NKG2A Down-Regulation by Dasatinib Enhances Natural Killer Cytotoxicity and Accelerates Effective Treatment Responses in Patients With Chronic Myeloid Leukemia

Abstract Primitive quiescent CD34+ cells in chronic myeloid leukemia (CML) are relatively resistant to the tyrosine kinase inhibitors imatinib and dasatinib, which may explain the persistence of detectable BCR-ABL transcripts following treatment with these agents. Conversely, allogeneic stem cell transplantation (SCT) can eradicate residual CML, suggesting that quiescent stem cells are eliminated by graft-versus-leukemia (GVL) effects. We studied the progeny of CD34+ 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 in 14 CML patients (8 chronic phase, 6 advanced phase) who subsequently received T cell depleted SCT from their HLA-identical sibling donors. Cycling CD34-negative and CD34+, and non-cycling quiescent CD34+ CML cells were isolated by fluorescence activated cell sorting. Fluorescence in situ hybridization in 4 representative CML patients revealed over 80% BCR-ABL positivity in both quiescent and cycling CD34+ and CD34-negative populations. Using real-time quantitative polymerase chain reaction, we found the expression of BCR-ABL, and leukemia-associated antigens (LAA), WT1, PR3 and ELA2, were the same in both cycling and quiescent CD34+ cell populations in CML. LAA expression was not significantly different when compared with similarly cultured CD34+ cells from healthy donors. Pre-SCT quiescent CD34+ cells from CML patients were lysed by natural killer (NK) cells from their donors but were less susceptible than their cycling CD34+ and CD34-negative counterparts. Purified donor NK cells (n=7) expanded after 11–13 days culture with interleukin-2 and irradiated EBV-LCL lysed quiescent CD34+ CML cells as well as their cycling CD34+ and CD34-negative progeny. Previous studies have demonstrated that bortezomib can sensitize malignant cells to NK-cell tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Lundqvist et al Cancer Res. 2006 Jul 15;66(14):7317–25). Addition of bortezomib 10nM to CD34+ cell cultures enhanced cytotoxic effects of expanded donor NK cells on quiescent CD34+ CML cells. As observed with other malignancies, this enhanced sensitivity to NK-cytotoxicity correlated with increased expression of TRAIL receptors DR4 and DR5 on the surface of CD34+ quiescent cells, compared with cycling CD34+ or CD34-negative cells. Bortezomib treatment did not significantly affect the expression of MHC Class I, MIC A/B or Fas (CD95) on CD34+ quiescent or cycling cells. These results suggest that adoptive transfer of in vitro expanded donor NK cells with concomitant administration of bortezomib to the recipient may enhance cytotoxicity to quiescent CD34+ cells and may improve NK-mediated GVL effects. This may be particularly applicable to CML patients who are increasingly transplanted in more advanced stage disease, and so are at a greater risk of relapse post-SCT.

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

Blood ◽

10.1182/blood.v116.21.3395.3395 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3395-3395

Author(s):

Agnes S.M. Yong ◽

Nicole Stephens ◽

Keyvan Keyvanfar ◽

Bipin N. Savani ◽

Rhoda Eniafe ◽

...

Keyword(s):

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Natural Killer ◽

Allogeneic Stem Cell Transplantation ◽

Myeloid Leukemia ◽

Bortezomib Treatment ◽

Cd34 Cells ◽

Nk Cytotoxicity

Abstract 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. Disclosures: No relevant conflicts of interest to declare.

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