Killing of Quiescent and Cycling Chronic Myeloid Leukemia CD34+ Cells by Autologous Ex-Vivo Expanded Natural Killer Cells Is Enhanced by Bortezomib Treatment.

Abstract 3395

Although non-cycling quiescent CD34+ chronic myeloid leukemia (CML) cells are more resistant to tyrosine kinase inhibitors (TKI) and cell-mediated immunity than their cycling counterparts, bortezomib treatment of these cells enhances the cytotoxic effect of allogeneic natural killer (NK) cells from HLA-identical sibling donors against them (Yong, et al, Blood 2009;113:875-82). To extend these observations for clinical application in CML patients ineligible for allogeneic stem cell transplantation, we studied the effect of autologous NK cells from patients with established CML against cycling and quiescent CD34+ CML cells. Purified NK cells from CML patients were cultured over 11–18 days, according to the technique previously reported for NK cells from healthy individuals, using irradiated EBV-LCLs as feeder cells, and interleukin-2. Autologous NK cells were expanded in 12 (6 chronic phase, 6 accelerated phase [AP]) of 14 CML patients with overt disease, achieving greater than 10-fold NK expansion in over 75% of patients. Expanded autologous NK cells were BCR-ABL negative by fluorescence in situ hybridization. In two patients with advanced CML (one blast crisis and another AP), autologous NK cells failed to expand. Using fluorescence activated cell sorting, the progeny of CD34+ CML cells after 4 days culture in serum-free media supplemented with interleukin-3, interleukin-6, stem cell factor, granulocyte-colony stimulating factor and Flt-3 ligand were isolated into cycling CD34-negative and CD34+, and non-cycling quiescent CD34+ populations. Expanded autologous NK cells lysed quiescent CD34+ cells from CML patients but these non-cycling cells were less susceptible to lysis than their cycling CD34+ and CD34-negative counterparts. Addition of the clinically achievable dose of 10nM bortezomib to CD34+ cell cultures significantly enhanced the cytotoxic effects of expanded autologous NK cells on cycling and quiescent non-cycling CD34+ CML cells by 20–40% compared to without pre-treatment. The increased sensitivity to autologous NK-cytotoxicity correlated with increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 on the surface of CD34+ quiescent cells, and was reversed by blocking TRAIL. Conversely, enhanced autologous NK-cytotoxicity against cycling CD34+ cells occurred independent of TRAIL and was mediated through upregulation of NKG2D ligands MICA/B, and reversed by NKG2D blockade. The direct pharmacologic effect of bortezomib on primitive CML progenitors is complementary to its ability to sensitize quiescent and cycling CD34+ CML cells to autologous NK cell cytotoxicity, and these findings support its further development as an adjunct treatment with adoptive transfer of autologous expanded NK cells in CML patients who are resistant to TKI and are not eligible for allogeneic stem cell transplantation.