Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells

Abstract

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Figure 1. Dual p97 and proteasome inhibition induces high levels of apoptosis and disrupts protein degradation in MMCs.

(A) A panel of human MMC lines were treated with the indicated concentrations of bortezomib (BTZ) or Eer1 for the indicated time. The proportion of live cells relative to DMSO-treated controls was determined by staining with Annexin V-FITC and PI (mean and SEM of 3 independent experiments). (B) MMC lines were treated with the indicated concentrations of BTZ and Eer1 for 48h (OPM-2, RPMI 8226) or 24h (U-266, KMS-11) and the proportion of live cells compared to controls determined as in A (*p<.05, **p<.001, two-sided student’s t-test). (C) BTZ-adapted AMO-1 MMCs co-cultured with human bone marrow MSCs, primary MMCs grown in the presence of IL-6, and healthy donor PBMNCs were subjected to single and dual inhibition with Bortezomib and Eer1 (the median of 3 technical replicates is shown). (D) Immunoblotting for ubiquitinated proteins and tubulin (loading control) carried out on whole cell extracts prepared from MMC lines treated with bortezomib, Eer1, or both inhibitors, for 24h (14h in KMS-11 cells due to their higher apoptotic sensitivity).

Figure 2. Combined inhibition of p97 and the proteasome dramatically affects ER configuration.

(A) RPMI 8226 myeloma cells were stained with ER Tracker Blue-White DPX following treatment for 24h with bortezomib (BTZ; 5nM), Eer1 (5µM), or both. Representative confocal microscopic images show minor ER alterations after BTZ treatment, transformation of tubule-lamellar into globular ER structures after treatment with Eer1, and widespread ER vacuolisation after dual treatment. (B) Representative electron microscopic images of OPM-2 cells after treatment with BTZ (5nM), Eer1 (5µM), or BTZ and Eer1, for 24h. Arrows indicate classical ER in control cells, black arrowheads indicate moderately dilated and disrupted ER in Eer1-treated cells, and open arrowheads indicate vacuolised ER with reduced ribosomes on the cytosolic ER surface. Another cell treated with Eer1 and BTZ is shown at lower magnification (right panel). Areas of dilated perinuclear space are indicated by asterisks. Nu, nucleus. (C) BTZ and Eer1 have different effects on ER volume as shown by staining of OPM-2 cells with BFA-BODIPY after treatment for 24h with BTZ (5nM), Eer1 (5µM), or with BTZ and Eer1. A representative histogram (left panel) and the mean and SEM of 6 experiments (right panel) are shown (*p<.05, **p<.001, two-sided student’s t-test). (D) Immunoblotting for lumenal ER chaperones and tubulin (loading control) was carried out on whole cell extracts prepared from OPM-2 cells treated as in (C).

Figure 3. Dual p97/proteasome inhibition deregulates key cell survival and protein translation control pathways in MMCs.

(A) Immunoblotting for the indicated survival- and apoptosis-related proteins was carried out on whole cell extracts from MMC lines and primary human fibroblasts prepared after treatment with either BTZ, Eer1, or both inhibitors, for 24h (14h in KMS-11 cells due to their higher apoptotic sensitivity to inhibitors). (B) Cell death induced by dual p97/proteasome inhibition is predominantly caspase-dependent and JNK-independent. The proportion of live cells was determined after treatment with BTZ (5nM) and Eer1 (5µM) with or without the pan-caspase inhibitor zVADfmk (50µM) or the JNK inhibitor SP600125 (10µM) for 24h (36h for OPM-2 cells; all values are the mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student’s t-test).

Figure 4. Structurally different ERAD inhibitors have comparable effects in MMCs.

(A) Representative microscopic images of parental and BTZ-adapted AMO-1 cells stained with ER Tracker Blue-White DPX after treatment for 18h with Eer1 (5µM), DBeQ (5µM) or Eer1/DBeQ plus Bortezomib (5nM). The images show small to medium-sized globular/vesicular ER after p97 inhibition and ER vacuolisation after dual ERAD inhibition. (B) The proportion of live RPMI8226 cells was determined after treatment with bortezomib (10nM), DBeQ (10µM), or both, for 4h followed by drug wash-out and a further 20h incubation in culture medium (mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student’s t-test). (C) Primary MMCs were isolated from a bone marrow aspirate, incubated in culture medium containing IL-6 (10ng/ml) for 6h, followed by addition of bortezomib (5nM), DBeQ (5µM), or both for 24h and analysis of apoptosis levels. Live cells (Annexin V-FITC and PI-negative) are shown in green in the left lower quadrant, early apoptotic (Annexin V-FITC positive, right lower quadrant) and dead (Annexin V-FITC and PI-positive, right upper quadrant) cells are shown in orange and red, respectively.