Targeting UPR Sensors: A Novel Approach To Promote Death of Multiple Myeloma Cells.

Multiple Myeloma (MM) is a plasma cell (PC) malignancy producing large amounts of monoclonal Igs requiring a highly developed endoplasmic reticulum (ER). A coordinated adaptative program called the unfolded protein response (UPR) allows cells to survive the stress consecutive to a transient protein overload of the ER. This process is referred to as "physiological UPR". If the stress is excessive or sustained, the UPR leads to cell death ("terminal UPR"). We have recently documented that the spontaneous death of non transformed human PC is initiated by an ER stress. Moreover the group of R. Sitia has established that the proteasomal activity decreases in the late phases of PC differentiation. These findings suggest that the imbalance between the high-rate of Igs secretion and the impaired proteolytic machinery predisposes PC to apoptosis. We have postulated that a dysregulated adaptative UPR contributes to the increased survival capacity of MM cells. We have undertaken a study aimed at promoting death of MM cells by disruption of ER homeostasis. Our approach has consisted in using RNA interference to knock-down the UPR sensors PERK, ATF6 and IRE1, in two MM cell lines (U 266 and NCI-H929). We show that the anti-UPR siRNAs induce a 70 to 80% reduction of the levels of expression of PERK, ATF6 and IRE1 and promote cell death of these two human MM cell lines. The PERK-targeted siRNA was consistently found to be the best apoptosis inducer. We found that invalidation of PERK leads to induction of a terminal UPR as revealed by the upregulation of GADD153/CHOP and of the spliced form of XBP-1. The death elicited by invalidation of the UPR sensors was accompanied by phosphatidylserine (PS) exposure and early loss of the mitochondrial transmembrane potential. By contrast, we found no evidence of DNA fragmentation and electronic microscopy revealed only a partial nuclear chromatin condensation. Cell death induced by UPR-targeted siRNAs operated in a caspase -independent fashion since it was not inhibited by the pan-caspase inhibitor z-VAD-fmk. Accordingly, the activated forms of caspase-3 and-4 were undetectable. However, calpain inhibitors and calcium chelators prevented the loss of the mitochondrial transmembrane potential and partially inhibited cell death. This suggests that the death signal initiated at the level of the ER is transmitted to the mitochondria via the calcium cysteine protease calpain. Transmission electron microscopy revealed that the PERK siRNA induces formation of numerous autophagic vacuoles, a result which was confirmed by the positive staining with monodansylcadaverine (MDC). Appearance of mature autophagic vacuoles preceeded acquisition of death morphology which was characterized by partial nuclear condensation and protein accumulation. The anti-UPR siRNAs induced the presence of immature and mature autophagic vacuoles but, in contrast, when acquiring death morphology, cells were devoid of autophagic vacuoles although they showed partial nuclear condensation. The autophagic specific inhibitor 3 methyladenine reduced both PS exposure and MDC staining induced by PERK siRNA. This suggests that UPR-targeted siRNAs induce death of MM cells by autophagy. Altogether, our data demonstrate that disruption of ER homeostasis by extinction of UPR sensors induce death of MM cells. They also reveal that a connection exists between ER stress and autophagy in MM cells.