δ: D23 large pre-B cells (CD19+CD43+BP1+).
Mentions: To determine the role of the Ikaros family during B cell differentiation, exon 5 of the Ikzf1 gene (defined hereafter as IkE5), encoding two Ikaros DNA binding zinc fingers, was floxed (IkE5fl/fl; Fig. 1a) and deleted from either the common lymphoid progenitor or the downstream definitive pro-B cell precursor using CD2-Cre or CD19-Cre transgenes, respectively (Supplementary Fig. 1a). Deletion of IkE5 generates Ikaros protein isoforms that lack DNA binding activity and are structurally similar to those encountered in human B-ALL (Ik6)24 (Fig. 1b, pre-B IkE5Δ/Δ. These mutant Ikaros isoforms act in a dominant-negative fashion by dimerizing with co-expressed family members, including Aiolos, and interfering with their DNA binding activity30,32. We confirmed the dominant-negative phenotype by combining the Ikzf3 (Aiolos gene) homozygous and the Ikzf1 heterozygous mutations (Ikzf3−/−Ikzf1+/−). Deletion of IkE5 or the combined Ikzf3−/−Ikzf1+/− mutations caused a similar block and expansion of large pre-B cells (CD19+CD43+BP1+; Fig. 1c, d and Supplementary Fig. 1b, c). These normally represent a minor population but were now found in numbers that were similar to those of all bone marrow (BM) B cells in wild type (WT) mice (Fig. 1d). As in WT, the majority of mutant large pre-B cells were in cell cycle (Fig. 1e). The few immature B (CD19+IgM+) cells detected in the IkE5fl/flCD2-Cre mice (Fig. 1d) had not deleted IkE5fl/fl (Supplementary Fig. 1d), indicating that transition from the large to the small pre-B cell is absolutely dependent on the DNA binding activities of Ikaros gene family members expressed at this stage of differentiation.

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