B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3 thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling4,5. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival6. We tested the hypothesis that targeted hyperactivation—above a maximum threshold—will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR–ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
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We thank R. W. Hendriks for encouragement and critical discussions, A. Park and C. Lin and all the members of the Müschen laboratory, L. Tian and B. Scott for their support. This work is supported by grants from the National Institutes of Health/National Cancer Institute through R01CA137060, R01CA139032, R01CA169458, R01CA172558 and R01CA157644 (to M.M.), ECOG-ACRIN grants CA180820 and CA180794 (to E.P.), grants from the Leukemia and Lymphoma Society (to M.M.), the California Institute for Regenerative Medicine through TR02-1816 (M.M.), and the William Lawrence and Blanche Hughes Foundation. M.M. is a Scholar of The Leukemia and Lymphoma Society and a Senior Investigator of the Wellcome Trust. M.R. is supported by the DFG through EXC294, TRR130 and SFB746.
The authors declare no competing financial interests.
Extended data figures and tables
a, Flow cytometry staining for cell-surface Igα (CD79A) and Igβ (CD79B) was performed for patient-derived Ph+ ALL cases (n = 8) and B-cell leukaemia/lymphomas lacking oncogenic tyrosine kinases (n = 4). b, c, Normal mouse pre-B cells or BCR–ABL1-transformed pre-B ALL cells were retrovirally transduced with CD8–Igα–GFP or empty vector (GFP) controls (EV). Relative changes of transduced (GFP+) populations were monitored by flow cytometry. d, Tyrosine phosphorylation of Syk, Src/Lyn, Btk and Plc-γ2 was studied in BCR-ABL1 ALL cells that were transduced with Igα–GFP or GFP empty vector controls, using β-actin as loading control. Data (c, d) are representative of three independent experiments. e, Human Ph+ ALL cells were transduced with GFP-tagged vectors for LMP2A–ITAM or empty vector. Relative changes of transduced (GFP+) populations were monitored by flow cytometry (n = 3). f, LMP2A–ITAM or an empty vector was expressed in three cases of human Ph+ ALL cells and effects on LMP2A expression and phosphorylation of SYK, SRC, BTK and PLC-γ2 were measured by western blot (n = 3). g, BCR-ABL1-transformed ALL cells were transduced with GFP-tagged SykMyr or an empty vector, and these cells were treated with the SYK inhibitor PRT (2.5 μmol l−1) or vehicle either 1 day before transduction (PRT-pre), or 1 day after transduction (PRT-post), or pre-treated, then washed out for 1 day after transduction, and treated again with PRT. The relative changes of transduced (GFP+) cells were monitored by flow cytometry. Error bars represent means ± s.d. from three independent experiments (b, e, g).
a, BCR-ABL1-transformed pre-B ALL cells were transduced with myristoylated (active) forms of Btk, Syk or empty vector controls (EV). Vectors were GFP-tagged and fractions of GFP+ cells were monitored over time. b, Btk−/− BCR-ABL1-transformed pre-B ALL cells were transduced with myristoylated (active) Btk or empty vector (both tagged with GFP). Fractions of GFP+ cells were monitored over time. c, Csk is a negative regulator of Src family kinase activity. Csk-AS transgenic mice express an analogue (3IB-PP1) sensitive form instead of endogenous Csk. For inducible activation of Src kinase activity, we transformed pre-B cells from Csk-AS transgenic mice with BCR-ABL1. Addition of 3IB-PP1 (10 μmol l−1) released Csk-mediated inhibition and induced increased phosphorylation of Src family kinases at Y416, but did not increase Syk Y352 phosphorylation (western blot). d, Pre-B cells from analogue-sensitive Csk-AS transgenic and wild-type mice were transformed with BCR-ABL1 and treated with 3IB-PP1. Cell viability in response to 3IB-PP1 treatment was monitored over time. Error bars (a, b, d) represent means ± s.d. from three independent experiments. e, Rag1−/− pro-B cells were expanded in the presence of 10 ng ml−1 Il-7 and transduced with an empty vector (GFP), constitutively active SYK (TEL-SYK–GFP), or a kinase-dead mutant of SYK (TEL-SYK(K402A)–GFP). Then Il-7 was removed from cell cultures and the effect of Il-7 removal on cell viability was studied. Rag1−/− pro-B cells transduced with empty vector or TEL-SYK(K402A)–GFP underwent apoptosis, whereas pro-B cells transduced with constitutively active SYK remained viable (data not shown). Four days after Il-7 removal, pro-B cells with constitutively active SYK had acquired growth factor (Il-7) independence, whereas pro-B cells with empty vector and TEL-SYK(K402A)–GFP remained dependent on Il-7. Data (c, e) are representative of three independent experiments.
a, Microarray data for 62 ITIM-bearing receptors are ranked based on the ratio of messenger RNA levels in Ph+ ALL compared to normal pre-B cells and mature B-cell lymphomas. b, Fluorescence-activated cell sorting (FACS) dot plots for double staining of PECAM1, CD300A, LAIR1 and BTLA with CD19 are shown for normal bone marrow pre-B cells (n = 1), Ph+ ALL cells (n = 8) and non-tyrosine-kinase-driven B-cell lymphoma (n = 4). c, Normal bone marrow mononucleated cells from bone marrow biopsies of healthy donors (n = 3), patient-derived Ph+ ALL (n = 11) and non-tyrosine-kinase-driven B-cell lymphoma (n = 11) were analysed by flow cytometry for surface expression of the ITIM-bearing inhibitory receptors PECAM1, CD300A, LAIR1, BTLA, CEACAM1, CD22, FCRL2. Additional staining for CD72 and LILRB5 did not show significant differences between Ph+ ALL cells and normal pre-B cells (data not shown). Statistical analysis of mean fluorescence intensities (MFIs) for normal pre-B cells (n = 3), Ph+ ALL (n = 11) and non-tyrosine-kinase-driven B-cell lymphoma (n = 11) showed significantly increased expression levels of PECAM1, LAIR1 and CD300A in Ph+ ALL compared to normal pre-B cells and non-tyrosine-kinase-driven B-cell lymphoma. P values were calculated using unpaired, two-tailed Student’s t-test.
Extended Data Figure 4 Higher than median expression levels of ITIM-bearing inhibitory receptors predict poor outcomes in patients with pre-B ALL.
a–c, mRNA levels for PECAM1, CD300A and LAIR1 were measured in 207 patients with paediatric ALL (COG P9906). PECAM1, CD300A and LAIR1 mRNA levels for ALL cells from 124 patients that had no detectable minimal residual disease (MRD negative; black) on day 29 in their bone marrow were compared to mRNA levels in 67 patients with positive MRD (red) at the time of bone marrow biopsy (day 29). On the basis of higher or lower than median expression levels of PECAM1, CD300A and LAIR1, patients were segregated into two groups (High, n = 104; Low, n = 103; plots in middle and right). Overall survival (OS; middle) and relapse-free survival (RFS; right) probabilities were estimated by Kaplan–Meier survival analyses. P values were calculated by Mann–Whitney–Wilcoxon test (left panels; MRD status) and log-rank test (middle and right panels; overall survival and relapse-free survival). d, e, ITAM-based agonists (CD79A, CD79B, IGHM) and ITIM-based inhibitors (PECAM1, CD300A, LAIR1) of pre-BCR signalling were combined into a six-gene outcome predictor based on ‘ITAM’ and ‘ITIM’ signatures and validated in two clinical trials for adults with Ph+ ALL (ECOG E2993) and children with ALL (COG P9906). P values were calculated by log-rank test. f, Lair1 deletion was confirmed by flow cytometry. g, Expression of checkpoint molecules Arf, p53, p21 and p27 was measured by western blot in the presence and absence of Pecam1 and Cd300a and upon inducible deletion of Lair1 in BCR-ABL1 pre-B ALL cells. h, Accumulation of ROS was measured by staining with 2′7′-dichlorofluorescein diacetate (DCF) in BCR-ABL1 pre-B ALL cells (grey histograms for control; red for gene deletion). Data are representative of three independent experiments (f–h).
Extended Data Figure 5 Consequences of genetic deletion of ITIM-bearing receptors in pre-B ALL cells.
a, b, B-cell precursors from the bone marrow of Pecam1−/− and Cd300a−/− as well as Lair1fl/fl mice and wild-type controls were propagated with Il-7 and transduced with an empty vector control (EV; normal B-cell precursors) or transformed with BCR-ABL1 to model Ph+ ALL. Lair1fl/fl pre-B and BCR-ABL1 leukaemia cells were transduced with 4-OHT-inducible retroviral Cre. Cell cycle progression of normal pre-B cells (EV) and BCR-ABL1 ALL cells was measured by BrdU staining (a). Propensity to cellular senescence was measured by staining of normal pre-B and BCR-ABL1 ALL cells for senescence-associated β-galactosidase (b). a, Numbers indicate percentage of cells in each cell cycle phase. b, Numbers indicate percentage of β-galactosidase-positive cells. c, Lair1fl/fl BCR-ABL1 ALL cells were transduced with 4-OHT-inducible Cre (Cre-ERT2) or an empty vector control (ERT2). Viability was measured by flow cytometry after 4-OHT treatment. d, Effects of inducible deletion of Lair1 on phosphorylation levels of Ptpn6 and Inpp5d were measured by western blot. e, Lair1fl/fl ALL cells were transduced with 4-OHT-inducible Cre. After antibiotic selection, ALL cells were transduced with a GFP-tagged empty vector control or GFP-tagged overexpression vectors for constitutively active forms of Ptpn6 (lacking autoinhibitory SH2 domain), Inpp5d (membrane-anchored by CD8) and Ptpn11 (constitutively active D61A mutation). Expression levels of Ptpn6, Inpp5d and Ptpn11 were measured by western blot using β-actin as loading control. The transduced cells were used for Cre-mediated deletion of Lair1 to determine if expression of constitutively active Ptpn6, Inpp5d and Ptpn11 can rescue leukaemia cell survival. Data (a, d, e) are representative of three independent experiments. Data (b, c) represent means ± s.d. from three independent experiments.
a, Protein levels of PTPN6 and INPP5D were measured by western blot in CD19+ bone marrow pre-B cells from healthy donors (n = 3), patient-derived Ph+ ALL (n = 8) and B-cell leukaemia/lymphoma (n = 4) lacking an oncogenic tyrosine kinase. Additional western blot analyses compared expression levels of PTPN6 and INPP5D in patient-derived Ph+ ALL (n = 5) and patient-derived chronic phase CML cells (n = 5). b, c, Bone marrow cells were isolated from Ptpn6+/fl or Inpp5dfl/fl mice and pre-B cells were propagated with Il-7 (10 ng ml−1). Ptpn6+/fl and Inpp5dfl/fl pre-B cells were then transformed with BCR-ABL1 retrovirus and subsequently transduced with 4-OHT-inducible Cre (Cre-ERT2) or an empty vector control (ERT2). Addition of 4-OHT induced nuclear translocation of Cre and Cre-mediated excision of Ptpn6+/fl (one allele) and Inpp5dfl/fl alleles as verified here by genomic PCR (b, left, for Ptpn6+/fl; c, left, for Inpp5dfl/fl). Near-complete deletion of the Ptpn6+/fl (one allele) and Inpp5dfl/fl floxed alleles was observed after 3 and 4 days, respectively, at the genomic level (left). Kinetics of protein depletion upon heterozygous deletion of Ptpn6 and homozygous deletion of Inpp5d (Inpp5dΔ70) was studied by western blot (right) using β-actin as loading control. d, e, Effects of Cre-mediated inducible deletion of Ptpn6 (d) or Inpp5d (e) on BCR-ABL1-transformed pre-B ALL cell viability were measured by flow cytometry at the times indicated. Numbers denote percentages of viable cells (determined by forward scatter (FSC) and propidium iodide (PI) uptake). Data are representative of three independent experiments (d, e).
Extended Data Figure 7 Functional consequences of inducible Ptpn6 or Inpp5d deletion in pre-B ALL cells.
a, The effects of deletion of Ptpn6 or Inpp5d on cellular ROS levels were measured by flow cytometry using DCF in BCR-ABL1 pre-B ALL cells (grey histograms for control; red for gene deletion). b, Whether ROS accumulation in response to deletion of Ptpn6 or Inpp5d results in wide-spread cysteine-oxidation and, hence, inactivation, of multiple other PTP active sites was determined by western blot using antibodies against oxidized PTP active sites. c, Protein levels of the checkpoint molecules Arf and p53 were measured by western blot in BCR-ABL1 ALL cells before (empty vector (EV)) and after (Cre) deletion of Ptpn6 and Inpp5d. Data are representative of three independent experiments (a–c). d, e, Functional readouts for inducible deletion of Ptpn6 and Inpp5d include measurement of proliferation (BrdU incorporation) (d) and colony formation capacity in methylcellulose (colony-forming unit (c.f.u.) assay) (e). BrdU assays (flow cytometry) and c.f.u. data (images from colonies on plates) are presented in Fig. 3d, e. Quantitative and statistical analysis for BrdU incorporation (d) and c.f.u. assays (e) are depicted here as bar charts. P values were calculated by unpaired, two-tailed Student’s t-test. Error bars (d, e) represent means ± s.d. from three independent experiments.
Extended Data Figure 8 Deletion of the ITIM-bearing receptors Pecam1, Cd300a or Lair1 has no significant effects on myeloid CML-like cells.
a, b, Myeloid progenitor cells from the bone marrow of Pecam1−/− and Cd300a−/− mice as well as age-matched wild-type controls were propagated in the presence of Il-3, Il-6 and Scf and transformed with retroviral BCR-ABL1. After 7 days, outgrowth of myeloid-lineage CML-like leukaemia was observed. One-hundred-thousand Pecam1−/− and Cd300a−/− CML-like cells as well as wild-type controls were plated in methylcellulose. Colonies were counted two weeks later (a). P values were calculated by unpaired, two-tailed Student’s t-test (b). c–e, Myeloid progenitor cells from the bone marrow of Lair1fl/fl mice were propagated in the presence of Il-3, Il-6 and Scf and transformed with retroviral BCR-ABL1. After 7 days, outgrowth of myeloid-lineage CML-like leukaemia was observed and CML-like phenotype was verified by flow cytometry using antibodies against B220/CD19 (negative), Sca-1/c-Kit and CD13 (c). CML-like cells were transduced with 4-OHT-inducible Cre (Cre-ERT2) and empty vector controls (ERT2) and deletion of Lair1 was verified by measurement of Lair1 surface expression (d). After adding 4-OHT, cell viability of Lair1fl/fl CML cells carrying ERT2 or Cre-ERT2 was monitored over 9 days by flow cytometry and is plotted in e. Data (a, b, e) represent means ± s.d. from three independent experiments.
Extended Data Figure 9 Deletion of Ptpn6 or Inpp5d specifically affects B-cell-lineage ALL cells but not normal pre-B cells, myeloid progenitors or myeloid leukaemia.
a, b, Bone marrow mononuclear cells were isolated from Ptpn6+/fl and Inpp5dfl/fl mice. Myeloid progenitor cells were propagated with Il-6 (25 ng ml−1), Il-3 (10 ng ml−1) and Scf (50 ng ml−1) and propagated as common myeloid progenitor cells (CMPs) or transformed with BCR-ABL1 to induce myeloid CML-like leukaemia. Pre-B cells were expanded in the presence of Il-7 (10 ng ml−1) and either propagated as pre-B-cell cultures or transformed by BCR-ABL1 to induce Ph+ ALL-like leukaemia. Lineage identity and >95% purity of cell populations was verified by flow cytometry. Ptpn6+/fl and Inpp5dfl/fl CMPs, pre-B cells, CML-like and Ph+ ALL-like leukaemia cells were then transduced with 4-OHT-inducible Cre (Cre) or an empty vector control (EV). Addition of 4-OHT induced nuclear translocation of Cre and Cre-mediated excision of Ptpn6+/fl (one allele) (a) or Inpp5dfl/fl alleles (b). Effects of inducible deletion on cell viability were measured by flow cytometry at the times indicated. Error bars (a, b) represent means ± s.d. from three independent experiments.
Extended Data Figure 10 The inhibitory receptor Lair1 and the phosphatases Ptpn6 and Inpp5d are specifically required by B-cell lineage leukaemia cells.
a, b, B-cell lineage BCR-ABL1 ALL cells were engineered with a doxycycline-inducible vector system for expression of Cebpa, which results in downregulation of B-cell antigens and myeloid-lineage differentiation as measured by flow cytometry (a) and western blot (b). Data (a, b) are representative of three independent experiments. c–e, BCR-ABL1-driven Lair1fl/fl, Ptpn6fl/fl and Inpp5dfl/fl B-cell lineage ALL cells (CD19+ Mac1−) were reprogrammed into myeloid-lineage (CD19− Mac1+) leukaemia cells by addition of doxycycline. Cell cultures were then transduced with 4-OHT-inducible GFP-tagged Cre and viability was measured in B-cell (gated on CD19+ Mac1−) and myeloid-lineage (gated on CD19− Mac1+) populations. f, Structure of the INPP5D small-molecule inhibitor 3AC. g, Patient-derived Ph+ ALL (n = 3) and chronic-phase CML cells (n = 3) were treated with 3AC (10 μmol l−1) for 15 min, and phosphorylation of SYK was measured by western blot, using β-actin as loading control. h, Dose–response curves are shown for five patient-derived cases of ALL (LAX2, LAX9, BLQ1, BLQ5 and PDX2, red curves) and five cases of B-cell leukaemia/lymphoma (lacking an oncogenic tyrosine kinase; KARPAS-422, MHH-PREB-1, JEKO-1, MN-60 and JJN-3, grey curves). i, Dose–response curves are shown for the treatment of six patient-derived cases of Ph+ ALL that have acquired global resistance to TKI treatment (LAX2, BLQ5, BLQ11) or partial resistance (ICN1, LAX9, PDX59). Dose–response curves for the TKI imatinib are shown in grey and for the INPP5D inhibitor 3AC in red (concentration plotted on same scale for both agents). Error bars (c–e, h–i) represent means ± s.d. from three independent experiments.
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Chen, Z., Shojaee, S., Buchner, M. et al. Signalling thresholds and negative B-cell selection in acute lymphoblastic leukaemia. Nature 521, 357–361 (2015). https://doi.org/10.1038/nature14231
Integrative multi-omics and drug response profiling of childhood acute lymphoblastic leukemia cell lines
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