Prolonged unfolded protein reaction is involved in the induction of chronic myeloid leukemia cell death upon oprozomib treatment

Abstract

To select the most efficient chemical to induce apoptosis in leukemia cells, a multidrug screen was applied on bone marrow mononuclear cells from chronic myeloid leukemia (CML) patients. Oprozomib (Cpd 21) was chosen for the subsequent experiments. The isobaric tags for relative and absolute quantitation (iTRAQ) was then performed to identify the responsible pathway relative to apoptosis and the results showed that endoplasmic reticulum (ER) chaperones were upregulated. Apoptosis was attributed to a joint effect of calcium leakage andPERK and IRE1α phosphorylation. The PERK branch was responsible for the first wave of cell death that occurred within 24 hours. The later wave of apoptosis was mediated by IRE1α, which transmit apoptotic signals through the ASK-JNK-BIM axis. Release of Ca2+ from ER into cytosol resulted in activation of calpain, which, in turn, cleaved caspase-12. Our data also explained the selective killing effects of oprozomib on CML cells, which relied on proteasome activity. The present study demonstrated that prolonged inhibition of proteasome to trigger unfolded protein response could be an alternative strategy for treating CML in light of tyrosine kinase inhibitors resistance.

Keywords: CML; ER stress; apoptosis; proteasome inhibitor; unfolded protein reaction.

FIGURE 1

Drug screening identified oprozomib as the most potent agent to induce apoptosis in chronic myeloid leukemia (CML) bone marrow cells. A, Viability of bone marrow mononuclear cells derived from five CML patients was determined in the presence of 43 compounds. Cells were treated with indicated compound at 1 μM for 48 h. Viability was tested by Cell Counting Kit‐8 (CCK‐8). B, IC50 of four compounds for 48 h was determined by CCK‐8. C, Apoptosis induced by four compounds after 48 h treatment at average IC50 was measured by flow cytometry (FCM). D, Cytotoxicity of four compounds on bone marrow mononuclear cells (BM‐MNC) from healthy donors. Cells treated with indicated compounds, each compound was used at average IC50 that calculated from 3 patient‐derived cells (fig 2B). E, Oprozomib (Cpd 21) triggered processing of caspase‐3 and downstream substrate poly ADP ribose polymerase (PARP)

FIGURE 2

Oprozomib exposure inhibited proteasome activity and launched unfolded protein response (UPR). A, CT‐L activity of proteasome was suppressed by oprozomib. B, Oprozomib increased ubiquitin‐tagged proteins after blockage of proteasome activity. C, Pathway enrichment of the upregulated proteins. has 04141 (marked by red color) was selected for further investigation. D, Parallel reaction monitoring validation of differentially expressed proteins that enriched in pathway has 04141. E,F, NMS‐873 treatment resulted in rise of ubiquitin‐tagged proteins in a time‐dependent fashion. G, Oprozomib triggered UPR activation. Data from three parallel tests are presented as mean ± SD. *P < 0.05, **P < 0.01

FIGURE 3

PERK branch of unfolded protein response (UPR) was responsible for the first wave cell death. A, Disassociation of PERK and IREα with Bip started within 6 h in the presence of oprozomib. B, Flowchart for investigating the role shift of UPR from cytoprotection to cytotoxicity. C, Phosphorylation of PERK and IREα at different time points. D, Phosphorylation of PERK became irreversible if the oprozomib treating time lasted for more than 4 h. Lane 2, w/d 20 stands for after oprozomib withdrawal; the cells were cultured in drug‐free medium for another 20 h. E, PERK dephosphorylation after 4‐h (upper) or 8‐h (lower) treatment. The indicated time at the top represents the duration of drug‐free culture after oprozomib withdrawal. F, Apoptosis induction at indicating treating time (aubergine) or time of drug‐free culture according to the description in (B). G, eIF2α phosphorylation and ATF4, CHOP upregulation induced by oprozomib; the effect was blocked by co–treatment with salubrinal (Sbl) (300 nM). H, Salubrinal inhibited apoptosis at 12 h but failed at 24 h. For western blot, cells from patients were separated and treated as stated in Figure 1C; the lysates were collected and stored at −80° and the test was conducted using a pool of lysates from several patients, presented in Table S2. For apoptosis assay, Flow cytometry (FCM) test was conducted for individual patients after treatment if the cells were sufficient. Data from three parallel tests are presented as mean ± SD. **P < 0.01

FIGURE 4

Activation of IREα branch. A, Association of IREα with ASK1 and TRAF2 was improved at 36 and 16 h, respectively, post–oprozomib administration. B, Phosphorylation of ASK1 and TRAF2. Upregulation (C) and phosphorylation (D) of Bim were measured at 48 h post–oprozomib administration, which was blocked by selonsertib (Slt) (500 nM), an ASK1 inhibitor. E, Combination of selonsertib and salubrinal further inhibited oprozomib‐induced apoptosis in comparison with selonsertib or salubrinal alone (48 h). F, Upregulation and phosphorylation of Bim were not affected by P53 blockage (48 h). G, Upregulation and phosphorylation of Bim tested in K562 cells in a time and dose‐dependent fashion. H, Activation of ASK1 and JNK at 48 h was independent of endonuclease activity of IREα. I, Kira6 but not STF083010 significantly reduced apoptosis induction by oprozomib (48 h). Data from three parallel tests are presented as mean ± SD. **P < 0.01

FIGURE 5

ITPR2‐mediated Ca2+ efflux. Cytosolic Ca2+ was detected by Fluo‐3 staining using flow cytometry (A) and a fluorescent microscope (B) at 48 h post–oprozomib treatment. Lentivirus vector containing siRNA‐targeted ITPR2 (LV‐ITPR2) was applied to downregulate the ITPR2 and subsequently inhibited Ca2+ release. C, LV‐ITPR2 suppressed the calpain activity that increased as a result of Ca2+ accumulation in cytosol. D, Caspase‐12 was the downstream target of Ca2+ Calpain signal. E, LV‐ITPR2 block in part apoptosis. F, ITPR2 expression induction in the presence of oprozomib was time‐dependent and became obvious at 24 h post–oprozomib exposure. As a consequence, cytosolic Ca2+ increased remarkably at this time (G). H, Induction of apoptosis mediated by ITPR2 occurred at 24 h post–oprozomib addition. Data from three parallel tests are presented as mean ± SD. *P < 0.05, **P < 0.01

FIGURE 6

Comparison of proteasome activity between heathy donors and chronic myeloid leukemia (CML) patients. CT‐L activity of proteasome in bone marrow mononuclear cells (BM‐MNC) (A) and peripheral blood mononuclear cells (PB‐MNC) (B) from CML patients was significantly higher than for heathy counterparts. C, No difference in CT‐L activity of proteasome was observed between BM‐MNC and PB‐MNC from 10 CML patients. D, Comparison of Bip, p‐PERK, and p‐IRE1α levels in three chronic myeloid leukemia (CML) patients with three heathy donors. Data from three parallel tests are presented as mean ± SD. *P < 0.05, **P < 0.01, n.s., not significant