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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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Ikaros-deficient pre-B cells grow only on stromaa, Flow cytometric analysis of sorted large pre-B cells (CD19+CD43+BP1+) cultured for 7 days stromal-free with limiting serum and IL-7. Differentiation of WT and IkE5Δ/Δ large pre-B cells is monitored by stage-specific markers. Arrows indicate the direction of pre-B cell differentiation as depicted in Supplementary Fig. 1a. b, Growth of WT and IkE5Δ/Δ large pre-B cells in high, low, and no (5, 0.05, and 0 ng/ml, respectively) IL-7 concentrations under stromal-free conditions (left) or with OP9 BM stroma (right). The mean absolute number of cells obtained in stromal-free (n=5) and stromal-containing (n=4) cultures with replicates for each experiment is shown in a line graph ± s.d. Asterisks denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). c, Mean percentage ± s.d. of apoptotic (AnnexinV+) WT and IkE5Δ/Δ large pre-B cells in stromal-free cultures as in Fig. 2b, left panel. d, Cell cycle stage distribution (mean percentage ± s.d. of cells in S+G2+M) of WT and IkE5Δ/Δ large pre-B stromal cultures as in Fig. 2b, right panel. Asterisks in c and d denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). e, Cell cycle kinetics of WT and IkE5Δ/Δ large pre-B cells grown on stroma as measured by BrdU pulse-chase. The mean fluorescence intensity (MFI) of BrdU staining is shown at 45 min of pulse and after 48 h of chase.
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Figure 2: Ikaros-deficient pre-B cells grow only on stromaa, Flow cytometric analysis of sorted large pre-B cells (CD19+CD43+BP1+) cultured for 7 days stromal-free with limiting serum and IL-7. Differentiation of WT and IkE5Δ/Δ large pre-B cells is monitored by stage-specific markers. Arrows indicate the direction of pre-B cell differentiation as depicted in Supplementary Fig. 1a. b, Growth of WT and IkE5Δ/Δ large pre-B cells in high, low, and no (5, 0.05, and 0 ng/ml, respectively) IL-7 concentrations under stromal-free conditions (left) or with OP9 BM stroma (right). The mean absolute number of cells obtained in stromal-free (n=5) and stromal-containing (n=4) cultures with replicates for each experiment is shown in a line graph ± s.d. Asterisks denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). c, Mean percentage ± s.d. of apoptotic (AnnexinV+) WT and IkE5Δ/Δ large pre-B cells in stromal-free cultures as in Fig. 2b, left panel. d, Cell cycle stage distribution (mean percentage ± s.d. of cells in S+G2+M) of WT and IkE5Δ/Δ large pre-B stromal cultures as in Fig. 2b, right panel. Asterisks in c and d denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). e, Cell cycle kinetics of WT and IkE5Δ/Δ large pre-B cells grown on stroma as measured by BrdU pulse-chase. The mean fluorescence intensity (MFI) of BrdU staining is shown at 45 min of pulse and after 48 h of chase.

Mentions: The developmental defect in IkE5Δ/Δ large pre-B cells was further evaluated in in vitro cultures11,34. Under differentiation-inducing conditions (i.e. seven days of stromal-free culture in low concentrations of serum and IL-7), the majority of WT large pre-B cells exited the cell cycle and differentiated into small pre-B (CD19+CD2+IgM−) and immature B (CD19+IgM+CD2+) cells, whereas mutant large pre-B cells (CD19+CD43+BP1+) remained undifferentiated (Fig. 2a). An increase in the concentration of IL-7 promoted the proliferative expansion of WT large pre-B cells but had little effect on their mutant counterparts. In the absence of stroma, survival of IkE5Δ/Δ pre-B cells was greatly compromised compared to WT pre-B cells even in the presence of high concentrations of IL-7, with high levels of apoptosis detected from early time points of culture (Fig. 2b, left panel, and Fig. 2c).


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)

Ikaros-deficient pre-B cells grow only on stromaa, Flow cytometric analysis of sorted large pre-B cells (CD19+CD43+BP1+) cultured for 7 days stromal-free with limiting serum and IL-7. Differentiation of WT and IkE5Δ/Δ large pre-B cells is monitored by stage-specific markers. Arrows indicate the direction of pre-B cell differentiation as depicted in Supplementary Fig. 1a. b, Growth of WT and IkE5Δ/Δ large pre-B cells in high, low, and no (5, 0.05, and 0 ng/ml, respectively) IL-7 concentrations under stromal-free conditions (left) or with OP9 BM stroma (right). The mean absolute number of cells obtained in stromal-free (n=5) and stromal-containing (n=4) cultures with replicates for each experiment is shown in a line graph ± s.d. Asterisks denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). c, Mean percentage ± s.d. of apoptotic (AnnexinV+) WT and IkE5Δ/Δ large pre-B cells in stromal-free cultures as in Fig. 2b, left panel. d, Cell cycle stage distribution (mean percentage ± s.d. of cells in S+G2+M) of WT and IkE5Δ/Δ large pre-B stromal cultures as in Fig. 2b, right panel. Asterisks in c and d denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). e, Cell cycle kinetics of WT and IkE5Δ/Δ large pre-B cells grown on stroma as measured by BrdU pulse-chase. The mean fluorescence intensity (MFI) of BrdU staining is shown at 45 min of pulse and after 48 h of chase.
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Figure 2: Ikaros-deficient pre-B cells grow only on stromaa, Flow cytometric analysis of sorted large pre-B cells (CD19+CD43+BP1+) cultured for 7 days stromal-free with limiting serum and IL-7. Differentiation of WT and IkE5Δ/Δ large pre-B cells is monitored by stage-specific markers. Arrows indicate the direction of pre-B cell differentiation as depicted in Supplementary Fig. 1a. b, Growth of WT and IkE5Δ/Δ large pre-B cells in high, low, and no (5, 0.05, and 0 ng/ml, respectively) IL-7 concentrations under stromal-free conditions (left) or with OP9 BM stroma (right). The mean absolute number of cells obtained in stromal-free (n=5) and stromal-containing (n=4) cultures with replicates for each experiment is shown in a line graph ± s.d. Asterisks denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). c, Mean percentage ± s.d. of apoptotic (AnnexinV+) WT and IkE5Δ/Δ large pre-B cells in stromal-free cultures as in Fig. 2b, left panel. d, Cell cycle stage distribution (mean percentage ± s.d. of cells in S+G2+M) of WT and IkE5Δ/Δ large pre-B stromal cultures as in Fig. 2b, right panel. Asterisks in c and d denote significant differences between WT and mutant cells (*P < 0.05, **P < 0.01, two-tailed Student's t-test). e, Cell cycle kinetics of WT and IkE5Δ/Δ large pre-B cells grown on stroma as measured by BrdU pulse-chase. The mean fluorescence intensity (MFI) of BrdU staining is shown at 45 min of pulse and after 48 h of chase.
Mentions: The developmental defect in IkE5Δ/Δ large pre-B cells was further evaluated in in vitro cultures11,34. Under differentiation-inducing conditions (i.e. seven days of stromal-free culture in low concentrations of serum and IL-7), the majority of WT large pre-B cells exited the cell cycle and differentiated into small pre-B (CD19+CD2+IgM−) and immature B (CD19+IgM+CD2+) cells, whereas mutant large pre-B cells (CD19+CD43+BP1+) remained undifferentiated (Fig. 2a). An increase in the concentration of IL-7 promoted the proliferative expansion of WT large pre-B cells but had little effect on their mutant counterparts. In the absence of stroma, survival of IkE5Δ/Δ pre-B cells was greatly compromised compared to WT pre-B cells even in the presence of high concentrations of IL-7, with high levels of apoptosis detected from early time points of culture (Fig. 2b, left panel, and Fig. 2c).

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