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

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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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Figure 1: The differentiation of pre-B cells is dependent on the Ikaros family.
Figure 2: Ikaros-deficient pre-B cells grow only on stroma.
Figure 3: A stroma-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikaros.
Figure 4: Signaling pathways in wild-type and Ikaros-deficient pre-B cells.
Figure 5: Increase in integrin signaling mediates the adhesion of IkE5Δ/Δ pre-B cells to a stromal niche.
Figure 6: Inhibition of FAK interferes with the survival of IkE5Δ/Δ pre-B cells.
Figure 7: Cooperation between integrin signaling and growth-factor signaling supports the survival and proliferation of IkE5Δ/Δ pre-B cells.
Figure 8: Leukemogenic potential of IkE5Δ/Δ pre-B cells.

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Acknowledgements

We thank B. Czyzewski for mouse husbandry; K. White, J.M. Park, B. Morgan, R. Bakshi, E. Alonzo and J. Seavitt for critical review of the manuscript; R. Bakshi for assistance with statistical analysis; and R. Arya for assistance with confocal microscopy. High-throughput DNA sequencing was done at the Bauer Center for Genomic Research (Harvard University). Supported by the US National Institutes of Allergy and Infectious Diseases (American Recovery and Reinvestment Act supplement to 5R01AI 42254-14) and the National Cancer Institute (5R01CA162092-20 to K.G. and CA090576 to R.A.V.E.).

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I.J., T.Y., N.J., X.Q. and J.Z. did the experiments and edited the manuscript; I.J. and T.Y. created figures; and R.A.V.E. and K.G. supervised the research and wrote the manuscript.

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Correspondence to Richard A Van Etten or Katia Georgopoulos.

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Integrated supplementary information

Supplementary Figure 1 Analysis of B-lymphoid differentiation in Ikaros-mutant BM.

a, Schematic representation of B cell differentiation as defined by stage-specific markers. Dotted lines indicate differentiation stages with CD2- or CD19-Cre activity, red lines the differentiation block associated with germline or conditional Ikaros gene mutations, and red arrow the stage from which B-ALL is derived. b-c, Representative flow cytometric analyses of wild-type (WT), IkE5fl/fl CD19-Cre (b) and Ikzf3−/− Ikzf1+/− (c) BM cells as described in Fig. 1d, demonstrating a consistent block at the large pre-B cell stage. IkE5fl/fl CD19-Cre, n=9; Ikzf3−/− Ikzf1+/−, n=3. d, Deletion analysis of the Ikzf1 locus in pro-B cells (CD19+CD43+c-Kit+BP1) and immature B cells (CD19+IgM+) sorted from BM of IkE5fl/fl CD2-Cre mice.

Supplementary Figure 2 Analysis of B-lymphoid differentiation in immunoglobulin κ-chain−reconstituted Ikaros-mutant pre-B cells.

Flow cytometric analysis of BM B cells from WT, D23, IkE5fl/fl CD2-Cre and IkE5fl/fl CD2-Cre:D23 and intracellular staining for IgM and Igκ in large pre-B cells (CD19+CD43+BP1+).

Supplementary Figure 3 Characterization of adherent and nonadherent wild-type pre-B cells.

a, The mean pro-apoptotic index (percentage of Annexin V+ cells) of WT and IkE5Δ/Δ adherent (left panel) and non-adherent (right panel) pre-B cells propagated on OP9 stroma with 5 ng/ml of supplemental IL-7. Asterisk denotes significant changes in apoptosis between WT and mutant pre-B cells (n=2, *P < 0.05). b, Representative cell cycle profiles of WT adherent and WT non-adherent pre-B cells grown as in Fig. 2a. WT non-adherent pre-B cells were further subdivided according to FSC. The ratio of small vs. large non-adherent WT pre-B in IL-7 cultures increases over time (data not shown). The progressive loss in proliferation in the WT non-adherent pre-B cell phase seen even in the presence of IL-7, suggests a need for stromal contact for maintenance of pre-B cell proliferation. Withdrawal of IL-7 accelerates this process with the ratio of small-non-cycling/large-cycling non-adherent pre-B cells increasing dramatically within 24 hrs (data not shown).

Supplementary Figure 4 Signaling pathways in wild-type and Ikaros-deficient pre-B cells.

a. Schematic representation of signaling pathways operating downstream of pre-BCR and IL-7R and supporting pre-B cell proliferation, survival and differentiation. Signaling effectors assayed for expression and activity in Fig. 4a, b are shown. b, Ca2+ flux (Ca2+ Green/Fura Red) after ionomycin treatment of WT and IkE5Δ/Δ adherent and WT non-adherent pre-B cells, n=2. c, Total Blk expression is shown for WT and IkE5Δ/Δ adherent and non-adherent pre-B cells, with total Akt (T-Akt) as loading control.

Supplementary Figure 5 Lack of circulating IkE5Δ/Δ pre-B cells and decrease in phosphorylated FAK by FAK inhibitor.

a, Flow cytometric analysis of peripheral blood from wild-type (WT) and IkE5fl/fl CD19-Cre mice for large pre-B (CD19+CD43+) and small pre-B cells (CD19+CD43); n=2 for each genotype. b, FAK inhibitor treatment reduces p-FAK staining in BM IkE5Δ/Δ pre-B cells, as described in Fig. 6b.

Supplementary Figure 6 Model of pre-BCR, growth factor, and integrin signalling interactions operating during pre-B cell differentiation.

Augmentation of integrin signaling by IkE5Δ/Δ mutation blocks cells in a stromal-dependent, self-renewing and highly proliferative state where they are unable to differentiate, from which B-ALL arises.

Supplementary Figure 7 Clinicopathological characterization of lymphoid tumors from recipients of IkE5Δ/Δ pre-B cells.

a, Immunophenotypic analysis of precursor B-cell acute lymphoblastic leukemia/lymphoma derived from IkE5Δ/Δ pre-B cells demonstrates a similar large pre-B cell surface phenotype (CD19+CD43+BP1+CD2) to the original transplanted population. b, Analysis of parental WT and IkE5Δ/Δ pre-B cell populations (non-adherent and adherent), showing polyclonal Igh rearrangements similar to that observed in WT splenocytes. The PCR-based D-J rearrangement assay described in Fig. 1f was used to determine clonality. PCR products were probed with a JH-specific probe. c, PCR analysis of V-D-J rearrangements in lymphoid tumors from NSG recipients of IkE5Δ/Δ pre-B cells, as described in Fig. 1f. Forward primers from specific VH regions (558, Q52, 7183) were used in conjunction with a common reverse primer from JH3 (Fig. 1f). Note that lymphoid tumors from mice #391, 393, and 394 (from IkE5Δ/Δ CD19-Cre donor) had monoclonal Igh rearrangement while #392 tumor had clonal rearrangement of both Igh alleles. d, Southern blot analysis of Igh gene rearrangements in tissues of leukemic NSG recipients of IkE5Δ/Δ pre-B cells, as in panel c. The position of two germline (GL) Igh bands (present in control BM myeloid cells, “C”) is denoted by arrowheads. The tissue origin of the sample is indicated (Sp, spleen; LN, lymph node). Common rearrangements between tumors from IkE5Δ/Δ CD19-Cre recipients are indicated by asterisks. Rearrangements in IkE5Δ/Δ CD2-Cre recipients #385 and 386 may not be detected by this probe.

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Joshi, I., Yoshida, T., Jena, N. et al. Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia. Nat Immunol 15, 294–304 (2014). https://doi.org/10.1038/ni.2821

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