N-Cadherin-Mediated Microenvironmental Interactions Protect CML Stem Cells from Imatinib Mediated Apoptosis.

The therapeutic success of Imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by incomplete elimination of malignant hematopoietic stem cells (HSC). The bone marrow microenvironment supports the self-renewal, proliferation and survival of HSC. We investigated whether microenvironmental interactions protected CML HSPC from elimination by IM. CML CD34+CD38− primitive and CD34+CD38+ committed progenitors were cultured for 96 hours with IM (5μM) and with or without irradiated hTERT-immortalized primary human marrow stromal cells. Stroma coculture effectively protected CML progenitors from IM-induced apoptosis (20±6% apoptosis of CD38− cells without stroma and 4±1% apoptosis with stroma, p=0.04, n=4; 13±4% apoptosis of CD38+ cells without stroma and 7±1% apoptosis with stroma, p=0.04, n=4). Stroma coculture did not affect IM-mediated inhibition of CML progenitor proliferation. CML CD34+ cells cultured with and without stroma were injected into irradiated NOD-SCID-IL2Rγ- chain deleted (NSG) mice. Prior coculture with stroma prior to injection resulted in significantly increased engraftment of CML cells in the BM and spleen of NSG mice both at 4 weeks (short-term repopulation) and 10 weeks (long-term repopulation) after transplantation, both with and without IM treatment (without IM, 53±9% human cells in BM after 4 weeks with stroma and 27±5% without stroma, p=0.02, n=4; with IM, 13±2% with stroma and 5±2% without stroma, p=0.01, n=4). Q-PCR analysis showed that stroma coculture was associated with increased engraftment of BCR-ABL+ cells both with and without IM treatment. Culture of CML CD34+ cells in Transwell inserts (0.45μm pores) over stromal layers resulted in partial reversal of protective effect of stroma, suggesting that direct stromal contact contributed to protection of CML progenitors from IM-induced apoptosis (13±2% apoptosis with stroma, 20±5% with Transwell, and 30±3% without stroma). N-cadherin, a cell-cell adhesion protein expressed on HSPC and stromal cells, may contribute to drug resistance in Ph+ lymphoblastic leukemia. Blocking antibodies (Ab) to N-cadherin partially reversed the protective effect of stroma on CML cells treated with IM (33±2% apoptosis with anti-N-cadherin Ab, 26±4% without Ab, p=0.01, n=3). Similarly an N-cadherin-blocking cyclical peptide enhanced apoptosis of IM-treated CML progenitors cultured on stroma (31±4% apoptosis with N-cadherin peptide, 26±3% with control peptide, p=0.03, n=3). CML progenitors cultured on N-cadherin coated plates showed modestly reduced apoptosis compared with controls indicating that N-Cadherin may also directly transduce anti-apoptotic signals (24±4% apoptosis with N-cadherin, 28±3% with BSA, p=0.001, n=4). A minority of CML progenitors expressed N-cadherin on the cell surface. Expression was higher in CD38− cells (17%) compared with CD38+ cells (6%). The percentage of cell surface N-cadherin +ve cells increased after IM treatment in the presence of stroma but not after culture with stroma or IM alone (no stroma no IM 6.5%, stroma alone 6.8%, IM alone 9.9%, stroma and IM both17.8%). We show that purified surface N-cadherin +ve CML CD34+ cells are capable of engrafting NSG mice (0.9±0.4% human cells for N-cadherin +ve and 0.6±0.2% for N-cadherin -ve cells, p=0.12, n=4). Interestingly, surface N-cadherin -ve cells also expressed N-cadherin RNA on Q-PCR, and antibody labeling after fixation and permeablization indicated that a large proportion of these cells expressed intracellular N-cadherin protein, suggesting that they could potentially express surface N-cadherin. In conclusion, our studies indicate that the marrow microenvironment enhances CML HSPC preservation and protects CML HSC from pro-apoptotic effects of IM treatment through direct cell-cell contact-mediated interactions mediated at least in part through N-cadherin. Measures aimed at interfering with N-cadherin mediated microenvironmental interactions could enhance eradication of residual malignant HSC in CML patients receiving IM treatment.