Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster I
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, apoptosis, Translational Research, CLL, Lymphomas, B Cell lymphoma, Combination therapy, Diseases, Therapies, Lymphoid Malignancies, Biological Processes
Methods: Tumor cell lines and patient-derived acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells were treated with several protein synthesis inhibitors and UPR-inducers. Synergy was determined as fold-increase over additivity. Mechanistic studies were performed using western blots, shRNA-mediated knock-down, and CRISPR/Cas9-mediated knock-out. In vivo synergy was tested using intravenously injected xenografts.
Results: 2-deoxyglucose (2-DG), which competitively inhibits protein glycosylation in the endoplasmic reticulum (ER), induced UPR in B cell lymphoma cell lines. An additional block of protein synthesis by CD22-targeted immunotoxin Moxetumomab pasudotox (Moxe) prevented the upregulation of the protective binding immunoglobulin protein (BiP), although UPR signaling remained active. In line, 2-DG synergistically enhanced activity of protein synthesis inhibitors Moxe, cycloheximide, and puromycin suggesting a conserved mechanism of action. Furthermore, drug synergy was not only reversed by addition of mannose, which restores proper glycosylation, but was also mimicked by the two other UPR-inducers tunicamycin and bortezomib indicating UPR as cause of synergy. Mechanistically, Moxe and 2-DG induced synergistic mitochondrial apoptosis, which correlated with synergistic cleavage of PARP, synergistic reduction of BID, and additive fall in MCL-1. Knock-down of several UPR-associated proteins identified Inositol-requiring enzyme 1α (IRE1α) as responsible protein for synergy. Knock-out of IRE1α reversed synergistic cell death, PARP cleavage, and reduction of BID but not the fall of MCL-1 confirming IRE1α as key mediator of synergy. Suggesting broad applicability, immunotoxins and 2-DG induced synergy in seven distinct hematologic and three solid tumor entities, while healthy blood cells were insensitive towards the combination. Because UPR-induced cell death depends on a persisting stress signal and 2-DG has a short half-life, exposure time in vivo was increased by four injections of 2-DG per day. Against a systemic mantle cell lymphoma model, only high frequent 2-DG treatment, but not daily single injections, enhanced Moxe activity by 3-fold. The in vivo synergy was reproduced by combination with tunicamycin and bortezomib. Furthermore, the combination of 2-DG and Moxe induced a 35-fold synergy against a systemic Burkitt lymphoma model and an up to 37-fold synergy against two patient-derived B-ALL xenografts of the Burkitt’s type.
Conclusion: Blocking UPR counter-regulation by simultaneous inhibition of protein synthesis induces synergistic cell death in several malignancies. Synergy depends on IRE1α-mediated deregulation of BID which enhances MCL-1-mediated mitochondrial apoptosis. The broad applicability suggests a cancer cell-specific vulnerability which, together with a cell-targeted arrest of protein synthesis by immunotoxins, generates a unique therapeutic window supporting clinical evaluation.
Disclosures: Mackensen: BMS/Celgene: Honoraria; Miltenyi Biomedicine: Honoraria; Kite/Gilead: Honoraria; Novartis: Honoraria. Mueller: BMS: Consultancy, Honoraria; Abbvie: Consultancy; Janssen: Consultancy, Honoraria; Kite/Gilead: Honoraria; Novartis: Honoraria; MedImmune: Research Funding.
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