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. 2014 Jun;124(6):2585-98.
doi: 10.1172/JCI73448. Epub 2014 May 8.

Inhibition of ER stress-associated IRE-1/XBP-1 pathway reduces leukemic cell survival

Chih-Hang Anthony Tang, Sujeewa Ranatunga, Crystina L Kriss, Christopher L Cubitt, Jianguo Tao, Javier A Pinilla-Ibarz, Juan R Del Valle, Chih-Chi Andrew Hu

Inhibition of ER stress-associated IRE-1/XBP-1 pathway reduces leukemic cell survival

Chih-Hang Anthony Tang et al. J Clin Invest. 2014 Jun.

Abstract

Activation of the ER stress response is associated with malignant progression of B cell chronic lymphocytic leukemia (CLL). We developed a murine CLL model that lacks the ER stress-associated transcription factor XBP-1 in B cells and found that XBP-1 deficiency decelerates malignant progression of CLL-associated disease. XBP-1 deficiency resulted in acquisition of phenotypes that are disadvantageous for leukemic cell survival, including compromised BCR signaling capability and increased surface expression of sphingosine-1-phosphate receptor 1 (S1P1). Because XBP-1 expression requires the RNase activity of the ER transmembrane receptor IRE-1, we developed a potent IRE-1 RNase inhibitor through chemical synthesis and modified the structure to facilitate entry into cells to target the IRE-1/XBP-1 pathway. Treatment of CLL cells with this inhibitor (B-I09) mimicked XBP-1 deficiency, including upregulation of IRE-1 expression and compromised BCR signaling. Moreover, B-I09 treatment did not affect the transport of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumor-bearing mice suppressed leukemic progression by inducing apoptosis and did not cause systemic toxicity. Additionally, B-I09 and ibrutinib, an FDA-approved BTK inhibitor, synergized to induce apoptosis in B cell leukemia, lymphoma, and multiple myeloma. These data indicate that targeting XBP-1 has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma.

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Figures

Figure 1
Figure 1. XBP-1 deficiency decelerates leukemic progression in Eμ-TCL1 mice.
(A) CD5–/B220+ B cells purified from 6-week-old XBP-1WT/Eμ-TCL1 (XBP-1WT/TCL1) and XBP-1KO/Eμ-TCL1 (XBP-1KO/TCL1) mice were stimulated with LPS for a course of 3 days and lysed for analysis of indicated proteins by immunoblots. Data shown in immunoblots are representative of 3 independent experiments. (B–D) Splenocytes isolated from XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice at the ages of 5, 9, and 12 months were stained with CD3-APC-Cy7, IgM-PE-Cy7, B220-FITC, CD5-APC, and DAPI. Gated live CD3–IgM+ B cell populations were analyzed for the expression of B220 and CD5. (E) The percentages of CD5+B220+ CLL cells in splenocytes of XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice at the ages of 5, 9, and 12 months were plotted as mean ± SEM (n = 5 in each age group). (F) CD5+B220+ CLL cells purified from spleens of XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice were lysed to analyze for the expression of indicated proteins. Data shown in immunoblots are representative of 3 independent experiments. (G) Spleens from 12-month-old age-matched XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 littermates and a WT mouse. (H) Kaplan-Meier analysis of overall survival of XBP-1KO/Eμ-TCL1 mice (n = 18). Four mice from the XBP-1KO/Eμ-TCL1 group were censored (circled in red), as they were removed for other studies.
Figure 2
Figure 2. XBP-1 deficiency compromises activation of the BCR and synthesis of secretory IgM in Eμ-TCL1 B cells.
(A) XBP-1KO/Eμ-TCL1 B cells respond ineffectively to activation via the BCR. XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 B cells were treated with LPS for 3 days, stimulated with F(ab′)2 anti-mouse IgM to crosslink the BCR for indicated times, and lysed for analysis of indicated proteins by immunoblots. (B and C) WT B cells and CLL cells were isolated from 12-month-old WT, XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice. Purified cells were radiolabeled for 15 minutes, chased for indicated times, and lysed. Intracellular and extracellular IgM were immunoprecipitated from lysates (B) and culture medium (C), respectively, using an anti-κ antibody. Immunoprecipitates were analyzed on an SDS-PAGE gel. Data are representative of 3 independent experiments.
Figure 3
Figure 3. XBP-1 deficiency delays immunophenotypic changes in leukemia.
(A) Splenocytes isolated from approximately 9-month-old XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice were stained with monoclonal antibodies against CD3, IgM, CD5, and CD43. The expression of CD43 on the surface of CD5– B cells and CD5+ CLL cells were analyzed on gated CD3–IgM+ B cell populations of the spleens. Data are representative of 3 independent experiments. (B–G) Splenocytes isolated from approximately 9-month-old XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice were stained with monoclonal antibodies against CD3, IgM, CD5, and an additional marker indicated in each panel: (B) B220, (C) CD21, (D) CD22, (E) CD23, (F) IgD, and (G) S1P1. The expressions of the indicated markers on the surface of CD5– B cells and CD5+ CLL cells were analyzed on gated CD3–IgM+ B cell populations of the spleens. Data are representative of 3 independent experiments. (H) B cells purified from XBP-1WT/Eμ-TCL1 and XBP-1KO/Eμ-TCL1 mice were stimulated with LPS for 3 days and lysed for analysis of the expression of S1P1, p97, and actin by immunoblots. (I) Splenocytes isolated from a 14-month-old XBP-1KO/Eμ-TCL1 mouse were stained with monoclonal antibodies against CD3, IgM, CD5, and S1P1 and similarly analyzed.
Figure 4
Figure 4. Development of potent inhibitors to target the IRE-1/XBP-1 pathway.
(A) Recombinant human IRE-1 (hIRE-1) was expressed in insect cells and purified using Ni-NTA column chromatography. Purified hIRE-1 was analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue G-250. (B) A diagram depicting the mini-XBP1 stem-loop RNA and its cleavage by hIRE-1. IRE-1 inhibitors block hIRE-1 from cleaving the XBP1 stem-loop RNA substrate. (C) The Michaelis-Menten curve for hIRE-1 showing catalytic RNase activity in a FRET assay. Initial reaction rates are plotted as a function of different XBP1 stem-loop RNA concentrations in the presence of 5 nM hIRE-1. (D) Structures of IRE-1 inhibitors with in vitro IC50 values obtained from FRET-suppression assays. (E) Dose-response curves were generated from FRET-suppression assays. Dose-response experiments were carried out a minimum of 3 times on different days, and data were plotted as mean ± SEM. The IC50 values were calculated from the mean inhibition value at each concentration. Shown here are representative curves for selected IRE-1 inhibitors. (F) LPS-stimulated XBP-1–deficient B cells were treated with B-I06 or B-I07 (control) for 24 hours and lysed for immunoprecipitations using an anti-biotin antibody and protein G–conjugated agarose. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted for IRE-1.
Figure 5
Figure 5. IRE-1 inhibitors with masked aldehyde moieties potently suppress XBP-1s expression and leukemic growth.
(A) WaC3 CLL cells were treated with indicated compounds for 24 hours and lysed for RNA extraction. Human unspliced XBP1 (XBP1u), spliced XBP1 (XBP1s), and actin were detected by RT-PCR using specific primers. Data are representative of 3 experiments. (B) Mouse B cells were stimulated with LPS for 48 hours to allow for XBP-1s expression, and then treated with indicated inhibitors for 24 hours. Lysates were immunoblotted for XBP-1s and p97. Data are representative of 3 experiments. (C) LPS-stimulated B cells were treated with 0, 1.25, 2.5, 5, 10, 20, 40, 80, or 160 mM B-H09 for 24 hours. Equal amounts of lysates were immunoblotted for XBP-1s. The XBP-1s protein band intensity was determined using ImageJ. The percentage of inhibition was determined by comparing with the untreated group. Data from 3 experiments were plotted as mean ± SEM. (D) Mouse CD3–IgM+CD5+ Eμ-TCL1 CLL cells were treated with DMSO or indicated inhibitors for 3 days and subjected to XTT assays. Percentages of growth were determined by comparing inhibitor-treated with DMSO-treated groups. Data from 4 identical experimental groups were plotted as mean ± SD. Results are representative of 3 experiments. (E and F) Primary CLL cells from 2 human patients were treated with DMSO or indicated inhibitors (20 μM), subjected to XTT assays, and similarly analyzed. Data from 4 identical experimental groups were plotted as mean ± SD. Results are representative of 3 experiments.
Figure 6
Figure 6. B-I09 does not affect synthesis, assembly and transport of critical B cell integral membrane proteins.
(A and B) To reveal the effect of B-I09 on membrane-bound IgM (mIgM), XBP-1WT/μS KO B cells were stimulated with LPS for 2 days and subsequently treated with DMSO (control) or B-I09 (20 μM) for 1 additional day. DMSO-, or B-I09–treated XBP-1WT/μS KO B cells and DMSO-treated XBP-1KO/μS KO B cells were radiolabeled for 15 minutes, chased for indicated time, and lysed. Intracellular mIgM and κ light chain were immunoprecipitated from lysates using an anti-κ antibody (A). Secreted free κ chains were immunoprecipitated from culture medium using an anti-κ antibody (B). Immunoprecipitates were analyzed by SDS-PAGE. Data are representative of 3 independent experiments. (C) Lysates similar to those in A were immunoprecipitated using an antibody against the class I MHC heavy chain (HC), and immunoprecipitates were analyzed by SDS-PAGE. Data are representative of 3 independent experiments. (D) Using lysates similar to those in A, we performed immunoprecipitations using an anti-Igβ antibody to retrieve the Igα/Igβ heterodimers. Immunoprecipitated Igα/Igβ proteins were eluted from the beads and treated with endo-H or PNGase F before being analyzed by SDS-PAGE. μM, membrane-bound μ chain; CHO, high mannose-type glycans; CHO*, complex-type glycans; NAG, N-acetylglucosamines. Data are representative of 3 independent experiments.
Figure 7
Figure 7. B-I09 suppresses leukemic growth and is well tolerated in mice.
(A) Pharmacokinetic analysis of B-I09 is described in Methods (n = 3; mean ± SEM). The terminal half-life (T1/2), time of peak concentration (Tmax), maximum concentration (Cmax), and AUC versus time calculated using zero to infinity (AUCinf) of B-I09 in mouse plasma are indicated. (B) CLL-bearing Eμ-TCL1 mice were intraperitoneally injected with DMSO (n = 3) or B-I09 (50 mg/kg in DMSO, n = 8) daily for the first 5 days of each of 3 weeks. Blood was collected to measure lymphocyte numbers using a HemaTrue Hematology Analyzer (HESKA). Data were compared with lymphocyte counts prior to B-I09 injections and plotted as mean ± SEM. (C) Lymphocyte counts in the peripheral blood of B-I09–treated Eμ-TCL1 mice (n = 8) were plotted as mean ± SEM. (D) PBMCs from B-I09 mice, before and after injections, were lysed for analysis of indicated proteins. (E) Splenocytes from DMSO- or B-I09–injected Eμ-TCL1 mice were stained with IgM-PE-Cy7, B220-FITC, CD5-APC, annexin V–PE, and 7-AAD. Gated IgM+B220+CD5+ splenic CLL cells were analyzed for annexin V– and/or 7-AAD–positive populations. (F) Percentages of apoptotic cells in gated IgM+B220+CD5+ CLL populations from spleens of DMSO-injected (n = 3) or B-I09–injected (n = 8) Eμ-TCL1 mice were plotted as mean ± SEM. (G) Weight of DMSO-injected (n = 3) or B-I09–injected (n = 8) Eμ-TCL1 mice was plotted as mean ± SD. (H) Paraffin-embedded sections of indicated organs from Eμ-TCL1 mice receiving 3 weeks of injections with DMSO or B-I09 were stained with H&E. Scale bars: 80 μm.
Figure 8
Figure 8. B-I09 mimics genetic Xbp1 knockout in compromising BCR signaling, and exerts a strong synergistic effect with ibrutinib.
(A) WT and (B) Eμ-TCL1 B cells were treated with DMSO or B-I09 (20 μM) in the presence of LPS (20 μg/ml) for 2 days, stimulated with F(ab′)2 anti-mouse IgM for indicated times to activate the BCR, and lysed for analysis of indicated proteins by immunoblots. Data are representative of 3 experiments. (C) Eμ-TCL1 B cells were stimulated with LPS for 2 days and subsequently treated with DMSO, B-I09 (20 μM), ibrutinib (10 μM), or B-I09 in combination with ibrutinib for another day. LPS-stimulated XBP-1KO/Eμ-TCL1 B cells serve as controls. After stimulation with F(ab′)2 anti-mouse IgM for 5 minutes, cells were lysed for analysis of indicated proteins by immunoblots. Data are representative of 3 experiments. (D) Eμ-TCL1 CLL cells were treated with DMSO (control), B-I09 (10 μM), ibrutinib (1 μM), or a combination of both for 3 days and subjected to XTT assays. Percentages of growth were determined by comparing inhibitor-treated groups with control groups. Data from 4 identical experimental groups were plotted as mean ± SD. Results are representative of 3 experiments. (E–G) Dose-dependent growth inhibition curves of MEC1, MEC2, and WaC3 human CLL cells treated for 48 hours with B-I09, ibrutinib, or a combination were determined by CellTiter Blue assays. The concentration ranges for B-I09 and ibrutinib were 3.9 μM∼100 μM and 1.56 μM∼40 μM, respectively. Data from 2 experimental repeats were plotted as mean ± SEM.
Figure 9
Figure 9. B-I09 synergizes with ibrutinib to induce apoptosis in human CLL, MM, and MCL cell lines.
(A–C) MEC1, MEC2 and WaC3 human CLL cells were treated with DMSO (control), B-I09 (20 μM), ibrutinib (10 μM), or a combination of both for 4 days, and subjected to XTT assays. Percentages of growth were determined by comparing inhibitor-treated groups with control groups. Data from 4 identical experimental groups were plotted as mean ± SD. Results are representative of 3 independent experiments. (D) MEC1 and MEC2 human CLL cells were treated with DMSO (control) or B-I09 (20 μM) for 48 hours and lysed for RNA extraction and RT-PCR. Human XBP1u, XBP1s, and actin were detected using specific primers. Results are representative of 3 independent experiments. (E) Human MEC2 CLL cells were treated for 72 hours with DMSO (control), B-I09 (20 μM), ibrutinib (10 μM), or the combination of B-I09 and ibrutinib. Cells were lysed for analysis of indicated proteins by immunoblots. Data are representative of 3 independent experiments. (F and G) MM cell lines (F) and MCL cell lines (G) were treated with DMSO or the combination of B-I09 (20 μM) and ibrutinib (10 μM) for a course of 4 days and subjected to XTT assays at the end of each day. Percentages of growth were determined by comparing treated groups with control groups. Each data point derived from 4 independent groups receiving exactly the same treatment was plotted as mean ± SD. Results are representative of 3 independent experiments.
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