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

Abstract

Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)-like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243-induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

Graphical abstract

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.