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The vascular niche in chronic myeloid leukemia
essential mediators of engraftment of chronic myeloid leukemia (CML)-initiating cells. However, the mechanism for overexpression of CD44 on leukemia-initiating cells (LIC) in CML mediating engraftment, as previously described by us,8 has not been established. CD44, known to mediate the transport of acute myeloid leukemia cells to stem cell-supportive niches,9 also acts as an E-selectin ligand on colon cancer10 and breast cancer cells.11
GMI-1271 is a specific small molecule antagonist of E- selectin with a dissociation constant of 0.54 mM. Co- administration of GMI-1271 was recently demonstrated to overcome resistance to bortezomib in E-selectin li- gand-enriched multiple myeloma cells,12 and GMI-1271 is currently being tested in clinical trials in combination with chemotherapy in patients with acute myeloid leukemia. It is surmised that - similar to mobilization by granulocyte colony-stimulating factor13,14 - GMI-1271- mediated mobilization of LSC may break LSC dormancy and, thereby, lead to improved eradication by tyrosine kinase inhibitors or chemotherapy. We had previously shown that targeting the osteolineage compartment of the BM microenvironment can lead to successful reduc- tion of LSC in CML.15 Imatinib, a tyrosine kinase inhibitor targeting BCR-ABL1, the oncoprotein causing CML, does not eradicate LSC.16,17 We hypothesized that treatment with GMI-1271 may lead to non-adhesion of CML-initi- ating cells to the BM endothelium and in combination with imatinib may be better at eliminating LSC in CML than imatinib alone.
Indeed, in this study we show that inhibition of E- selectin leads to a dissociation of BCR-ABL1+ cells from the endothelium. Concomitantly, this leads to increased leukemic cell proliferation and upregulation of the hematopoietic transcription factor and proto-oncogene Scl/Tal1, whose overexpression is frequently found in T- cell acute lymphoblastic leukemia.18 SCL/TAL1, itself phosphorylated by pAKT downstream of BCR-ABL1, is demonstrated to be a negative transcriptional regulator of CD44. Collectively, these data illustrate the reciprocal link between external cues from the BM microenvironment and LSC-intrinsic effects which, in turn, can influence LSC expression of adhesion molecules essential for interaction with the BM microenvironment and, thereby, modify therapy response. This is a concept which could influence future treatment strategies.
Methods
Additional methods are described in the Online Supplementary Material.
Mice
BALB/c or FVB mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Rag-2-/- CD47-/- IL-2 receptor γ-/- (C57/Bl6 background) and Tie2-GFP mice (FVB back- ground) were bred in our institute. All murine studies were approved by the local animal care committee (Regierungspräsidium Darmstadt).
Statistical analysis
Statistical significance between different treatment groups was assessed by the Student t-test or analysis of variance (ANOVA) test (with a Tukey test as a post-hoc test) using Prism Version 6 soft- ware (GraphPad, La Jolla, CA, USA). Differences in survival were
assessed by Kaplan-Meier nonparametric tests (log-rank or Wilcoxon tests). Data are presented as the mean ± standard devi- ation and differences are considered statistically significant when P≤0.05.
Results
GMI-1271 decreases the contact time of human chronic myeloid leukemia cells with bone marrow endothelium
In order to test whether inhibition of E-selectin by GMI- 1271 decreases adhesion of human CML cells to BM endothelium, we injected leukocytes from five different human CML patients into Rag-2-/-CD47-/-IL-2 receptor γ-/- mice19 treated with vehicle or GMI-1271 and imaged the calvarium of the injected mice by in vivo microscopy (Figure 1A and Online Supplementary Movie). This revealed a significantly reduced contact time of leukemia cells from four out of five patients with BM endothelium in mice treated with GMI-1271 (P<0.0001, Figure 1B and Online Supplementary Figure S1A-D). As anticipated, this result suggested that GMI-1271 leads to non-adhesion of human cells to the BM endothelium. Furthermore, in an in vitro adhesion assay of human CML cells plated on E-selectin, a smaller number of human CML cells adhered to E- selectin in the presence of GMI-1271 than in the presence of vehicle (P=0.0013) (Figure 1C).
Inhibition of E-selectin affects survival in mice with chronic myeloid leukemia
Next, we tested the effect of inhibition of E-selectin in combination with imatinib mesylate, considered the stan- dard of care for CML, on the survival of mice with CML. Treatment of murine recipients of BCR-ABL1-transduced BM in the retroviral transduction/ transplantation model of CML-like myeloproliferative neoplasia15,20 with vehicle, imatinib, GMI-1271 or a combination of imatinib and GMI-1271 (Online Supplementary Figure S2A) revealed a modest, but significant prolongation of survival in mice that received the combination therapy compared to mice that received imatinib alone (P=0.028) (Figure 1D) with all mice succumbing to BCR-ABL1+ leukemia and pulmonary infiltration by mature neutrophils, as described elsewhere.8,15 Leukocyte counts (P=0.0008) (Online Supplementary Figure S2B), BCR-ABL1+ (GFP+) CD11b+ myeloid cells (P=0.031) (Online Supplementary Figure S2C) and spleen weights (P=0.024) (Online Supplementary Figure S2D) were significantly reduced in mice with CML treated with imatinib plus GMI-1271, when compared to vehicle- treated mice. However, the direct comparison of treat- ment with imatinib plus GMI-1271 vs. imatinib alone did not reveal a statistically significant difference. A signifi- cant reduction of GFP (BCR-ABL1)+ Lin- c-Kit+ (LK) cells, which harbor the LSC fraction in this model,21 was observed in mice treated with imatinib and GMI-1271, when compared to vehicle-treated mice (P=0.001) (Online Supplementary Figure S2E). Lin- GFP+ (BCR-ABL1+) cells from CML mice (P=0.001) (Figure 1E) or BCR-ABL1+ BaF3 cells (P=0.002) (Online Supplementary Figure S2F) injected into Tie2-GFP mice, which express green fluorescent pro- tein (GFP) under the TEK receptor tyrosine kinase (Tie2)- promoter, were found significantly further away from the endothelium, but not the bone (Online Supplementary Figure S2G-H), when mice had been treated with GMI-
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