Block of Counter-Regulation By Inhibition of Protein Synthesis Sensitizes Cancer Cells to IRE1α-Mediated Apoptosis Following Unfolded Protein Response

Background: Cellular stress responses like the unfolded protein response (UPR) decide over the fate of an individual cell. To prevent cell death, UPR induces expression of protective proteins that aim to restore homeostasis. A similar strategy is used by cancer cells to induce resistance against UPR-inducing drugs. Therefore, we hypothesized that a simultaneous block of protein synthesis prevents UPR counter-regulation and thus, enhances UPR-induced apoptosis.

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.

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.