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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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Increase in integrin signaling mediates adhesion of IkE5Δ/Δ pre-B cells to a stromal nichea, Pathway analysis of genes upregulated in IkE5Δ/Δ relative to WT large pre-B cells. Analysis was performed with a signature of upregulated genes shared by ex vivo mutant pre-B cells prior to and after limited stromal expansion. Pathways enriched for integrins and integrin signaling effectors are highlighted in red. b, Upregulated expression of components of the integrin-actin cytoskeleton pathway in primary and cultured WT and IkE5Δ/Δ pre-B cells as defined in Figs. 1 and 3. Hierarchical clustering of normalized gene expression values across different conditions is shown. c, Cell surface expression of integrins α5, β6, and activated β1 in ex vivo sorted and in vitro cultures of large pre-B cells. MFI for integrin staining is provided. d-f, Increase in FAK activation measured by flow cytometry, immunoblot and confocal microscopy. d, Flow cytometric analysis of p-FAK expression in ex-vivo and in vitro cultured large pre-B cells. MFI for p-FAK is indicated. e, Confocal immunofluorescence microscopy detection of activated p-FAK (red channel), GFP-expressing OP9 stroma (green channel), and nuclei (DAPI, blue channel). Scale bar, 25 μm. f, Immunoblot analysis of total FAK and activated p-FAK, with Btk as a loading control as in Fig. 4a. g, Adhesion of WT and IkE5Δ/Δ adherent pre-B cells to fibronectin-coated plates (left panel) in the presence of the fibronectin-derived RGD peptide or the RGE mutant variant (right panel). Asterisks denote significant differences in adhesion between mutant pre-B cells in the presence or absence of RGD or RGE peptides (n=3; *P < 0.05, two-tailed Student's t-test). h, Chemotaxis of WT (circle) and IkE5Δ/Δ (square) pre-B cells measured in a transwell migration assay in the presence of SDF1. The mean percentage of cells recovered at the bottom of the well in two independent studies is shown.
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Figure 5: Increase in integrin signaling mediates adhesion of IkE5Δ/Δ pre-B cells to a stromal nichea, Pathway analysis of genes upregulated in IkE5Δ/Δ relative to WT large pre-B cells. Analysis was performed with a signature of upregulated genes shared by ex vivo mutant pre-B cells prior to and after limited stromal expansion. Pathways enriched for integrins and integrin signaling effectors are highlighted in red. b, Upregulated expression of components of the integrin-actin cytoskeleton pathway in primary and cultured WT and IkE5Δ/Δ pre-B cells as defined in Figs. 1 and 3. Hierarchical clustering of normalized gene expression values across different conditions is shown. c, Cell surface expression of integrins α5, β6, and activated β1 in ex vivo sorted and in vitro cultures of large pre-B cells. MFI for integrin staining is provided. d-f, Increase in FAK activation measured by flow cytometry, immunoblot and confocal microscopy. d, Flow cytometric analysis of p-FAK expression in ex-vivo and in vitro cultured large pre-B cells. MFI for p-FAK is indicated. e, Confocal immunofluorescence microscopy detection of activated p-FAK (red channel), GFP-expressing OP9 stroma (green channel), and nuclei (DAPI, blue channel). Scale bar, 25 μm. f, Immunoblot analysis of total FAK and activated p-FAK, with Btk as a loading control as in Fig. 4a. g, Adhesion of WT and IkE5Δ/Δ adherent pre-B cells to fibronectin-coated plates (left panel) in the presence of the fibronectin-derived RGD peptide or the RGE mutant variant (right panel). Asterisks denote significant differences in adhesion between mutant pre-B cells in the presence or absence of RGD or RGE peptides (n=3; *P < 0.05, two-tailed Student's t-test). h, Chemotaxis of WT (circle) and IkE5Δ/Δ (square) pre-B cells measured in a transwell migration assay in the presence of SDF1. The mean percentage of cells recovered at the bottom of the well in two independent studies is shown.

Mentions: A comparative genome-wide transcriptional analysis of primary and cultured WT and IkE5Δ/Δ pre-B cells was performed to reveal potential pathways that might support the aberrant adhesion and growth properties of the mutant population. A signature of genes was deduced that was differentially expressed between both freshly isolated IkE5Δ/Δ and WT large pre-B cells and between IkE5Δ/Δ and WT adherent pre-B cells cultured in vitro (Fig. 5a, b). Up-regulated genes in IkE5Δ/Δ pre-B cells were highly enriched in pathways involved in focal adhesion and remodeling of the actin cytoskeleton (Fig. 5a). Integrins (e.g. Itga1, Itga5, Itgb1) as well as other structural and signaling components of focal adhesions (e.g. Ptk2, Vcl, Actn1, Cttn, Dock1, Rogdi) were shared by many of these pathways (Fig. 5b). The increase in integrin expression was validated at the protein level in both primary and cultured cells. Furthermore, expression of the active isoform of integrin β1 (detected with an activation-specific anti-integrin β1 antibody and elevated levels of phosphorylated focal adhesion kinase (p-FAK), a key downstream effector of integrin signaling, indicated that not only expression but also activation of integrin signaling were elevated in IkE5Δ/Δ pre-B cells (Fig. 5c-f). Although not as pronounced as in IkE5Δ/Δ pre-B cells, significantly higher amounts of FAK and p-FAK were also observed in WT adherent relative to non-adherent pre-B cells, indicating that integrin signaling is also active in WT adherent pre-B cells (Fig. 5d-f).


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)

Increase in integrin signaling mediates adhesion of IkE5Δ/Δ pre-B cells to a stromal nichea, Pathway analysis of genes upregulated in IkE5Δ/Δ relative to WT large pre-B cells. Analysis was performed with a signature of upregulated genes shared by ex vivo mutant pre-B cells prior to and after limited stromal expansion. Pathways enriched for integrins and integrin signaling effectors are highlighted in red. b, Upregulated expression of components of the integrin-actin cytoskeleton pathway in primary and cultured WT and IkE5Δ/Δ pre-B cells as defined in Figs. 1 and 3. Hierarchical clustering of normalized gene expression values across different conditions is shown. c, Cell surface expression of integrins α5, β6, and activated β1 in ex vivo sorted and in vitro cultures of large pre-B cells. MFI for integrin staining is provided. d-f, Increase in FAK activation measured by flow cytometry, immunoblot and confocal microscopy. d, Flow cytometric analysis of p-FAK expression in ex-vivo and in vitro cultured large pre-B cells. MFI for p-FAK is indicated. e, Confocal immunofluorescence microscopy detection of activated p-FAK (red channel), GFP-expressing OP9 stroma (green channel), and nuclei (DAPI, blue channel). Scale bar, 25 μm. f, Immunoblot analysis of total FAK and activated p-FAK, with Btk as a loading control as in Fig. 4a. g, Adhesion of WT and IkE5Δ/Δ adherent pre-B cells to fibronectin-coated plates (left panel) in the presence of the fibronectin-derived RGD peptide or the RGE mutant variant (right panel). Asterisks denote significant differences in adhesion between mutant pre-B cells in the presence or absence of RGD or RGE peptides (n=3; *P < 0.05, two-tailed Student's t-test). h, Chemotaxis of WT (circle) and IkE5Δ/Δ (square) pre-B cells measured in a transwell migration assay in the presence of SDF1. The mean percentage of cells recovered at the bottom of the well in two independent studies is shown.
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Figure 5: Increase in integrin signaling mediates adhesion of IkE5Δ/Δ pre-B cells to a stromal nichea, Pathway analysis of genes upregulated in IkE5Δ/Δ relative to WT large pre-B cells. Analysis was performed with a signature of upregulated genes shared by ex vivo mutant pre-B cells prior to and after limited stromal expansion. Pathways enriched for integrins and integrin signaling effectors are highlighted in red. b, Upregulated expression of components of the integrin-actin cytoskeleton pathway in primary and cultured WT and IkE5Δ/Δ pre-B cells as defined in Figs. 1 and 3. Hierarchical clustering of normalized gene expression values across different conditions is shown. c, Cell surface expression of integrins α5, β6, and activated β1 in ex vivo sorted and in vitro cultures of large pre-B cells. MFI for integrin staining is provided. d-f, Increase in FAK activation measured by flow cytometry, immunoblot and confocal microscopy. d, Flow cytometric analysis of p-FAK expression in ex-vivo and in vitro cultured large pre-B cells. MFI for p-FAK is indicated. e, Confocal immunofluorescence microscopy detection of activated p-FAK (red channel), GFP-expressing OP9 stroma (green channel), and nuclei (DAPI, blue channel). Scale bar, 25 μm. f, Immunoblot analysis of total FAK and activated p-FAK, with Btk as a loading control as in Fig. 4a. g, Adhesion of WT and IkE5Δ/Δ adherent pre-B cells to fibronectin-coated plates (left panel) in the presence of the fibronectin-derived RGD peptide or the RGE mutant variant (right panel). Asterisks denote significant differences in adhesion between mutant pre-B cells in the presence or absence of RGD or RGE peptides (n=3; *P < 0.05, two-tailed Student's t-test). h, Chemotaxis of WT (circle) and IkE5Δ/Δ (square) pre-B cells measured in a transwell migration assay in the presence of SDF1. The mean percentage of cells recovered at the bottom of the well in two independent studies is shown.
Mentions: A comparative genome-wide transcriptional analysis of primary and cultured WT and IkE5Δ/Δ pre-B cells was performed to reveal potential pathways that might support the aberrant adhesion and growth properties of the mutant population. A signature of genes was deduced that was differentially expressed between both freshly isolated IkE5Δ/Δ and WT large pre-B cells and between IkE5Δ/Δ and WT adherent pre-B cells cultured in vitro (Fig. 5a, b). Up-regulated genes in IkE5Δ/Δ pre-B cells were highly enriched in pathways involved in focal adhesion and remodeling of the actin cytoskeleton (Fig. 5a). Integrins (e.g. Itga1, Itga5, Itgb1) as well as other structural and signaling components of focal adhesions (e.g. Ptk2, Vcl, Actn1, Cttn, Dock1, Rogdi) were shared by many of these pathways (Fig. 5b). The increase in integrin expression was validated at the protein level in both primary and cultured cells. Furthermore, expression of the active isoform of integrin β1 (detected with an activation-specific anti-integrin β1 antibody and elevated levels of phosphorylated focal adhesion kinase (p-FAK), a key downstream effector of integrin signaling, indicated that not only expression but also activation of integrin signaling were elevated in IkE5Δ/Δ pre-B cells (Fig. 5c-f). Although not as pronounced as in IkE5Δ/Δ pre-B cells, significantly higher amounts of FAK and p-FAK were also observed in WT adherent relative to non-adherent pre-B cells, indicating that integrin signaling is also active in WT adherent pre-B cells (Fig. 5d-f).

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