Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche

Abstract

We investigated adhesion pathways that contribute to engraftment of breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-induced chronic myelogenous leukemia (CML)-like myeloproliferative neoplasia in a mouse retroviral transduction/transplantation model. Compared with normal stem/progenitor cells, BCR-ABL1(+) progenitors had similar expression of very late antigen-4 (VLA4), VLA5, leukocyte functional antigen-1, and CXCR4 but lower expression of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. Whereas vascular cell adhesion molecule-1 and P-selectin were not required, deficiency of E-selectin in the recipient bone marrow endothelium significantly reduced engraftment by BCR-ABL1-expressing stem cells following intravenous injection, with leukemogenesis restored by direct intrafemoral injection. BCR-ABL1-expressing cells deficient for PSGL-1 or the selectin ligand-synthesizing enzymes core-2 β1,6-N-acetylglucosaminyltransferase or fucosyltransferases IV/VII were impaired for engraftment, and destruction of selectin ligands on leukemic progenitors by neuraminidase reduced engraftment. BCR-ABL1-expressing L-selectin-deficient progenitors were also defective in homing and engraftment, with leukemogenesis rescued by coexpression of chimeric E/L-selectin. Antibody to L-selectin decreased the engraftment of BCR-ABL1-transduced stem cells. These results establish that BCR-ABL1(+) leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than do normal stem cells. Selectin blockade is a novel strategy to exploit differences between normal and leukemic stem cells that may be beneficial in autologous transplantation for CML and perhaps other leukemias.

Figures

Figure 1

The expression ofL-selectin and PSGL-1 are decreased on c-Kit+Lin–BCR-ABL1+stem/progenitor cells. Flow cytometric analysis of the normalized expression level of adhesion molecules (x-axis) on (left) BM, (center) PB, and (right) spleen cells from a mouse with BCR-ABL1–induced CML-like disease (black lines), gated on c-Kit+Lin–GFP+ cells, vs normal c-Kit+Lin– BM cells (gray): (A) α4-integrin, (B) α5-integrin, (C) α4β7-integrin, (D) LFA-1 (αLβ2), (E) CXCR4, (F) PSGL-1, and (G) L-selectin. Staining with PE-conjugated isotype antibody is indicated by the dotted histograms in the BM samples. Data are representative of 3 independent experiments on mice euthanized 3 weeks after transplantation.

Figure 2

Recipient P-selectin and VCAM-1 are not required for engraftment of BCR-ABL1–expressing leukemic stem cells. (A) Kaplan-Meier survival curve for WT (B6129 F2, solid line, n = 33), VCAM-1 KO (Vcam1flox/flox/TIE2Cre) mutant (dotted line, n = 5), or P-selectin KO (Selp−/−) mutant (dashed line, n = 5) recipients of BCR-ABL1–transduced B6129 F2 BM. Here and in subsequent figures, the curve for the WT donor/recipient combination represents cumulative results from all transplants in this study. All mice that died prior to day 56 developed CML-like MPN. The difference in overall survival between the 3 cohorts was not significant (P = .81 for WT vs VCAM-1 KO and P = .96 for WT vs P-selectin KO; Mantel-Cox tests). (B) Increased clonality of CML-like disease in Vcam1 and Selp mutant recipients. Genomic DNA from spleen (spl) or PB of leukemic mice was analyzed by (upper) Southern blot with a GFP probe to detect distinct proviral integration events and (lower) subsequently with an ABL1 probe to allow determination of the total proviral content of each sample, shown at the bottom. Con 1 and Con 2 are control DNAs from cell lines that each contain a single BCR-ABL1 provirus, whereas WT 1 and WT 2 are spleen DNA samples of WT recipients with BCR-ABL1–induced CML-like disease. Lanes 12 to 14 show the clonality of disease in P-selectin mutant recipients, whereas lanes 20 to 22 are representative leukemia samples from VCAM-1 mutant recipients.

Figure 3

Recipient E-selectin is required for efficient engraftment of BCR-ABL1+stem cells. (A-B) Kaplan-Meier curves for (A) mortality due to CML-like leukemia and (B) overall survival of B6129 F2 control (solid line, n = 33) or E-selectin KO (dotted line, n = 15) recipients of BCR-ABL1–transduced BM from WT B6129F2 donors, administered via an i.v. route. The difference in overall survival between the 2 cohorts was significant (P = .021, Mantel-Cox test). (C) Engraftment of E-selectin mutant recipients, assessed by Southern blotting of leukemic cell DNA as in Figure 2B. Note that E-selectin KO i.v. leukemia samples in lanes 7, 10, and 13 had proviral copy number ≥2, indicating engraftment by leukemia-initiating cells with multiple proviral integrations per cell. (D) Intrafemoral transplantation increases engraftment of E-selectin mutant recipients. Kaplan-Meier survival curve for B6129 F2 control (solid line, n = 8) or E-selectin KO (dotted line, n = 13) recipients of BCR-ABL1–transduced BM from WT B6129 F2 donors, administered by direct i.f. injection. All mice that succumbed within 2 months of transplantation developed CML-like leukemia. There was no significant difference in overall survival between the 2 groups (P = .62, Mantel-Cox test).

Figure 4

Selectin ligands on BCR-ABL1–expressing leukemic stem cells contribute to engraftment. (A-B) Kaplan-Meier curves for (A) mortality due to CML-like leukemia and (B) overall survival of B6129 F2 WT recipients of BCR-ABL1–transduced BM from B6129 F2 WT control (solid line, n = 33), PSGL-1 KO (Selplg−/−; dotted line, n = 8), and PSGL-1/CD44 double KO (Selplg−/−Cd44−/−; dashed line, n = 14) donors. Only 5 of 8 recipients of PSGL-1–deficient BM developed CML-like leukemia. Two mice succumbed at 127 days after transplant to BCR-ABL1–induced T-cell acute lymphoblastic leukemia, whereas another died of delayed graft failure (see text). The overall survival of PSGL-1 KO recipients was not significantly different from recipients of BCR-ABL1–transduced WT BM. (C) Deficiency of PSGL-1 and CD44 in donor BM leads to decreased engraftment of BCR-ABL1–induced CML-like leukemia. Southern blot of genomic DNA from BM of recipients of BCR-ABL1-transduced WT (lanes 2-11) and PSGL-1/CD44 double KO (lanes 12-22) donor BM, hybridized with a GFP probe. Recipients in lanes 6 and 15 to 21 did not develop clinical disease, whereas the recipient in lane 22 succumbed to histiocytic sarcoma. *Background bands. (D-E) Kaplan-Meier curves for (D) mortality due to CML-like leukemia and (E) overall survival for WT recipients of WT (solid line, n = 33), Core2GlcNAcT-I KO (Gcnt1−/−; dashed line, n = 6), and FucT-IV/FucT-VII double KO (Fut4−/−/Fut7−/−; dotted line, n = 6) BCR-ABL1–transduced BM. The difference in survival between recipients of WT and FucT-IV/FucT-VII double KO BM was of borderline significance (P = .13, Mantel-Cox test), whereas that of Core2GlcNAcT-I KO recipients was not. One recipient in the FucT-IV/FucT-VII double KO cohort had circulating BCR-ABL1–expressing myeloid cells early after transplant, but these leukocytes disappeared, and the animal ultimately engrafted with BCR-ABL1– cells (data not shown). (F) Donor Core-2 and Fuc-T IV/VII deficiency leads to a reduction of clonality of CML-like leukemia. Genomic DNA from the indicated tissues was analyzed by Southern blot as in Figure 2B. Lanes 5 to 8 and 9 to 12, respectively, contain the samples of WT recipients of BCR-ABL1–transduced Core-2 KO or Fuc-T IV/VII double KO BM. Note the reduction of clonality in lanes 5 to 12 to 1 or 2 clones. Proviral copy number is indicated below.

Figure 5

Neuraminidase treatment of donor BM leads to impaired engraftment of BCR-ABL1+leukemia-initiating cells. (A) Kaplan-Meier curve for survival of Balb/c recipients of BCR-ABL1–transduced BM incubated without NA (0 NA, solid line, n = 5), treated with NA at 6.25 (dotted line, n = 6) or 12.5 (dashed line, n = 6) mU/mL, or with heat-inactivated (HI) NA (dotted-dashed line, n = 5) prior to transplantation into WT recipients. All recipients died of CML-like leukemia. (B) NA treatment leads to decreased engraftment of BCR-ABL1–induced CML-like leukemia. Southern blot of genomic DNA from BM of recipients of BCR-ABL1–transduced BM incubated without NA (lanes 2-6), with 6.25 mU/mL NA (lanes 7-10), with 12.5 mU/mL NA (lanes 11-16), or with heat-inactivated NA (lanes 16-19). *Background band. (C) NA treatment selectively reduces leukemic stem cell engraftment. (Left) Ratio of mRFP+ to GFP+ BM myeloid (Mac-1+) cells from recipients of NA-treated BM transduced with BCR-ABL1 retrovirus, assessed at the time of morbidity or death. (Right) mRFP+ to GFP+ ratio of BM myeloid cells from recipients of BM transduced with parental retrovirus, assessed at 65 days after transplantation. The reduction in the mRFP+:GFP+ ratio at 12.5 mU/mL NA was significant for recipients of BCR-ABL1–expressing cells (P = .0063, Student t test) but not for normal HSCs.

Figure 6

L-selectin contributes to engraftment of BCR-ABL1+leukemic stem cells. (A) Kaplan-Meier–style curves for (A) mortality due to CML-like leukemia and (B) overall survival of WT B6129 F2 recipients of BCR-ABL1–transduced BM from WT (solid line, n = 33) and L-selectin KO (Sele−/−; dotted line, n = 16) donors transduced with BCR-ABL1 retrovirus and of L-selectin KO BM transduced with retrovirus coexpressing BCR-ABL1 and chimeric E/L-selectin (dashed line, n = 7). The difference in survival between recipients of WT and L-selectin KO BM was significant (P = .05, Mantel-Cox test), whereas there was no significant difference between WT and L-selectin KO + E/L-selectin (P = .45). Interestingly, the majority of mice in the E/L-selectin rescue cohort had evidence of malignant ascites and/or pleural effusions in addition to organomegaly, which might reflect an increased tendency of leukocytes expressing the stabilized selectin to traffic to serosal surfaces. (C) Serial flow cytometric analysis of a representative B6129 F2 WT recipient of BCR-ABL1–transduced L-selectin KO BM on days 14, 19, 27, 34, 41, and 54 after transplantation, with the PB leukocyte count indicated. (D) Flow cytometric analysis of a representative B6129 F2 WT recipient of L-selectin KO BM transduced with retrovirus coexpressing BCR-ABL1 and human E/L-selectin chimera, where human E-selectin expression is detected with monoclonal antibody CL-37 (y-axis). (Upper) Serial analysis of PB, showing rapid accumulation of circulating myeloid cells expressing human E-selectin. PB from an untransplanted WT mouse is shown in the last panel. (Lower) Analysis of leukocytes from spleen, pleural effusion, and ascites at necropsy. (E) Restoration of polyclonal engraftment by coexpression of E/L-selectin. Genomic DNA from the indicated tissues (asc, ascites; PE, pleural effusion) was analyzed by Southern blot using a probe from the human E-selectin gene. Brackets indicate different tissues from the same mouse. The BM donor was WT in lanes 4 to 8 and L-selectin KO in lanes 9 to 18. The BCR-ABL1 retrovirus used for transduction coexpressed GFP in lanes 3 to 5 and human E/L-selectin in lanes 6 to 18. Note the increased clonality in most recipients in lanes 9 to 18. Proviral copy number is indicated below. *Bands from the endogenous murine E-selectin gene.

Figure 7

L-selectin blockade decreases engraftment of BCR-ABL1+leukemia-initiating cells. (A) L-selectin–deficient leukemic progenitors have a defect in BM homing. Irradiated recipient mice (n = 3) were injected with a mixture of BM cells from mice with BCR-ABL1–induced CML-like disease induced from WT donor cells (expressing GFP) and Sell−/− donor cells (expressing NGFR) and euthanized 2 hours later. (Upper left) Flow cytometric analysis of c-Kit+Lin– normal BM stained with isotype-PE antibody. (Upper right) Input mixture of leukemic WT and Sell−/− progenitors. (Lower) Corresponding c-Kit+Lin– populations isolated from BM of the 3 recipients, stained with PE-conjugated antibody against human NGFR. The percentage of GFP+ and NGFR+ cells is shown adjacent to the respective regions. (B) No defect in engraftment of Sell−/− HSC transduced with empty GFP virus. BM from WT and Sell−/− donors was transduced with empty retrovirus expressing GFP alone and equivalent numbers of cells transplanted into lethally irradiated WT recipients. Ten weeks after transplant, recipient BM was harvested, and the number of engrafting proviral clones determined by Southern blotting of BglII-digested DNA with a GFP probe. Lanes 2 to 4 are recipients of WT BM, whereas lanes 5 to 7 are recipients of Sell−/− BM. The probe also faintly detects a common sequence (*) in mouse genomic DNA. (C) Anti–L-selectin antibody treatment impairs engraftment of BCR-ABL1–transduced progenitors. Southern blot analysis with a GFP probe of genomic DNA of leukemic BM from recipients of BCR-ABL1–transduced WT BM that was untreated (lanes 2-4), isotype control antibody–treated (lanes 5-7), and anti–L-selectin antibody–treated (lanes 8-12). Con1 and Con2 are control DNAs containing 1 and 2 proviral clones, respectively.