LAUSR

Chronic myeloid leukemia (CML) results from hematopoietic stem cell (HSC) transformation by the BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKI), although effective in inducing remissions in CML, fail to eradicate leukemia stem cells (LSC) which persist as source of relapse. LSC resistance to TKI-treatment occurs through kinase-independent mechanisms, which include alterations in intrinsic cell-regulatory mechanisms as well as signals from the bone marrow (BM) microenvironment that support LSC persistence. HSC have been shown to be regulated by C-X-C motif chemokine ligand 12 (CXCL12)-expressing bone marrow niches, but the nature and regulatory role of BM niches for LSC remains poorly understood.Here, we used CXCL12-GFP mice and CXCL12f/fmice crossed with Cre lines targeting specific CXCL12-expressing cells to investigate the contribution of CXCL12-expressing populations to LSC regulation. We found that targeted deletion of CXCL12 from Prx1+ mesenchymal stromal cells (MSC) reduced normal HSC numbers. In contrast, deletion of CXCL12 from Prx1+ MSC in the setting of CML, resulted in increased leukocytosis, neutrophilia, BM cellularity and LSC numbers, and reduced survival, compared to control CML mice. CXCL12 deletion from Prx1+ MSC was found to enhance LSC cycling. Despite increased cycling, the expanded LSC from these mice maintained their in vivo repopulating capacity. To evaluate the effect of CXCL12 deletion on LSC and MSC distribution, we performed 3D imaging of BM volumes from Prx1-Cre mice crossed with tdTomato reporter mice. CML development resulted in formation of large pathological tissue niches harboring an abnormally high density of MSCs as well as c-Kit+ leukemia progenitor cells. However, these MSC and leukemic progenitor clusters were not observed in Prx1-Cre+CXCL12fl/fl mice, indicating that formation of tissue structures with colocalized leukemic progenitors is dependent on CXCL12 expression in MSC. We performed gene expression analysis of LSC from Prx1-Cre+CXCL12f/f and Cre-negative mice. Gene expression analysis revealed enrichment of cell cycling and MYC related genes in CML LSC from Prx1-Cre+CXCL12f/f mice, and downregulation of Polycomb Repressive Complex 2 (PRC2) target genes, indicating increased PRC2 activity. We confirmed that EZH2 expression and H3K27 trimethylation were increased in LSC from Prx1-Cre+CXCL12f/f mice. Treatment with the EZH2 inhibitor, GSK 343, resulted in significant reduction in WBC, neutrophils, BM cellularity and LSC in Prx1-Cre+CXCL12f/f mice, but not control CML mice. These results support a role for increased PRC2 activity in LSC expansion in mice with CXCL12 deletion from Prx1+ MSC. We evaluated the effect of CXCL12 deletion from Prx1+ MSC on LSC sensitivity to treatment with the TKI Nilotinib. Treatment of Cre-negative CML mice with Nilotinib reduced WBC counts, spleen cellularity, and splenic LSC numbers, but did not reduce BM cellularity or LSC numbers. On the other hand, Nilotinib treatment significantly reduced BM cellularity and LSC numbers, and enhanced survival, in Prx1-Cre+CXCL12f/f CMLmice. Transplantation of BM from TKI-treated Prx1-Cre+CXCL12f/f mice to irradiated normal recipients resulted in significantly reduced long-term donor engraftment and donor LSC numbers compared to vehicle-treated mice. These results indicate that CXCL12 deletion from Prx1+ MSC leads to enhanced sensitivity of CML LSC to elimination by TKI treatment. In conclusion, our studies show that deletion of CXCL12 from Prx1+ MSC niches leads to loss of MSC clustering and colocalization with leukemic progenitors, and to loss of quiescence and expansion of LSC, dependent on enhanced PRC2 activity. CXCL12 deletion from MSC also increases TKI-mediated targeting of resistant, quiescent, self-renewing CML LSC. Strategies to inhibit CXCL12-mediated niche interactions represent a promising approach for LSC depletion to enhance opportunities for cures in CML. No relevant conflicts of interest to declare.