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Targeting ubiquitin-activating enzyme induces ER stress–mediated apoptosis in B-cell lymphoma cells

Scott Best, Taylor Hashiguchi, Adam Kittai, Nur Bruss, Cody Paiva, Craig Okada, Tingting Liu, Allison Berger and Alexey V. Danilov

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    TAK-243 blocks ubiquitin conjugation and induces ER stress and the UPR in DLBCL cells. (A) DLBCL cell lines were treated with the indicated doses of TAK-243 for 4 hours. Proteins were lysed and subjected to immunoblotting. (B) DLBCL cell lines were treated with TAK-243 for 24 hours. Apoptosis was determined by Annexin-V staining. Data are presented as mean ± standard error (SE). *P < .05 compared with untreated control. (C-D) DLBCL cells were treated with the indicated doses of TAK-243 for 4 hours (C) or as indicated (D), and proteins were lysed and subjected to immunoblotting. (E) OCI-LY3 cells were treated with TAK-243 for 4 hours. Total RNA was isolated, reverse transcribed, and subjected to RT-PCR with the indicated probes (in duplicates). Results were normalized to GAPDH levels. Data are presented as mean ± SE. *P < .05 compared with untreated control. GCB, germinal center B-cell–like.

  • Figure 2.

    TAK-243 exhibits in vivo antiproliferative and cytotoxic activity against DLBCL tumors in mice. OCI-LY3 cells were xenografted in mice as described in Materials and methods. Mice were treated with 10 or 20 mg/kg TAK-243 or vehicle control. (A) Tumor growth was measured daily. (B-D) At the end of the experiment, mice were euthanized 2 hours after the final TAK-243 dose, and tumor cells were analyzed for apoptosis (B), proliferation (C), and expression of ER stress markers by RT-PCR (D). Tumor size was normalized to pretreatment values (represented by 0 on the y-axis). *P < .05; **P < .01.

  • Figure 3.

    TAK-243 induces DNA damage and cell cycle arrest in DLBCL cells. OCI-LY19 (A) and OCI-LY3 (B) cells were treated with TAK-243, and proteins were lysed at the indicated time points and subjected to immunoblotting. (C-D) OCI-LY19 cells were treated with TAK-243 for 24 hours. Flow cytometry was used to measure DNA content in cells stained with propidium iodide. DMSO, dimethyl sulfoxide.

  • Figure 4.

    TAK-243 induces rapid ER stress and exhibits enhanced cytotoxicity compared with bortezomib. DLBCL cells were treated with TAK-243 or bortezomib. (A) Cells were lysed, and proteins were subjected to immunoblotting at the indicated time points. (B) Apoptosis was assessed after 24 hours by Annexin V staining. Data are presented as mean ± SE; *P < .05. (C-E) DLBCL cell lines were treated with TAK-243 or bortezomib. Cells were then subjected to immunoblotting. (F) OCI-LY3 cells were treated with 1000 nM TAK-243, 1000 nM bortezomib, or vehicle control for 4 hours and stained for LC3 (green) and 4′,6-diamidino-2-phenylindole (blue). Original magnification ×500.

  • Figure 5.

    MYC sensitizes DLBCL to TAK-243-induced apoptosis. (A-B) U-2932 were treated with TAK-243 as shown. MYC protein and RNA levels were evaluated by immunoblotting and RT-PCR. (C) OCI-LY3 cells were engineered to express MYC or vector control. MYC overexpression was confirmed by RT-PCR. Cells were treated with 300 nM TAK-243 for 24 hours, and apoptosis was assessed by Annexin V staining. (D) OCI-LY19 cells were engineered to express shMYC or vector control. MYC knockdown was confirmed by RT-PCR. Cells were treated with 300 nM TAK-243 for 24 hours, and apoptosis was assessed by Annexin V staining. (E-F) Cells manipulated to express MYC or shMYC were treated with 300 nM TAK-243 for 4 hours. Proteins were lysed and subjected to immunoblotting. Expression of ER stress genes was assessed by RT-PCR with the indicated probes. (G) OCI-LY19 cells were treated with SKT and TAK-243 for 4 hours. Proteins were lysed and subjected to immunoblotting. (H) OCI-LY19 cells were treated with SKT and TAK-243 for 24 hours. Apoptosis was assessed by Annexin V staining. Data are presented as mean ± SE. *P < .05; **P < .01.

  • Figure 6.

    TAK-243 induces ER stress and apoptosis in primary DLBCL cells. (A) Primary DLBCL cells were cocultured with CD40L-expressing or control stroma for 24 hours, then treated with then indicated concentrations of TAK-243 for additional 24 hours. Apoptosis of the CD19-positive B cells was assessed by Annexin V staining. Data are presented as mean ± SE. (B-C) DLBCL cells (n = 3) were cocultured with the CD40L-expressing stroma for 24 hours and then treated with the indicated concentrations of TAK-243 for 2 and 4 hours. (B) Cells were harvested for protein lysates (analyzed by immunoblotting) and RNA. (C) Transcript mRNA levels were quantitated by quantitative RT-PCR, and fold change was measured against cells treated with vehicle control. *P < .05; **P < .01.