Specialized microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However the niche interactions of leukemia stem cells (LSC) are largely unknown. Targeted expression of the BCR-ABL gene in murine hematopoietic stem and progenitor cells (HSPC), via a Tet-regulated SCL promoter, results in development of a chronic phase CML-like disorder (Blood 105:.324, 2005). We have employed this SCL-tTA-BCR/ABL mouse model to investigate the characteristics of LSC in CML. BCR-ABL transgenic mice were crossed with GFP transgenic mice to facilitate tracking of transplanted cells. We have reported that LSC capacity is restricted to a population of cells with LT-HSC phenotype (LSK Flt3-CD150+CD48-). BCR-ABL expression is associated with reduced numbers of LT-HSC in the BM and greatly increased numbers of LT-HSC in the spleen compared with controls(Blood 2009, 114: 858). These observations suggest that CML LT-HSC demonstrate altered niche requirements compared to normal LT-HSC. We therefore conducted additional studies to investigate whether abnormal localization of CML LT-HSC was related to reduced homing and/or reduced retention in the BM microenvironment. To evaluate LT-HSC homing, BCR-ABL+ and control LT-HSC (10,000 cells/mouse) were labeled with CFSE and injected by tail vein injection into wild type mice irradiated at 900cGy and CFSE+ cells in the BM and spleen of recipient mice were evaluated 4h after injection. We observed 32% reduction in homing of BCR-ABL+ LT-HSC to the BM of recipient mice compared to control LT-HSC (p=0.04), with similar homing to the spleen. To study trafficking from BM to extramedullary sites, BCR-ABL+ and control LT-HSC were injected directly into the right femur of irradiated congenic mice (1000 cells/mouse). Recipient mice were euthanized 2 and 4 weeks after injection and localization of LT-HSC, progenitors and WBC in the right femur, the contralateral (left) femur, and spleen analyzed by flow cytometry. We observed 4.9-fold increased numbers of BCR-ABL+ LT-HSC compared with control LT-HSC in the spleen(p=0.008) and 60% decreased numbers of BCR-ABL+ LT-HSC in the marrow compared with control LT-HSC at 4 weeks post-injection(p=0.048). Increased egress from BM to spleen was not related to BM hypercellularity. These results are consistent with enhanced egress of BCR-ABL+ LT-HSC from the BM to the spleen and/or enhanced growth in the spleen. No significant differences in expression of α4, α5, and α6 integrin and CD44 expression were seen in LT-HSC from the spleen and BM of BCR-ABL+ and control mice. A small population of β7 integrin expressing LT-HSC was seen in BM from BCR-ABL+ mice. Adhesion of LT-HSC from the spleen and BM of BCR-ABL+ and control mice to fibronectin coated wells was evaluated. LT-HSC from BM of BCR-ABL+ mice showed reduced adhesion to fibronectin after 2 hours(43±3%) compared to LT-HSC from control mice (57±3%, p=0.004), indicating impaired α4β1 and α5β1 integrin receptor function despite normal levels of receptor expression. BCR/ABL+ LT-HSC cells demonstrated higher CXCR4 expression and enhanced migration to CXCL12 (SDF-1) in a 3-hour transwell migration assay(20±5%), compared to control LT-HSC (9±3%, p=0.04). CXCL12-induced migration of BCR/ABL+ LT-HSC was completely blocked by the CXCR4 antagonist AMD3100. ELISA analysis of CXCL12 levels revealed 2.5-fold reduction in BM supernatants and 1.4-fold increase in splenic supernatants from BCR/ABL+ mice compared to control mice. We conclude that BCR-ABL expression results in significant reduction in LT-HSC homing to BM niches and markedly increased egress of LT-HSC from BM to the spleen through a combination of both intrinsic defects in LT-HSC adhesion and migration and leukemia-associated alterations in the BM and splenic microenvironments. Our results indicate LT-HSC-niche interactions are markedly perturbed in CML, potentially contributing to deregulated stem cell growth.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.