(A) Left: Crystal structure of the cytoplasmic portion (kinase domain and kinase extension nuclease, KEN domain) of yeast IRE1α dimer (PDB#:3LJ0), with ADP and quercetin (a flavonol that activates IRE1α endoribonuclease activity) bound. Right: Bottom view of the IRE1α dimer where the endoribonuclease domain and RNA substrate recognition site are located. Individual monomers are colored in blue and yellow. The 3 residues (H1061, R1056 and Y1043) critical for endoribonuclease activity are labeled in red. (B) Docking (left) and binding affinity (right) of doxorubicin to the endoribonuclease domain of IRE1α. The ZINC database ID# of doxorubicin is shown on the left. (C) Log [inhibitor] response curve of doxorubicin defined as the ratio of luciferase activity measured from HT1080-XBP1-luc cells and that from the HT1080-CMV-luc (a control for non-specific toxicity) cells treated with 300 nM thapsigargin and varying doses of doxorubicin for 12 hours.

(A) Western blot analysis of UPR signaling proteins in HT1080 cells treated with 300 nM thapsigargin together with varying doses of doxorubicin for 14 hours. XBP1s denotes spliced XBP1 and XBP1u denotes unspliced XBP1. β-actin was used as a loading control. (B) Western blot analysis of UPR signaling proteins in RPMI 8226 cells treated with varying doses of doxorubicin for 14 hours. β-actin was used as a loading control. (C) Top: 0-hour and 96-hour bioluminescent imaging of XBP1-luc transgenic mice injected with 7 mg/kg doxorubicin intraperitoneally. Color scheme shows the radiance level. Lower left panel: Treatment schedule with arrows indicating the time points for each treatment. Lower right panel: histograms summarize the bioluminescent signals normalized to 0 hour, which is set to 100%. (D) Expression levels of representative target genes of different UPR branches as well as 2 RIDD target genes in RPMI 8226 cells after no treatment or 1 μM doxorubicin treatment for 6 hours analyzed by qRT-PCR. Results represent average quantities of technical triplicates normalized to β-actin ± c.v. (coefficient of variation).

(A) Chemical structures of doxorubicin and etoposide. (B) Western blot analysis of the IRE1α-XBP1 branch in HT1080 cells treated with 300 nM thapsigargin together with varying doses of doxorubicin or etoposide for 14 hours. β-actin was used as a loading control. (C) Extent of DNA damage as measured by Western blot analysis of phospho-γH2AX in HT1080 cells treated with 300 nM thapsigargin together with varying doses of doxorubicin or etoposide for 14 hours. β-actin was used as a loading control. (D) Western blot analysis of the IRE1α-XBP1 branch in RPMI 8226 cells treated with varying doses of doxorubicin and etoposide for 14 hours. β-actin was used as a loading control. (E) XTT cell viability assay for RPMI 8226 multiple myeloma cells treated with varying doses of doxorubicin or etoposide for 24 hours. Values represent % viable cells normalized to no treatment (set to 100%) ± SD.

(A) XTT cell viability assay of RPMI 8226 and MM1.R MM cells and 4 other cancer cell lines (H1299 non-small cell lung carcinoma, HCT116 colorectal carcinoma, A172 glioblastoma and MCF7 mammary adenocarcinoma) treated with varying doses of doxorubicin for 24 hours. Values represent % viable cells normalized to no treatment (set to 100%) ± SD. (B) Western blot analysis of IRE1α and XBP1 proteins for the cell lines used in (A). (C) Doxorubicin inhibited endogenous XBP1 mRNA splicing in CD138⁺ cells freshly isolated from MM patients undergoing active therapy. CD138⁺ cells isolated from 6 MM patients were treated with 1 μM doxorubicin for 6 hours then total RNA was extracted and used for qRT-PCR analysis with primers specific for either spliced or total XBP1. Results represent average quantities of technical triplicates of spliced XBP1 (XBP1s) normalized to total XBP1 ± c.v. Results of CD138⁺ cells from both peripheral blood (PB) and bone marrow (BM) of patient 1 are shown.