Abstract

Abstract 1212

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.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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