Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

Abstract

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

Keywords: Breast cancer; Cell stress; Drug therapy; Oncology; Therapeutics.

Figure 1. MYC is necessary for activation of the IRE1/XBP1 pathway.

(A and B) Immunoblot of MYC and IRE1 in SUM159 cells (A) or BT549 cells (B) infected with lentiviruses encoding control scramble shRNA (shScr) or 2 distinct MYC shRNAs (shMYC-1 and shMYC-2). Actin and GAPDH were used as loading controls. (C–F) qRT-PCR analysis of IRE1 expression and XBP1 splicing in infected SUM159 cells (C and E) or BT549 cells (D and F). XBP1 s/t, the ratio of XBP1s to total XBP1t. The XBP1 s/t ratio was normalized to that of the scramble (shScr) control. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. All data are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey’s multiple comparison test.

Figure 2. MYC is sufficient for activation of the IRE1/XBP1 pathway.

(A) Schematic representation of the MCF10A^MYC-ER system. In the presence of 4-OHT, MYC-ER fusion protein translocates to the nucleus and transactivates the MYC target genes. (B) Immunoblot and XBP1 splicing assay (RT-PCR) of MCF10A^MYC-ER cells treated with different doses of 4-OHT for 24 hours. MYC-ER, XBP1s, and TBP were detected from nuclear extracts (NE) and IRE1 from whole cell lysates. TBP, actin, and GAPDH were used as loading control. (C and D) qRT-PCR analysis of the expression of IRE1, XBP1s, XBP1 t, XBP1 s/t (C), and XBP1 target genes (D) in MCF10A^MYC-ER cells treated with different doses of 4-OHT for 24 hours. (E and F) The tissue microarray containing specimens from 60 breast cancer patients was subjected to IHC for MYC and IRE1 (DAB staining, brown). (E) Representative photographs are shown indicating weak, moderate, and strong staining. Scale bars: 50 μm. (F) MYC H-score in tissue microarray samples with distinct IRE1 intensities. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. Tissue microarray was performed once, and all other data are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey’s multiple comparison test.

Figure 3. MYC binds to and regulates IRE1 proximal promoter and enhancer.

(A) Track view of MYC ChIP-seq density profile on IRE1 genomic region from published data sets. (B) Schematic diagram of the ChIP primer (P1–P4) locations across the IRE1 promoter region. (C and D) Chromatin extracts from SUM159 cells (C) and MC1 PDX tumors (D) were subjected to ChIP using anti-MYC antibody or normal IgG. Genomic regions of the IRE1 promoter were tested for enrichment of MYC binding. Data are presented relative to input and shown as mean ± SD of technical triplicates. (E) Schematic diagram of the ChIP primer (E1–E4) locations across the IRE1 enhancer region. (F and G) Chromatin extracts from SUM159 cells (F) and MC1 PDX tumors (G) were subjected to ChIP using anti-MYC antibody or normal IgG. Genomic regions of IRE1 intron were tested for enrichment of MYC binding. Data are presented relative to input and shown as mean ± SD of technical triplicates. (H and I) IRE1 promoter (H) or enhancer (I) luciferase reporter was transfected into SUM159 cells infected with lentiviruses encoding scramble control shRNA (shScr) or MYC shRNA (shMYC), and luciferase activity was measured 48 hours after transfection. pGL3-basic or pGL3-promoter is the empty vector control for IRE1 promoter or enhance reporter, respectively. Data are presented relative to Renilla readings and shown as mean ± SD of biological triplicates. All results shown are representative of 3 independent experiments. *P < 0.05; **P < 0.01, 2-tailed unpaired Student’s t test.

Figure 4. MYC interacts with XBP1s and regulates XBP1s transcriptional activity.

(A–C) ChIP assays of SUM159 cells (A) and MC1 PDX tumors (B and C) were performed using anti-MYC or anti-XBP1s antibodies to detect enriched gene-promoter fragments. IgG was used as mock control. Genomic region upstream of VEGFA lacking XBP1s- and MYC-binding sites was used as a negative control (Ctrl). Data are presented relative to input and shown as mean ± SD of technical triplicates. (D) ChIP-re-ChIP assay of SUM159 cells was performed using the anti-MYC antibody first (MYC ChIP). Eluents were subjected to a second ChIP assay using IgG (IgG reChIP) or anti-XBP1s antibody (XBP1s reChIP) to detect enriched gene-promoter fragments. ND, not detected by qPCR assay. Data are shown as mean ± SD of technical triplicates. (E and F) Flag-tagged MYC and HA-tagged XBP1s were coexpressed in 293T cells and coimmunoprecipitation was performed with anti-HA antibody (E) or anti-Flag antibody (F). The immunoblot was probed with anti-Flag and anti-HA antibodies. HA-GFP (E) or Flag-GFP (F) was used as control. (G and H) Nuclear extracts from TM-treated SUM159 cells were subjected to coimmunoprecipitation with anti-XBP1s antibody (G) or anti-MYC antibody (H). The immunoblot was probed with anti-XBP1s and anti-MYC antibodies. (I–L) UPRE or ERSE reporter was cotransfected with MYC or XBP1s or both expression plasmids into BT549 (I and K) or 293T (J and L) cells. Luciferase activity was measured 48 hours after transfection. In I–L, GFP expression plasmid was used as control. Data are presented relative to Renilla readings and shown as mean ± SD of biological triplicates. All results shown are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test (D) or 1-way ANOVA with Tukey’s multiple comparison test (I–L).

Figure 5. MYC hyperactivation is synthetic lethal with XBP1 inhibition.

(A) Clonogenic growth of MCF10A^MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ. (B) Immunoblot of MYC-ER in nuclear extracts of MCF10A^MYC-ER cells treated with 4-OHT for 24 hours. (C) Chemical structure of 8866. (D) XBP1-splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. (E) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. (F) Schematic diagram of fluorescence-based RNA cleavage assay. (G) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. (H) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. (I) Clonogenic growth of MCF10A^MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A^MYC-ER–GFP cells. In A and I, data are presented as mean ± SD of biological triplicates. *P < 0.05; **P < 0.01, 2-tailed unpaired Student’s t test (A) or 1-way ANOVA with Tukey’s multiple comparison test (I).

Figure 6. IRE1 RNase inhibitor 8866 suppresses growth of patient-derived tumors with high MYC expression.

(A) Immunostaining of MYC in human normal mammary gland or PDX tumors. Normal mammary gland without primary antibody incubation was used as negative control. Scale bars: 50 μm. (B) Immunoblot of MYC in PDX models. (C) Schematic of treatment strategy with 8866. (D) Tumor volume quantification of established MC1 PDX tumors in SCID/beige mice treated with vehicle or 8866. Data are presented as mean ± SEM. (E) Kaplan-Meier survival curve of MC1 PDX tumor–bearing mice from treatment start time in vehicle (n = 6) and 8866 (n = 7) treatment groups. (F) RT-PCR analysis of XBP1 splicing in vehicle-treated (n = 6) and 8866-treated (n = 7) MC1 PDX tumor samples harvested at the end of the experiment. (G) qRT-PCR analysis of XBP1 target gene expression in vehicle-treated and 8866-treated MC1 PDX tumor samples harvested at the end of the experiment. Data are presented as mean ± SD of biological replicates, and actin was used as internal control. (H) H&E, cleaved caspase-3, or CD31 immunostaining of MC1 PDX tumors harvested at the end of the experiment. Scale bars: 50 μm. (I and J) Quantification of CD31-positive cells (I) or cleaved caspase-3–positive cells (J) on tumor sections from vehicle-treated (n = 6) or 8866-treated (n = 7) MC1 PDX mice. (K–M) Tumor volume quantification (upper panel) and Kaplan-Meier survival curve (lower panel) of 4913 (K), 2147 (L), and 4195 (M) PDX tumor–bearing mice treated with vehicle or 8866. Data are presented as mean ± SEM. The log-rank test was used to test for the significant differences of survival between the groups (E, K–M). *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Bonferroni’s post test (D, K–M) or 2-tailed unpaired Student’s t test (G, I, J).

Figure 7. 8866 enhances MYC-overexpressing PDX and GEM tumor response to docetaxel chemotherapy.

(A) Schematic of treatment strategy with 8866 with or without docetaxel. (B) Tumor volume quantification of established MC1 PDX tumors in SCID/beige mice treated with vehicle, 8866, docetaxel, or 8866 plus docetaxel (n = 4). Combination treatment of tumor-bearing mice with 8866 plus docetaxel was stopped at day 42. Data shown are representative of 3 independent experiments and presented as mean ± SEM. (C) H&E, immunostaining of CD31 or cleaved caspase-3 (Casp-3), and TUNEL staining of MC1 PDX tumors in different treatment groups harvested 20 days after treatment. Representative images are shown. Scale bars: 50 μm. (D) Quantification of CD31-positive microvessels, cleaved caspase-3–positive cells, or TUNEL-positive cells on tumor sections from different treatment groups. Doc, docetaxel. 6–12 tumor areas from each group were counted. (E) Kaplan-Meier survival curve of MC1 PDX tumor–bearing mice from treatment start time in vehicle, 8866, docetaxel, or 8866 plus docetaxel treatment groups. (F) Immunoblot of Myc in tissue lysates of 2 p53-null GEM models. Actin and GAPDH were used as loading controls. (G and H) Tumor volume quantification of established 2153L (G, n = 6) and T11 (H, n = 5) tumors in BALB/c mice treated with vehicle, 8866, docetaxel, or 8866 plus docetaxel. (I) Model for the role of the IRE1/XBP1 pathway in MYC-driven breast cancer. Oncogenic MYC activates IRE1 transcription and forms a transcriptional complex with XBP1 to facilitate the resolution of MYC-induced proteotoxic stress and the restoration of ER homeostasis. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Bonferroni’s post test (B and H), 1-way ANOVA with Tukey’s multiple comparison test (D) or log-rank test (E).