ACUTE LYMPHOBLASTIC LEUKEMIA - THERAPY, EXCLUDING TRANSPLANTATION POSTER I
Drug-Induced, NKG2D-Dependent Sensitization of ALL Cell Lines to NK Recognition.
Laura Spence, MD*,1,
Sophie Hambleton, MD, PhD*,1,
Venetia Bigley, MD*,1,
Sarah Pagan*,1 and
Matthew Collin, MD, PhD2
1 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom
Poster Board II-1
The combination of tumor-sensitizing drugs with NK cell infusionis beginning to emerge as a novel anti-tumor therapy. A growingbody of in vitro studies show that drugs such as proteosomeinhibitors, histone deacetylase inhibitors and thiazolidinedionesare able to sensitize tumor cells but not their healthy counterpartsto NK-mediated lysis. Drug induced NK-sensitization has shownpromise in acute myeloid leukemias but no studies have yet proventhis principle in acute lymphoblastic leukemia (ALL); a tumorphenotype reported to be relatively NK-resistant. The mechanismsunderlying sensitization have not been fully identified butup regulation of ligands for TRAIL and the NK activating receptorNKG2D: MICA MICB and the UL16-binding proteins, may have a role.We set out to explore ALL susceptibility to NK cytotoxicityand whether this could be modulated by drug treatment. In contrastto published data, untreated ALL cell lines were positive forsurface expression of MICB and ULBP2. Median fluorescence intensityratios (mean ± SD; n = 6) for MICB detection on the celllines 697, NALM-6, BV173 and SEM were: 3.2 ± 0.9; 3.8± 1.3; 4.0 ± 0.5; 2.5 ± 0.9, respectivelyand for ULBP-2: 2.3 ± 0.4; 55 ± 4.9; 2.9 ±0.2; 1.8 ± 0.4, respectively. NALM-6 was also positivefor ULBP1 (3.3 ± 0.6) while all were negative for MICAand ULBP3. Susceptibility of untreated ALL lines to NK mediatedkilling was assessed by chromium release assay using an IL-2stimulated primary NK cell line. At effector to target ratio40:1, specific release was 2.3% with cell line 697, 12% withNALM-6, 36% with BV173 and 63% with SEM. These results correlatedwith CD107a exposure in a degranulation assay using IL-2 stimulatedperipheral blood lymphocytes: specific degranulation (% CD107a+target with effector minus %CD107a+ effector alone) was 0.68%(697), 7.1% (NALM-6), 10% (BV173) and 17% (SEM). There was nocorrelation between baseline expression of NKG2DL and susceptibilityto NK killing. Bortezomib, sodium valproate and troglitazonewere added to cell cultures at sub-IC50 doses for 48 hours andcompared with equimolar vehicle controls. Surface NKG2DL expressionwas measured by flow cytometry. On NALM-6 troglitazone treatmentincreased ULBP1 MFI by 2.0 ± 0.33 fold compared withvehicle control and increased percentage of ULBP1 positive cellsby 39.6% (paired t-test: p=0.063). Sodium valproate increasedMICA expression by 2.91 ± 1.18 fold and percentage ofMICA positive cells by 12.3% (p=0.0382). On BV173, sodium valproatetreatment increased ULBP2 MFI by 1.55 ± 0.07 fold andpercentage of ULBP2 positive cells by 8.6% (p=0.04). There wereno significant ligand changes after drug treatment on cell line697. No NKG2DL changes were seen after Bortezomib treatmenton any cell line. The functional significance of NKG2DL changeswas assessed by CD107a degranulation assay. NALM-6 treated for48 hours with drugs yielded the following fold increases inspecific degranulation of NK cells compared to NALM-6 vehiclecontrols: 5.02 ± 5.98 for Bortezomib (mean ± SD),2.4 ± 0.67 for Troglitazone and 1.44 ± 0.13 forValproate. Levels of NK degranulation with 697 were very low(<5%) and drug treatment had no effect. Finally, we demonstratedthat sensitization of NALM-6 was at least partly dependent onNKG2DL recognition, since blocking antibody to NKG2D reducedCD107a exposure by all three drugs. Compared with controls,blocking reduced CD107a expression by 59 ± 12% for Bortezomib,47 ± 1.1% for Troglitazone and 48 ±11% for valproate-treatedcells. This result was unexpected for Bortezomib as no changesin surface NKG2DL expression were detected after drug treatment.However, we are investigating the possibility that Bortezomibmay down-regulate HLA class I expression, thus reducing inhibitorysignaling upon NALM-6/NK interaction and unmasking an activationpathway that signals through NKG2D. In conclusion, we foundbasal levels of expression of NKG2DL on ALL cell lines. Therewas no correlation between NKG2DL expression and susceptibilityto NK lysis, although this was to be expected given that a wealthof other activating and inhibitory receptors that contributeto NK activation. Bortezimib, valproate and troglitazone inducedNKG2DL expression and sensitization to NK recognition in a cellline-specific manner. These drugs may therefore be useful toaugment conventional chemotherapy or immunotherapeutic approachesto ALL.
Disclosures: No relevant conflicts of interest to declare.
* Asterisk with author names denotes non-ASH members.