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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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A stromal-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikarosa, An adherent phase in pre-B cell differentiation as revealed in stromal cultures of WT and IkE5Δ/Δ large pre-B cells grown in the presence of IL-7 (5 ng/ml). Areas with adherent cells were marked with rectangles (left) and digitally magnified (right). Dotted circle marks the nucleus of OP9 stromal cells used as a stromal reference (scale bar, 30 μm). b, Ratio of adherent to non-adherent cells in WT and IkE5Δ/Δ pre-B cultures at day 2 (D2) and day 3 (D3) with 5 and 0.05 ng/ml of IL-7. The mean ratio is presented ± s.d. Asterisks denote significant differences between WT and mutant pre-B cells at each culture time point (***P < 0.0001, **P < 0.01, *P < 0.05, two-tailed Student's t-test). c, Comparative expression analysis of pre-B cell differentiation genes in adherent and non-adherent pre-B cells. Hierarchical clustering of normalized gene expression values across different conditions is shown. d, Flow cytometric analysis of adherent and non-adherent cells from WT and IkE5Δ/Δ large pre-B cell stromal cultures for CD25 and intracellular Igκ and Igκ. The percentages of positive cells relative to isotype control (grey curve) are indicated. e, Rates of propagation of WT adherent and non-adherent pre-B cell fractions grown with 5 ng/ml of IL-7. The mean number of cells generated by 5 × 104 adherent (dark blue) or non-adherent (light blue) WT pre-B cells after replating on OP9 stroma for 3 days of culture is shown in the top panel. The mean number of adherent and non-adherent subsets recovered from plating either WT adherent or non-adherent pre-B cell stromal cultures is shown in the bottom panel. Error bars indicate s.d. Asterisks indicate a statistically significant difference in the growth (top panel) of WT adherent and non-adherent B cells (*P< 0.05, **P < 0.01, two-tailed Student's t-test). f, Limiting dilution colony forming assay was performed as described previously29. The mean frequency of colony forming cells was calculated based on Poisson distribution and is presented in a line graph ± s.e. g, Re-association of WT and IkE5Δ/Δ pre-B cells after replating on stroma. The mean percentage ± s.d. of stromal-adherent cells, measured 3 hrs after replating is shown. Study was performed with two independent WT and mutant pre-B cell cultures (closed and open symbols), each assayed in ten grids/well. Binding to stroma was calculated per twenty grids and averaged for each cell type (*P < 0.0001, two-tailed Student's t-test).
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Figure 3: A stromal-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikarosa, An adherent phase in pre-B cell differentiation as revealed in stromal cultures of WT and IkE5Δ/Δ large pre-B cells grown in the presence of IL-7 (5 ng/ml). Areas with adherent cells were marked with rectangles (left) and digitally magnified (right). Dotted circle marks the nucleus of OP9 stromal cells used as a stromal reference (scale bar, 30 μm). b, Ratio of adherent to non-adherent cells in WT and IkE5Δ/Δ pre-B cultures at day 2 (D2) and day 3 (D3) with 5 and 0.05 ng/ml of IL-7. The mean ratio is presented ± s.d. Asterisks denote significant differences between WT and mutant pre-B cells at each culture time point (***P < 0.0001, **P < 0.01, *P < 0.05, two-tailed Student's t-test). c, Comparative expression analysis of pre-B cell differentiation genes in adherent and non-adherent pre-B cells. Hierarchical clustering of normalized gene expression values across different conditions is shown. d, Flow cytometric analysis of adherent and non-adherent cells from WT and IkE5Δ/Δ large pre-B cell stromal cultures for CD25 and intracellular Igκ and Igκ. The percentages of positive cells relative to isotype control (grey curve) are indicated. e, Rates of propagation of WT adherent and non-adherent pre-B cell fractions grown with 5 ng/ml of IL-7. The mean number of cells generated by 5 × 104 adherent (dark blue) or non-adherent (light blue) WT pre-B cells after replating on OP9 stroma for 3 days of culture is shown in the top panel. The mean number of adherent and non-adherent subsets recovered from plating either WT adherent or non-adherent pre-B cell stromal cultures is shown in the bottom panel. Error bars indicate s.d. Asterisks indicate a statistically significant difference in the growth (top panel) of WT adherent and non-adherent B cells (*P< 0.05, **P < 0.01, two-tailed Student's t-test). f, Limiting dilution colony forming assay was performed as described previously29. The mean frequency of colony forming cells was calculated based on Poisson distribution and is presented in a line graph ± s.e. g, Re-association of WT and IkE5Δ/Δ pre-B cells after replating on stroma. The mean percentage ± s.d. of stromal-adherent cells, measured 3 hrs after replating is shown. Study was performed with two independent WT and mutant pre-B cell cultures (closed and open symbols), each assayed in ten grids/well. Binding to stroma was calculated per twenty grids and averaged for each cell type (*P < 0.0001, two-tailed Student's t-test).

Mentions: A striking morphological difference was apparent between IkE5Δ/Δ and WT large pre-B cell OP9 stromal cultures. The majority of WT pre-B cells were round, light-refracting cells loosely attached to stroma, but the majority of mutant cells had a dark, flat morphology and appeared incorporated into the stromal layer (Fig. 3a, b). Dark, stromal-adherent pre-B cells were also present in WT cultures, but at a much lower frequency (Fig. 3a, b). The few IkE5Δ/Δ non-adherent cells displayed increased apoptosis (Supplementary Fig. 3a), indicating that in the absence of stromal contact, their survival was greatly compromised.


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)

A stromal-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikarosa, An adherent phase in pre-B cell differentiation as revealed in stromal cultures of WT and IkE5Δ/Δ large pre-B cells grown in the presence of IL-7 (5 ng/ml). Areas with adherent cells were marked with rectangles (left) and digitally magnified (right). Dotted circle marks the nucleus of OP9 stromal cells used as a stromal reference (scale bar, 30 μm). b, Ratio of adherent to non-adherent cells in WT and IkE5Δ/Δ pre-B cultures at day 2 (D2) and day 3 (D3) with 5 and 0.05 ng/ml of IL-7. The mean ratio is presented ± s.d. Asterisks denote significant differences between WT and mutant pre-B cells at each culture time point (***P < 0.0001, **P < 0.01, *P < 0.05, two-tailed Student's t-test). c, Comparative expression analysis of pre-B cell differentiation genes in adherent and non-adherent pre-B cells. Hierarchical clustering of normalized gene expression values across different conditions is shown. d, Flow cytometric analysis of adherent and non-adherent cells from WT and IkE5Δ/Δ large pre-B cell stromal cultures for CD25 and intracellular Igκ and Igκ. The percentages of positive cells relative to isotype control (grey curve) are indicated. e, Rates of propagation of WT adherent and non-adherent pre-B cell fractions grown with 5 ng/ml of IL-7. The mean number of cells generated by 5 × 104 adherent (dark blue) or non-adherent (light blue) WT pre-B cells after replating on OP9 stroma for 3 days of culture is shown in the top panel. The mean number of adherent and non-adherent subsets recovered from plating either WT adherent or non-adherent pre-B cell stromal cultures is shown in the bottom panel. Error bars indicate s.d. Asterisks indicate a statistically significant difference in the growth (top panel) of WT adherent and non-adherent B cells (*P< 0.05, **P < 0.01, two-tailed Student's t-test). f, Limiting dilution colony forming assay was performed as described previously29. The mean frequency of colony forming cells was calculated based on Poisson distribution and is presented in a line graph ± s.e. g, Re-association of WT and IkE5Δ/Δ pre-B cells after replating on stroma. The mean percentage ± s.d. of stromal-adherent cells, measured 3 hrs after replating is shown. Study was performed with two independent WT and mutant pre-B cell cultures (closed and open symbols), each assayed in ten grids/well. Binding to stroma was calculated per twenty grids and averaged for each cell type (*P < 0.0001, two-tailed Student's t-test).
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Figure 3: A stromal-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikarosa, An adherent phase in pre-B cell differentiation as revealed in stromal cultures of WT and IkE5Δ/Δ large pre-B cells grown in the presence of IL-7 (5 ng/ml). Areas with adherent cells were marked with rectangles (left) and digitally magnified (right). Dotted circle marks the nucleus of OP9 stromal cells used as a stromal reference (scale bar, 30 μm). b, Ratio of adherent to non-adherent cells in WT and IkE5Δ/Δ pre-B cultures at day 2 (D2) and day 3 (D3) with 5 and 0.05 ng/ml of IL-7. The mean ratio is presented ± s.d. Asterisks denote significant differences between WT and mutant pre-B cells at each culture time point (***P < 0.0001, **P < 0.01, *P < 0.05, two-tailed Student's t-test). c, Comparative expression analysis of pre-B cell differentiation genes in adherent and non-adherent pre-B cells. Hierarchical clustering of normalized gene expression values across different conditions is shown. d, Flow cytometric analysis of adherent and non-adherent cells from WT and IkE5Δ/Δ large pre-B cell stromal cultures for CD25 and intracellular Igκ and Igκ. The percentages of positive cells relative to isotype control (grey curve) are indicated. e, Rates of propagation of WT adherent and non-adherent pre-B cell fractions grown with 5 ng/ml of IL-7. The mean number of cells generated by 5 × 104 adherent (dark blue) or non-adherent (light blue) WT pre-B cells after replating on OP9 stroma for 3 days of culture is shown in the top panel. The mean number of adherent and non-adherent subsets recovered from plating either WT adherent or non-adherent pre-B cell stromal cultures is shown in the bottom panel. Error bars indicate s.d. Asterisks indicate a statistically significant difference in the growth (top panel) of WT adherent and non-adherent B cells (*P< 0.05, **P < 0.01, two-tailed Student's t-test). f, Limiting dilution colony forming assay was performed as described previously29. The mean frequency of colony forming cells was calculated based on Poisson distribution and is presented in a line graph ± s.e. g, Re-association of WT and IkE5Δ/Δ pre-B cells after replating on stroma. The mean percentage ± s.d. of stromal-adherent cells, measured 3 hrs after replating is shown. Study was performed with two independent WT and mutant pre-B cell cultures (closed and open symbols), each assayed in ten grids/well. Binding to stroma was calculated per twenty grids and averaged for each cell type (*P < 0.0001, two-tailed Student's t-test).
Mentions: A striking morphological difference was apparent between IkE5Δ/Δ and WT large pre-B cell OP9 stromal cultures. The majority of WT pre-B cells were round, light-refracting cells loosely attached to stroma, but the majority of mutant cells had a dark, flat morphology and appeared incorporated into the stromal layer (Fig. 3a, b). Dark, stromal-adherent pre-B cells were also present in WT cultures, but at a much lower frequency (Fig. 3a, b). The few IkE5Δ/Δ non-adherent cells displayed increased apoptosis (Supplementary Fig. 3a), indicating that in the absence of stromal contact, their survival was greatly compromised.

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