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. 2012 Jun 14;119(24):5772-81.
doi: 10.1182/blood-2011-07-366633. Epub 2012 Apr 26.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

Naoya Mimura  1 , Mariateresa Fulciniti, Gullu Gorgun, Yu-Tzu Tai, Diana Cirstea, Loredana Santo, Yiguo Hu, Claire Fabre, Jiro Minami, Hiroto Ohguchi, Tanyel Kiziltepe, Hiroshi Ikeda, Yutaka Kawano, Maureen French, Martina Blumenthal, Victor Tam, Nathalie L Kertesz, Uriel M Malyankar, Mark Hokenson, Tuan Pham, Qingping Zeng, John B Patterson, Paul G Richardson, Nikhil C Munshi, Kenneth C Anderson
Affiliations

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

Naoya Mimura et al. Blood. .

Abstract

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

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Figures

Figure 1
Figure 1
The significance of IRE1α-XBP1 pathway in MM cells. (A) Expression of IRE1α in MM cell lines was detected by Western blotting. Actin served as a loading control. (B) XBP1 mRNA was detected by RT-PCR. XBP1u was observed as a 152-bp band, and XBP1s was observed as a 126-bp band. β-actin served as a loading control. (C-E) RPMI 8226 cells were transduced with 2 independent shRNA lentivirus vectors (XBP1sh#1 and XBP1sh#2) to knockdown XBP1 gene expression. A vector-targeting luciferase was used as a control. (C) Three days after transduction, total RNA was extracted, and XBP1 mRNA was evaluated by real-time quantitative PCR in the graph. Data represent mean ± SD fold changes relative to β-actin mRNA in triplicate samples. Expression of XBP1u and XBP1s was examined by RT-PCR in the right panel. (D) Two days after transduction, cells were incubated for the indicated times. Growth of the cells was assessed by MTT assay of quadruplicate cultures, expressed as a percentage of 4-hour samples. Data represent mean ± SD. (E) Two days after transduction, cells were treated with bor-tezomib (4nM) or 17-AAG (250nM) for 48 hours. Cell proliferation was assessed by [3H]-thymidine uptake of quadruplicate cultures, expressed as a percentage of untreated control vector cells. Data represent mean ± SD.
Figure 2
Figure 2
MKC-3946 is an IRE1α endoribonuclease inhibitor, which triggers modest cytotoxicity in MM cells. (A) Formal chemical structure of MKC-3946. (B) RPMI 8226 cells were treated with or without Tm (5 μg/mL) in combination with MKC-3946 (0-10μM) for 3 hours. Total RNA was extracted; XBP1 and β-actin mRNA were evaluated by RT-PCR. (C) RPMI 8226 cells were treated with Tm (5 μg/mL) in the presence or absence of MKC-3946 (10μM) for the indicated times. Total RNA was extracted, and XBP1 and β-actin mRNA were evaluated by RT-PCR. Whole-cell lysates were subjected to Western blotting using anti-IRE1α, phospho-IRE1α (p-IRE1α), BiP/GRP78, and actin Abs. (D) RPMI 8226 cells were cultured with or without MKC-3946 (10μM) for 8 hours. XBP1 target genes, such as SEC61A1, p58IPK, and ERdj4, were determined by real-time quantitative PCR. Data represent mean ± SD fold changes relative to β-actin mRNA in triplicate samples. *P < .001. (E) MM cell lines were cultured with MKC-3946 (0-12.5μM) for 48 hours. Cell viability was assessed by MTT assay of triplicate cultures, expressed as a percentage of untreated control. Data represent mean ± SD. (F) Primary MM cells isolated from patients (Pt) were cultured with or without MKC-3946 (10μM) for 6 hours. Total RNA was extracted and subjected to RT-PCR for analysis of XBP1 splicing.
Figure 3
Figure 3
MKC-3946 blocks XBP1 splicing and enhances cytotoxicity induced by bortezomib or 17-AAG. (A) RPMI 8226 cells were treated with bortezomib (Bor; 10nM) or 17-AAG (AAG; 1μM), in the presence or absence of MKC-3946 (10μM) for the indicated times. Total RNA was extracted, and XBP1 and β-actin mRNA were evaluated by RT-PCR. Whole-cell lysates were subjected to Western blotting using anti-IRE1α, phospho-IRE1α (p-IRE1α), BiP /GRP78, and GAPDH Abs. (B) RPMI 8226 cells were treated with bortezomib (Bor; 10nM) in the presence or absence of MKC-3946 (10μM) for the indicated times. Nuclear extracts were subjected to Western blotting using XBP1 Abs. Lamin B served as a loading control. (C) RPMI 8226 and INA6 cells were treated with bortezomib or 17-AAG in combination with MKC-3946, 0μM (□), 5μM (▩), or 10μM (■), for 48 hours. Cell proliferation was assessed by [3H]-thymidine uptake of quadruplicate cultures, expressed as a percentage of untreated control. Data represent mean ± SD. (D) Primary MM cells isolated from 3 patients were treated with bortezomib or 17-AAG in combination with MKC-3946 0μM (□), 5μM (▩), or 10μM (■) for 36 hours. Cell viability was assessed by MTT assay of triplicate cultures, expressed as a percentage of untreated control. Data represent mean ± SD.
Figure 4
Figure 4
MKC-3946 enhances ER stress-mediated apoptosis induced by bortezomib or 17-AAG. (A) RPMI 8226 cells were treated with bortezomib (Bor; 10nM) in the presence or absence of MKC-3946 (10μM) for the indicated times. Whole-cell lysates and nuclear extracts were subjected to Western blotting using PERK, eIF2α, phospho-eIF2α (p-eIF2α), and ATF4 Abs. GAPDH and histone H3 serve as loading controls. (B) RPMI 8226 cells were treated with bortezomib (2.5nM) or 17-AAG (500nM) and INA6 cells were treated with bortezomib (2.5nM) or 17-AAG (125nM), in each case in combination with MKC-3946 (10μM) for 24 hours. Apoptotic cells were analyzed by flow cytometry using annexin V/PI staining. (C) RPMI 8226 and INA6 cells were treated with bortezomib (2.5nM) in the presence or absence of MKC-3946 (10μM) for 24 hours. Whole-cell lysates were subjected to Western blotting using anti-CHOP, PARP, caspase-3, and α-tubulin Abs. (D) RPMI 8226 cells were treated with MKC-3946 (10μM), bortezomib (10nM), or the combination for 8 hours. CHOP mRNA was determined by real-time quantitative PCR. Data represent mean ± SD fold changes relative to β-actin mRNA in triplicate samples. *P < .001.
Figure 5
Figure 5
MKC-3946 enhances MM cytotoxicity of bortezomib or 17-AAG, even in the presence of BMSCs or exogenous IL-6. (A-B) INA6 cells were treated with bortezomib (A) or 17-AAG (B) in combination with MKC-3946 0μM (□), 5μM (▩), or 10μM (■), in the presence or absence of BMSCs for 48 hours. (C-D) INA6 cells were treated with bortezomib (C) or 17-AAG (D) in combination with MKC-3946 0μM (□), 5μM (▩), or 10μM (■), with 2.5 ng/mL or 10 ng/mL of IL-6 for 48 hours. Cell proliferation was assessed by [3H]-thymidine uptake of quadruplicate cultures. Data represent the mean ± SD of [3H]-thymidine incorporation (CPM).
Figure 6
Figure 6
MKC-3946 inhibits XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells, alone or with bortezomib. (A) SCID mice were treated with Tm (1 mg/kg intraperitoneally) for 4 or 6 hours. Two hours after Tm was administered, mice were treated with MKC-3946 50 mg/kg intraperitoneally. After 4-hour exposure to MKC-3946, mice were killed. Livers were harvested, and total RNA was prepared. RT-PCR was performed using murine-specific XBP1 primers. Each lane represents a single mouse. The vertical line indicates a repositioned gel lane. (B-D) SCID mice were injected subcutaneously with 1 × 107 RPMI 8226 cells on day 0 and treated with 100 mg/kg MKC-3946 intraperitoneally daily (MKC-3946, n = 8), 0.15 mg/kg bortezomib intravenously twice a week (bortezomib, n = 8), or 100 mg/kg MKC-3946 intraperitoneally daily and 0.15 mg/kg bortezomib intravenously twice a week (combination, n = 8), for 21 days starting on day 1. A vehicle control group received intraperitoneal injections of vehicle and intravenous injection of saline (vehicle, n = 8). (B) Tumor volume was calculated from caliper measurements every 3 to 4 days, and data represent mean ± SE. (C) Survival in the plasmacytoma model was evaluated from the first day of treatment using Kaplan-Meier curves. (D) Total RNA was prepared from subcutaneous plasmacytoma harvested from each group of mice after 3 weeks of treatment. XBP1 and β-actin mRNA were examined using RT-PCR. The graph represents fold changes of XBP1s density relative to β-actin. (E) Growth of INA6 cells engrafted in human bone chips in SCID mice was monitored by serial serum measurements of shuIL-6R in the graph. Mice were treated with MKC-3946 100 mg/kg (n = 3) or control vehicle (n = 3), and shuIL-6R levels were determined weekly by ELISA. Error bars represent ± SE. Total RNA was prepared from tumor harvested from the MKC-3946– and control-treated SCID-hu mice at 3 weeks. XBP1 and β-actin mRNA were examined using RT-PCR in the right panel.
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