PS1316 UNFOLDED PROTEIN RESPONSE AFTER TREATMENT WITH 2‐DEOXYGLUCOSE CREATES SUBSTANTIAL SYNERGY WITH IMMUNOTOXIN MOXETUMOMAB PASUDOTOX IN B‐CELL LYMPHOMA AND LEUKEMIA XENOGRAFTS
Authors: Wagner F. , Meier C. , Krumbholz M. , Metzler M. , Mackensen A. , Müller F. 
Published: June 2019

Background:Compared with healthy B‐cells, lymphoma cells commonly present with increased rate of protein synthesis and of glycolysis. 2‐Deoxyglucose (2‐DG), an inhibitor of the key enzyme of glycolysis, irreversibly blocks glucose metabolism. Despite promising in vitro activity, 2‐DG alone showed little clinical activity against various cancers. Immunotoxins are fusion proteins of an antibody fragment and pseudomonas exotoxin and kill lymphoma cells by arresting protein synthesis. The CD22‐targeted immunotoxin Moxetumomab pasudotox (Lumoxiti) was approved for the treatment of relapsed/refractory hairy cell leukemia where it produces over all response rates of 86%. In various animal models of B‐cell acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL), efficacy of Moxetumomab substantially increases when serum levels are kept high by repeated or continuous administration, compensating for the short serum half‐life after bolus injection.Aims:By targeted inhibition of protein synthesis and of glycolysis, we aimed to induce synthetic lethality in aggressive B‐cell malignancies.Methods:MCL, ALL, Burkitt's lymphoma (BL) cell lines, and patient‐derived B‐ALL were treated in vitro with 2‐DG and Moxetumomab alone or in combination. Drug synergy was determined mathematically as fold‐increase over additive synergy. Biochemical studies were performed using western blot. A systemic JeKo‐1 and a newly established systemic Ramos xenograft model were used to test the combination in vivo.Results:Moxetumomab alone was cytotoxic in the low pico‐molar range against MCL, ALL, and BL cell lines. 2‐DG alone showed cytotoxicity against some cells which was completely reversed by mannose. In combination, 2‐DG enhances Moxetumomab highly synergistically, increasing cytotoxicity of Moxetumomab by three to nine‐fold. Tested against patient‐derived primary ALL of the Burkitt's type in vitro, the high synergy of Moxetumomab and 2‐DG was reproduced. As for cytotoxicity of 2‐DG, the synergistic effects of 2‐DG on Moxetumomab was blocked by mannose. Mechanistically, Moxetumomab alone and 2‐DG alone resulted in a fall of MCL1 protein levels in an additive manner which did not explain synergy. In addition to changes in MCL1 levels, 2‐DG induced unfolded protein response (UPR) as determined by the upregulation of BIP and CHOP. The addition of mannose blocked 2‐DG induced UPR but not the fall of MCL1. Synergy was reproduced when combining Moxetumomab and the UPR inducer tunicamycin. In vivo, 2‐DG or tunicamycin alone had no activity against systemic JeKo‐1 or Ramos xenografts. Moxetumomab alone reduced bone marrow tumor burden by 5‐fold in the JeKo‐1 and by 16‐fold in the Ramos xenograft model. In vivo, efficacy of Moxetumomab was enhanced in a dose dependent manner up to 3‐fold in the JeKo‐1 model and the combination achieved an MRD‐negative bone marrow status in the Ramos model.Summary/Conclusion:The combination of 2‐DG and Moxetumomab is well tolerated, produces substantial drug synergy, and may be an interesting combination for future clinical testing.