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The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development

Nature Cell Biology volume 17, pages 665–677 (2015)Cite this article

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Abstract

Conventional strategies are not particularly successful in the treatment of leukaemia, and identification of signalling pathways crucial to the activity of leukaemia stem cells will provide targets for the development of new therapies. Here we report that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are crucial for the development of acute myeloid leukaemia (AML). Inhibition of expression of the ITIM-containing receptor LAIR1 does not affect normal haematopoiesis but abolishes leukaemia development. LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signalling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. The LAIR1–SHP-1–CAMK1–CREB pathway sustains the survival and self-renewal of AML stem cells. Intervention in the signalling initiated by ITIM-containing receptors such as LAIR1 may result in successful treatment of AML.

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Figure 1: Lair1, a representative ITIM receptor, is essential for the growth of human leukaemia cell lines.
Figure 2: Knockdown of lair1 blocks xenograft of human leukaemia cell lines.
Figure 3: LAIR1 enhances development of MLL-AF9 mouse AML during serial transplantation.
Figure 4: LAIR1 deficiency exhausts tumour-initiating cells by apoptosis.
Figure 5: SHP-1 rescues the lair1-null AML phenotype.
Figure 6: SHP-1 functions as an adaptor to recruit CAMK1 in the LAIR1 signalling.
Figure 7: CREB is a transcription factor for LAIR1 signalling in AML cells.
Figure 8: LAIR1 signalling is essential for human leukaemia development.

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Acknowledgements

We would like to thank H. Saya from Keio University School of Medicine for the pMX-IG N-Myc vector. We appreciate the support of staff of the tissue bank at the Department of Hematopathology, the University of Texas MD Anderson Cancer Center. Support to C.C.Z. was from NIH grant 1R01CA172268, Leukemia & Lymphoma Society Awards 1024-14 and TRP-6024-14, CPRIT RP140402, March of Dimes Foundation grant 1-FY14-201, Robert A. Welch Foundation grant I-1834, and When Everyone Survives Foundation. J.W.T. is supported by grants from the V Foundation for Cancer Research, the William Lawrence and Blanche Hughes Fund, and the National Cancer Institute (4 R00CA151457-03), and the Leukemia & Lymphoma Society. X.X. is supported by Susan G. Komen Foundation and RO1GM087305. J.E.C. is supported by the intramural program of the National Institute of Allergy and Infectious Diseases. M.J.Y. is supported in part by NIH/NCI R01 CA164346, Ladies Leukemia League, Developmental Research Awards in Leukemia SPORE CA100632, and Center for Inflammation and Cancer, IRG, Center for Genetics and Genomics, Sister Institution Network Fund and Physician Scientist Award of the University of Texas MD Anderson Cancer Center.

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Authors and Affiliations

  1. Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Xunlei Kang, Zhigang Lu, Changhao Cui, Mi Deng, Yuqi Fan & Cheng Cheng Zhang

  2. Department of Hematopathology, Division of Pathology and Laboratory Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas 77030-4009, USA

    Baijun Dong & M. James You

  3. Department of Laboratory Medicine, Division of Pathology and Laboratory Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas 77030-4009, USA

    Xin Han

  4. Department of Physiology and Pharmacology, Oregon Health and Science University Knight Cancer Institute, Portland, Oregon 97239, USA

    Fuchun Xie & Xiangshu Xiao

  5. Cell and Developmental Biology, Oregon Health and Science University Knight Cancer Institute, Portland, Oregon 97239, USA

    Jeffrey W. Tyner

  6. Receptor Cell Biology Section, National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland 20852, USA

    John E. Coligan

  7. Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Robert H. Collins

  8. The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, USA

    M. James You

  9. School of Life Science and Medicine, Dalian University of Technology, Panjin, Liaoning 124221, China

    Changhao Cui

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  1. Xunlei Kang
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Contributions

X.K. performed most of the experiments, analysed data and contributed to writing the paper; Z.L. performed the TCGA and clustering analyses; C.C. performed the CAMK experiments; M.D. performed the LILRB knockdown experiments. Y.F., F.X. and X.X. performed the CREB inhibitor experiments and provided advice; B.D., X.H., R.H.C. and M.J.Y. collected the primary AML samples and provided advice; J.W.T. helped with experiments using leukaemia cell lines and contributed to paper writing; J.E.C. provided LAIR1-deficient mice and contributed to paper writing; C.C.Z. conceived, coordinated and supervised the project, designed experiments, analysed data and wrote the paper.

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Correspondence to Cheng Cheng Zhang.

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Integrated supplementary information

Supplementary Figure 4 Lair1, a representative ITIM receptor, is essential for the growth of human leukemia cell lines.

(a) Expression of certain human ITIM receptor mRNAs negatively correlates with the overall survival of AML patients. A total of 58 ITIM receptors were selected based on the criteria that (1) they are plasma membrane receptors and (2) they use ITIM as the main signalling motifs. Data were obtained from the TCGA AML database, and analysed without normalization (condition 1) or normalized to GADPH expression (condition 2), Affymetrix housekeeping gene expression (condition 3), or total mRNA (condition 4). More information about data analysis can be found in Methods. (b) Effects of inhibition of expression of indicated ITIM receptors using shRNAs as determined by real-time RT-PCR. Data are from a single experiment, representative of 3 independent experiments. (c) In silico analysis of the correlation between human lair1 mRNA expression and the overall survival of AML patients younger than 65 years old. Data were obtained from the TCGA AML database (n = 52 patient samples for each groups, p = 0.0271, log-rank test). (d) An in silico analysis of human lair1 mRNA expression in 43 human AML samples as described previously1. (e) SP-D is not highly expressed in the bone marrow environment compared with mRNA expression in lung epithelial cells, as determined by real-time RT-PCR. Data are from a single experiment, representative of 3 independent experiments. (f) Schematic summary of the LAIR1 chimeric receptor signalling reporter cells system. (g) In the LAIR1 chimeric receptor signalling reporter cells system, flow cytometry analysis demonstrated that, while the immobilized collagen 1 (1 μg ml−1) or anti-LAIR1 antibody induced LAIR1 activation (as shown by increased GFP induction), the immobilized or soluble SP-D (1 μg ml−1) was unable to do so (the upper panels are for control cells, and the lower panels are for the hLAIR1 reporter cells).

Supplementary Figure 5 Depletion of lair1 suppresses growth of human leukemia cell lines in vitro and in vivo.

(a) Representative images (from at least 3 similar images) showing the marked reduction of MV4-11 cell growth on treatment with shRNAs targeting lair1 at 4 days after viral infection. Scale bar is 50 μM. (b) No effects on 562 cell growth were observed on treatment with shRNAs targeting lair1. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (c) No significant cell cycle change was detected between control and lair1-deficient MV4-11 cells at 3 and 6 days after infection with virus encoding a control shRNA or virus encoding shRNA 226, respectively. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (d) GFP+ LAIR1high cells in the LAIR1 shRNA 226 knockdown samples isolated from the transplanted NSG mice contained much less LAIR1 shRNA sequences than the original GFP+ LAIR1low counterparts before transplantation. Real-time PCR was performed to quantitate the shRNA sequences from these populations by using the shRNA 226 specific primer (GCTAGTCCATCTGAGTCAG-forward) together with the vector primer (AAGCGAGCTTATCGATACCG-reverse). Data are from a single experiment, representative of 3 independent experiments. (e) Flow cytometry analysis showing the decreased engraftment of lair1-deficient MV4-11 cells in BM of individual recipient mice (scrambled control versus lair1-shRNA treated samples).

Supplementary Figure 6 Flow cytometry analysis showing the decreased engraftment of lair1-deficient 697 cells in BM of individual recipient mice (scrambled control versus lair1-shRNA treated samples).

Supplementary Figure 7 LAIR1 deficient mice maintain normal hematopoiesis.

(a) WT and lair1-null neonatal liver Lin− cells (1 × 105 cells) were transplanted together with 1 × 105 CD45.1 competitor cells into lethally irradiated (10 Gy) CD45.1 mice (n = 5 mice). Peripheral blood engraftments are shown at 6 and 20 weeks after transplantation. (b) Comparison of mutilineage contribution between WT and lair1-null cells at 20 weeks after transplantation (n = 5 mice). (c) WT and lair1-null BM cells (1 × 106 cells) from primary engraftments were transplanted together with 2 × 105 CD45.1 competitor cells into lethally irradiated (10 Gy) CD45.1 mice (n = 5 mice). Peripheral blood engraftments are shown at 4 and 16 weeks after transplantation. (d) Comparison of mutilineage contributions of WT and lair1-null cells at 16 weeks after secondary transplantation (n = 5 mice). (e) WT and lair1-null HSCs home similarly to the recipient BM. BM cells from WT or lair1-null mice (n = 5 mice) were labelled with carboxyfluorescein succinimidyl ester (CFSE), and 1 × 107 cells were transplanted into lethally irradiated recipients. After 12 h, the total percentage of CFSE+ cells in the BM, spleen, and liver and LT-HSCs (CFSE+Lin−Sca-1+Kit+Flk2−CD34− cells) in BM were determined by flow cytometry.

Supplementary Figure 8 LAIR1 enhances leukemia development in several mouse leukemia models during serial transplantation.

(a) No significant difference in AML development were observed on primary transplantation with MLL-AF9-infected WT or lair1-null hematopoietic progenitors in primary transplantation; survival curves are shown (n = 10 mice; p = 0.1121, log-rank test). The experiment was repeated three times with similar results. (b) Summary of percentages of YFP+ AML cells, YFP+Mac-1+Gr-1+, and YFP+Mac-1+Kit+ cells in BM of primary recipient mice transplanted with the WT or lair1-null MLL-AF9 AML cells (n = 10 mice), the experiment was repeated three times with similar results. (c) The clonal relationship between the transplanted cells was studied by southern blotting on genomic DNA isolated from 4 pairs of WT and lair1 null MLL-AF9 induced leukemia samples. The result demonstrates that the leukemias were oligoclonal. The southern blotting was performed using a probe for YFP. (d) Southern blotting analysis of the oliogocolnal nature of MLL-AF9 integration in genomes of 3 primarily and 2 secondarily transplanted mice. The secondarily transplanted AML mice contained multiple MLL-AF9 clones from different primary samples. The bands indicated by the red number coordinates between primary and secondary samples. (e) Survival curves of mice receiving N-Myc infected WT or lair1-null hematopoietic progenitors in primary transplantation (n = 10 mice,p = 0.4204, log-rank test). (f) Survival curves of mice receiving 3,000 pooled GFP+ BM cells that were collected from primary recipients transplanted with WT or lair1-null N-Myc B-ALL cells (n = 5 mice, p = 0.0031, log-rank test). (g–i) Leukemia development vanishes on secondary transplantation of lair1-null MLL-AF9 cells. Summary of percentages of YFP+ AML cells and YFP+Mac-1+Kit+, YFP+Mac-1+Gr-1+, YFP+B220+, and YFP+CD3+ cells in (f) BM, (g) PB, and (h) spleen of secondary recipient mice transplanted with the WT or lair1-null MLL-AF9 AML cells at day 28 post-transplant (mean ± s.e.m., Student’s t-test; n = 5 mice; (g) Mac1+/Gr1+ **p = 0.0015; B220+ **p = 0.0019; CD3+ ***p < 0.0001; (h) Mac1+/Gr1+ **p = 0.0031; B220+ **p = 0.0022; CD3+ **p = 0.0017; (i) Mac1+/Gr1+ **p = 0.0002; B220+**p = 0.0035; CD3+ ***p < 0.0001).

Supplementary Figure 9 SHP-1-CAMK1 rescues the lair1-null AML phenotype.

(a) Retrovirally-expressed SHP-1 increased CFU numbers of lair1-null AML cells in secondary plating. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (b) The expression of endogenous shp-1 was inhibited by Cre virus infection, as determined by Q-PCR at 48 h after infection. Data are from a single experiment, representative of 3 independent experiments. (c,d) SHP-1 inhibitors have little effect on (c) CFU activity of MLL-AF9 AML cells or (d) cell growth capacity of human AML leukemia cell line (MV4-11). Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (e) Retrovirally-expressed SHP-1 WT, C453S, and 4YF(278, 303, 538, 566), but not SHP-1 PTPc, increased CFU numbers of lair1-null AML cells in secondary plating. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (f) Retrovirally-expressed CAMK1 increased CFU numbers of lair1-null AML cells in secondary plating. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (g) IP-western assay of MLL-AF9 BM cells of wide-type mouse by precipitating SHP-1, followed by detection of CAMK1 or LAIR1. (h) Additional 4 times of western-blot analyses as in Fig. 4a, showing SHP-1 protein levels in both primarily and secondarily transplanted WT and LAIR1 null leukemic mice.

Supplementary Figure 10 Transcription factor CREB is necessary for LAIR1-mediated signalling in AML cells.

(a) Retrovirally-expressed WT CREB, but not CREB S129A, S133A, or the double mutant S129/S133A, increased CFU numbers of lair1-null AML cells. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (b,c) Treatment of (b) 697 or (c) U937 cells with CREB inhibitor XX15 inhibited growth. YFP+ WT AML cells (20,000) were sorted by flow cytometry, plated in 1.55-cm wells, and treated with the indicated concentration of XX15. Cell numbers were determined on days 1, 2, and 3 from triplicate wells. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (d) Retrovirally-expressed CREB S219/133A decreased the total SHP-1 levels in the WT AML mouse bone marrow samples as shown by western blotting.

Supplementary Figure 11 LAIR1–SHP-1–CAMK1 axis supports human AML development.

(a) LAIR1high primary AML cells have greater colony-forming ability difference in both first and second plating (samples B1 and B2), whereas no significant difference in colony-forming ability was detected between LAIR1high and LAIR1low cord blood mononuclear cells (sample A). Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (b) Endogenous shp-1 expression was inhibited using shRNAs (201, 698, 786) in MV4-11 cells as determined by Q-PCR at 48 h after infection. Data are from a single experiment, representative of 3 independent experiments. (c) No clear association between shp-2 expression and AML patient survival was observed. Data were obtained from the TCGA AML database (n = 82 patient samples for high or n = 83 patient samples for low, p = 0.9451, log-rank test). (d) Endogenous camk1 expression was inhibited using an shRNA in MV4-11 cells as determined by Q-PCR at 48 h after infection. Data are from a single experiment, representative of 3 independent experiments. (e) LAIR1 expression is independent of the selected human AML stem cell phenotypic markers. The expression of LAIR1 and phenotypic markers (CD34/CD38/CD90) were analysed by flow cytometry in four AML clinical samples. (f) LAIR1 knockdown decreased colony-forming ability in all seven tested primary human AML cells as determined by CFU assays. Data from one experiment with n = 3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. (g) The survival curves of mice receiving control or LAIR1-knockdown primary human patient AML cells (sample# 6). n = 9 mice; p < 0.0001, log-rank test. (h) Schematic summary of the novel signalling pathway mediated by the ITIM receptor LAIR1 in leukemia cells.

Supplementary Figure 12 Original uncropped images of western blots.

Supplementary Table 1 In silico analysis of the correlation between human ITIM domain-containing receptor mRNA expression and the overall survival of AML patients.
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Supplementary Table 2 shRNA targeting sequences for the indicated ITIM receptors.
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Supplementary Table 3 Clinical sample information.
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Kang, X., Lu, Z., Cui, C. et al. The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development. Nat Cell Biol 17, 665–677 (2015). https://doi.org/10.1038/ncb3158

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