Hsp90 Inhibitors Deliver a Death Signal Via the Endoplasmic Reticulum Stress Pathway in Myeloma.

Immunoglobulin production by plasma cells both defines them functionally and also provides a differential target for the therapy of their malignant counterparts. Plasma cells producing high levels of paraprotein are dependent upon the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We have evaluated a strategy aimed at manipulating the UPR response to deliver a novel death signal in myeloma. In order to study the apoptotic effects of the disruption of the UPR, a panel of myeloma cell lines were treated with the ER stress inducers (thapsigargin (TG) a Ca2+-ATPase inhibitor and tunicamycin (TM) an N-linked glycosylation inhibitor); the HSP90 inhibitors, 17-AAG and radicicol; and the proteasome inhibitor bortezomib, which disrupts misfolded protein disposal. The presence of misfolded proteins in the ER is detected by a complex of proteins embedded within the membrane comprised of BiP, PERK, IRE-1 and ATF6, each of which has distinct downstream effects that are mediated by their release from the complex and activation of the UPR. Treatment with TG and TM led to the activation of all three branches of the UPR as demonstrated by early splicing of XBP1 to XBP1s indicative of IRE1 activation, PERK activation as measured by transcriptional upregulation of CHOP (7-22 fold) and ATF6 splicing. 17-AAG was also capable of inducing splicing of XBP1 and the induction of CHOP (30 fold) whereas bortezomib resulted in induction of CHOP (25 fold) and minimal, late onset splicing of XBP1. Following treatment with all the drugs expression levels of BiP mRNA were upregulated (3-10 fold), however due to high basal levels of BiP protein we were unable to detect any further rises in this protein. The ER-resident HSP90 analogue, Grp94, underwent minimal transcriptional upregulation (2–3 fold) in response to HSP90 inhibitors but larger responses were noted following treatment with TM and TG (3–5 fold). HSP90 protein expression remained constant following exposure to all drugs. In contrast a time-dependent upregulation of the anti-apoptotic HSP70 was noted in response to 17-AAG, radicicol and bortezomib treatment; TG and TM failed to affect levels of HSP70. Levels of the transcript of EDEM1, an ER stress inducible membrane protein that accelerates the degradation of misfolded protein in the ER by strengthening the ERAD machinery, were induced by all drugs with an increase in the transcript levels of between 2 and 4 fold after 24 hours. All drugs were capable of inhibiting proliferation as demonstrated by MTT assay. In addition TG, bortezomib, 17AAG and radicicol also induced myeloma cell death as demonstrated by trypan blue and Annexin V/PI staining. A distinct pattern of activation of caspases in response to the drugs was also established. Bortezomib activated both the intrinsic (caspases 9 and 3) and the extrinsic (caspases 8 and 3) caspase pathways, whilst 17-AAG appears to mediate cell death via the intrinsic pathway alone. In contrast, TM and TG failed to activate either the extrinsic or intrinsic pathways within 24 hours and within this time frame appeared to induce cell death by a caspase-independent mechanism. In conclusion as well as inducing apoptosis via the intrinsic caspase death pathway, HSP90 inhibitors also induce myeloma cell death via ER stress and the UPR death pathway. Our results confirm that the unfolded protein response is an exciting new pathway that can be therapeutically targeted in myeloma.