TNF-α-Induced Bone Marrow Stromal Progenitor Alterations Enhance Leukemic Stem Cell Growth and Treatment Resistance Via Increased CXCL1-CXCR2 Signaling

Mesenchymal cells within the bone marrow (BM) microenvironment play an important role in regulation of hematopoietic stem cell (HSC) fate. However, the effects of leukemia development on distribution and function of mesenchymal cell subpopulations are not well understood. Here we used the SCL-tTA-BCR-ABL transgenic CML mouse model to examine how CML development affected murine BM mesenchymal subpopulations within a recently delineated skeletal stem cell (SSC) hierarchy, and evaluate how mesenchymal subpopulations affected HSC and leukemia stem cell (LSC) growth.

We observed a significant increase in bone-forming "Thy" and stroma-forming "6C3" progenitor subsets in CML compared to normal BM. CML Thy and 6C3 progenitors demonstrated increased proliferation and CFU-F potential. In addition, CML Thy cells exhibiting increased osteogenic potential whereas CML 6C3 cells showed increased adipogenic potential compared to their normal counterparts.

CML LSC and normal HSC were cocultured with Thy and 6C3 cells, purified from CML and normal mice for 3 days and transplanted into irradiated normal recipients to evaluate long-term engraftment. Normal HSC engraftment was enhanced by coculture with normal Thy and 6C3 cells but not by their CML counterparts. On the other hand, LSC engraftment was enhanced by CML 6C3 cells compared to normal 6C3 cells, but not by CML Thy cells compared to normal Thy cells. These results indicate that CML stromal progenitors demonstrate enhanced support of LSC and reduced support of normal HSC.

Q-PCR analysis showed that expression of major hematopoietic regulatory molecules, including CXCL12, G-CSF, SCF, IL-1, IL-6, and IGF-1, was significantly reduced in CML 6C3 and Thy progenitors.RNA-Seq analysis demonstrated that expression of TNFaand NF-kbrelated gene sets was significantly increased in CML compared with normal 6C3 cells. Ligand-receptor interactome analysis, based on differential gene expression in LSC and normal HSC, and CML and normal 6C3 cells, revealed upregulation of the chemokines CXCL1 and CXCL5 in CML 6C3 cells, and of their cognate receptor CXCR2 in LSC. We have previously shown that TNFalevels are increased in CML compared to normal BM (Zhang et al., 2012). Here we found that treatment of WT mice with TNFα led to expansion of 6C3 cells and increased CXCL1 expression on 6C3 cells. In contrast, treatment of CML mice with anti-TNFα antibodies led to reduction in 6C3 cell numbers and reduced CXCL1 expression in 6C3 cells. These results support a critical role for TNFα signaling in expansion and increased CXCL1 expression by stromal progenitors in CML BM.

We evaluated the role of paracrine CXCL1-CXCR2 signaling in growth and TKI resistance of LSC. Treatment with CXCL1 and CXCL5 resulted in expansion of LSC and leukemic progenitors respectively, and this effect was blocked by the CXCR2 inhibitor SB225002. Treatment with SB225002 significantly reduced proliferation of LSC cocultured with CML 6C3 cells. SB225002 administration to mice engrafted with CML cells resulted in significant reduction in peripheral blood WBC counts, neutrophil percentage, and leukemic short-term HSC (STHSC) and granulocyte-macrophage progenitors (GMP) in the BM. The combination of SB225002 and the TKI Nilotinib (50mg/kg) resulted in significantly greater reduction in peripheral blood WBC and neutrophils, and in BM LSC compared to Nilotinib or SB225002 alone.SB225002 treatment also significantly reduced proliferation and enhanced apoptosis of human CML CD34+CD38- cells cocultured with human CML BM mesenchymal stromal cells, and that the combination of SB225002 and Nilotinib significantly enhanced apoptosis and inhibited proliferation of CML CD34+CD38- cells compared to Nilotinib alone.

We conclude that increased TNFα signaling results in expansion of stromal progenitors in CML BM, which differentially support LSC compared to normal HSC. TNFα-signaling leads to overexpression of CXCL1 by stromal progenitors, which interacts with CXCR2 overexpressed on LSC to enhance their growth. Inhibition of CXCR2 signaling reduces LSC proliferation and survival, and enhances LSC elimination in combination with TKI. These observations support further exploration of targeting of CXCL1-CXCR2 interactions as a novel and effective strategy to target BM microenvironment-protected TKI-resistant LSC.