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The Supplementary Methods show the Synthesis of DIM-C-pPhCF3+MeSO3- (BI1071); Figure S1 shows BI1071 induces apoptosis in different cancer cell lines; Fig. S2 shows BI1071 induces apoptosis in different breast cancer cell lines; Fig. S3 shows BI1071 induces apoptosis in cancer cell lines but not in non-transformed cell lines; Fig. S4 shows BI1071 did't induce the body weight loss; Fi.g S5 shows overexpression of Nur77-LBD enhances the killing effect of BI1071; Fig. S6 shows BI1071 has no effect on other nuclear receptors; Fig. S7 shows the imparied effect of BI1071 could be rescued by re-expression of Bcl-2; Fig. S8 shows BI1071 binding to Nur77 promotes Nur77 interaction with Bcl-2 and mitochondrial localization

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily, induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2 from an antiapoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria. However, small molecules that can bind directly to Nur77 to trigger its mitochondrial localization and Bcl-2 interaction remain to be explored. Here, we report our identification and characterization of DIM-C-pPhCF3+MeSO3− (BI1071), an oxidized product derived from indole-3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2, and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2–dependent manner. Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis. Our results identify BI1071 as a novel Nur77-binding modulator of the Nur77-Bcl-2 apoptotic pathway, which may serve as a promising lead for treating cancers with overexpression of Bcl-2.

Supplementary Figures S1-S6. Z compounds selectively activate Gal4-DBD-RXRα-LBD transcriptional activity in a dose-dependent manner (S1); Nitro group is required for Z-10 and Z-12 binding to RXRα-LBD (S2); Z-10 and Z-12 bind RXRα in a unique manner (S3); Z-10 and Z-12 inhibit TNFα activation of NFκB in RXRα/tRXRα dependent manner (S4); Z-10 and Z-12 inhibit tRXRα-mediated TNFα activation of NFκB (S5); Z compounds and TNFα synergistically induce cancer cell apoptosis in a tRXRα-dependent manner (S6).

Supplementary Table S1. Z serial compounds.

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

Supplementary Table S3. Z-10 and its derivatives.

Supplementary Table S2. Z compounds and their carboxyl derivatives.

Supplementary Methods. Description of additional methods and procedures used in the study.

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily, induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2 from an antiapoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria. However, small molecules that can bind directly to Nur77 to trigger its mitochondrial localization and Bcl-2 interaction remain to be explored. Here, we report our identification and characterization of DIM-C-pPhCF3+MeSO3− (BI1071), an oxidized product derived from indole-3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2, and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2–dependent manner. Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis. Our results identify BI1071 as a novel Nur77-binding modulator of the Nur77-Bcl-2 apoptotic pathway, which may serve as a promising lead for treating cancers with overexpression of Bcl-2.

Retinoid X receptor alpha (RXRα) and its N-terminally truncated version, tRXRα, are widely implicated in cancer development and represent intriguing targets for cancer prevention and treatment. Successful manipulation of RXRα and tRXRα requires the identification of their modulators that could produce therapeutic effects. Here, we report that a class of nitrostyrene derivatives bind to RXRα by a unique mechanism, of which the nitro group of nitrostyrene derivatives and Cys432 of RXRα are required for binding. The binding results in the potent activation of Gal4-DBD-RXRα-LBD transactivation. However, the binding inhibits the transactivation of RXRα homodimer, which might be due to the distinct conformation of RXRα homodimer induced by these nitrostyrene derivatives. Two RXRα point mutants with Cys432 substituted with Tyr and Trp, respectively, could mimic the bindings of two nitrostyrene derivatives and have the ability of autotransactivation. In studying the functional consequences of the binding, we show that these nitrostyrene derivatives could potently inhibit the TNFα/NFκB signaling pathway in a tRXRα-dependent manner. tRXRα promotes TNFα-induced NF-κB activation through its interaction with TRAF2 and enhances TNFα-induced ubiquitination of RIP1, which is strongly inhibited by nitrostyrene derivatives. The inhibition of TNFα-induced NF-κB activation results in the synergistic effect of the combination of nitrostyrene derivatives and TNFα on the induction of cancer cell apoptosis. Together, our results show a new class of RXRα modulators that induce apoptosis of cancer cells through their unique binding mode and new mechanism of action. Cancer Res; 75(10); 2049–60. ©2015 AACR.