Microenvironmental Protection of CML Stem and Progenitor Cells From Tyrosine Kinase Inhibitors Through N-Cadherin and Wnt Signaling
Authors: Zhang Bin , McDonald Tinisha , Holyoake Tessa L. , Moon Randall T , Campana Dario , Shultz Leonard , Bhatia Ravi 
Published: October 2019

Abstract
Abstract 912
BCR-ABL tyrosine kinase inhibitors (TKI) do not eliminate leukemia stem cells (LSC) in chronic myeloid leukemia (CML), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, indicating that additional kinase-independent mechanisms contribute to LSC preservation. We investigated the role of signals from the bone marrow (BM) microenvironment in protecting chronic phase (CP) CML stem/progenitor cells from TKI treatment. Culture with human BM mesenchymal stromal cells (MSC), immortalized by ectopic expression of telomerase, significantly inhibited apoptosis in CP CML CD34+CD38- primitive and CD34+CD38+ commited progenitors treated with imatinib (5μM, p<0.01), nilotinib (5μM, p<0.01), or dasatinib(150nM, p<0.05). MSC coculture reduced TKI-mediated apoptosis in both dividing and non-dividing CD34+CD38- cells, defined using CFSE labeling, and increased the percentage of undivided CD34+CD38- cells (p<0.05). MSC coculture also enhanced colony forming ability of CML CD34+CD38- (p<0.05) and CD34+CD38+ (p<0.05) cells treated with TKI. Importantly MSC coculture, with or without imatinib treatment, significantly enhanced engraftment of CML CD34+ cells in immunodeficient NSG mice, both at 4 weeks (without IM: 27.2±5% human CD45+ cells without stroma, 52.8±8% with stroma, p<0.001; with IM: 4.9±2% without stroma, 10.1±2% with stroma, p<0.05) and at 10 weeks post-transplant (without IM : 1.48±0.2% human CD45+ cells without stroma, 2.37±0.4% with stroma, p=0.07; with IM : 0.74±0.2% without stroma, 1.2±0.3% with stroma, p<0.05). CML progenitors cultured in a transwell insert over MSC showed increased apoptosis following TKI exposure compared to cells cultured in direct contact with MSC (p<0.05). An increased proportion of CML progenitors adhered to MSC after 4 days of TKI treatment (22±4% without IM, 42±5% with IM, p<0.05). Antibody or peptide (NCDH) mediated blockade of the N-Cadherin receptor reduced adhesion of CML progenitors to MSC (p<0.05), and increased apoptosis of TKI-treated CML progenitors cocultured with MSC (p<0.05), indicating an important role for N-Cadherin in MSC-mediated protection of CML progenitors from TKI. Although N-Cadherin expression measured by flow cytometry, Western blot and Q-RT-PCR was lower in CML compared to normal CD34+CD38- and CD34+CD38+ cells, we observed significantly increased N-Cadherin expression in CML cells remaining after 4 days culture with IM and MSC (p<0.05). We observed enhanced β-catenin activity in CML progenitors cocultured with MSC, with and without TKI treatment, as manifested by increased β-catenin protein levels and nuclear localization, enhanced expression of β-catenin target genes, and increased TCF/LEF reporter activity. β-catenin levels and target gene expression were increased primarily in MSC-adherent CML progenitors compared to non-adherent cells, and were significantly reduced by antibody or peptide-mediated inhibition of N-Cadherin-mediated adhesion. Using the Duolink labeling technology, we demonstrated increased protein-protein interactions between N-cadherin and β-catenin in CML progenitors cocultured with MSC, with or without TKI treatment. Finally, we showed that addition of exogenous Wnt1 protected CML progenitors from TKI treatment, whereas inhibition of Wnt receptors by DKK1, or inhibition of β-catenin signaling by the small molecule inhibitor ICG001, enhanced apoptosis of CML progenitors cocultured with MSC, suggesting an important role for exogenous Wnt signaling in MSC-mediated protection of CML progenitors from TKI-induced apoptosis. Microarray analysis of gene expression confirmed enrichment of Wnt/β-catenin and Cadherin related gene sets in CML CD34+ cells cultured on MSC with or without IM, as well as enrichment of genes related to hematopoietic stem cell (HSC) self-renewal, HSC quiescence, and cytokine signaling. In conclusion, our studies reveal an important role for exogenous Wnt-β-catenin signaling, and a close interplay between N-Cadherin and β-catenin, in microenvonment-mediated resistance of CML stem and progenitor cells to TKI treatment. These findings support further development of novel treatment strategies to eradicate residual LSC in TKI-treated CML patients through inhibition of Wnt secretion or blockade of Wnt or N-Cadherin receptors.

Disclosures:
No relevant conflicts of interest to declare.