Sensitizing primary acute lymphoblastic leukemia to natural killer cell recognition by induction of NKG2D ligands

Abstract

Natural killer (NK) cell immunosurveillance may be impaired by malignant disease, resulting in tumor escape and disease progression. Therapies that enhance NK cytotoxicity may therefore prove valuable in remission-induction and maintenance treatment regimens. Acute lymphoblastic leukemia (ALL) has previously been considered resistant to NK cell lysis and not tractable to this approach. Our study demonstrates that bortezomib, valproate and troglitazone can up-regulate NK activating ligands on a B-ALL cell line and on a proportion but not all adult primary B-ALL samples. Drug-treated ALL cells trigger higher levels of NK degranulation, as measured by CD107a expression, and this effect is dependent on signaling through the NK activating receptor NKG2D. These results suggest that bortezomib, valproate and troglitazone may have clinical utility in sensitizing ALL to NK mediated lysis in vivo.

Figures

Figure 1. Induction of NK Ligands on NALM-6

(A) MICA, MICB, ULBP1, ULBP2, DR4, DR5 and HLA expression was assessed on NALM-6 treated with vehicle control (upper) and with Bortezomib (lower) by flow cytometry. Live cells were gated for analysis based on forward and side scatter parameters. Histograms show fluorescence of unstained cells (grey line) compared with fluorescence of cells stained with specific antibody (black line). Representative data from one of 3 experiments is shown. (B) ΔMFI (mean fluorescence intensity in stained minus mean fluorescence intensity in unstained) showing mean +/- SD from 3 independent experiments with i) Bortezomib ii) Valporate and iii) Troglitazone treated NALM-6. (*p

Figure 2. Enhancement and Blockade of NK Degranulation

(A) Representative gating strategy used to identify the percentage of CD107a expressing NK cells in a degranulation assay where healthy donor PBMC are combined with a target cell. (B) CD107a expression by the NK cells where PBMC are combined with: i) medium alone (negative control), ii) MHC class I negative cell line K562 (positive control), iii) vehicle-treated NALM-6, iv) Bortezomib-treated NALM-6. NK degranulation against vehicle, drug, and drug in the presence of NKG2D blocking antibody is compared for (C) Bortezomib, (D) Valproate and (E) Troglitazone (*p

Figure 3. Induction of NK Ligand Expression on Primary ALL Blasts

(A-C) Scatter plots showing effect of drug treatment on NKG2D ligand expression (ΔMFI above isotype control) for each of 3 primary ALL samples treated in duplicate with: A) 50nM Bortezomib, B) 1mM Valproate and C) 5μM Troglitazone compared with vehicle controls. Ligands MICA, MICB, ULBP1 and ULBP2 as indicated (*p

Figure 4. Induction of NK Degranulation with Primary ALL Blasts

A) Induction of NK degranulation by 10 primary ALL samples (1-10) treated with 50nM Bortezomib (B), 1mM Valproate (V), 5μM Troglitazone (T) or vehicle control for 16 hours. Fold change in % specific degranulation = the ratio of % specific degranulation with and without drug. B-D) Cumulative analysis of data in A) shown for each drug treatment (shaded bars) compared with untreated controls (open bars). E-G) NK degranulation against 3 primary ALL samples treated as above, with 20µM src kinase inhibitor PP2 (shaded bars) vs no kinase inhibitor (open bars): D) 50nM Bortezomib, E) 1mM Valproate, F) 5μM Troglitazone (*p