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Supplementary Tables S1-S6 and Supplementary Figures S1-S2. Table S1: Data collection and refinement statistics, related to Figure 1B; Table S2: Summary of cell line related information; Table S3: BGB-283 is a reversible B-RAF inhibitor; Table S4. Kinase inhibition profile of BGB-283. BGB-283 selectively inhibited multiple kinases at concentration of 10 μM; Table S5: IC50 values of BGB-283 in inhibiting various kinases; Table S6. Anti-tumor efficacy of BGB-283 in CRC xenograft models; Figure S1: BGB-283 is a slow-off B-RAF inhibitor showing potent anti-EGFR activity in multiple cell lines; Figure S2. The effect of BGB-283 on body weight of nude mice subcutaneously implanted with human cancer xenografts.

Supplementary Methods, Figures 1-9 from Modulation of Gene Expression and Tumor Cell Growth by Redox Modification of STAT3

The mutation of K‐RAS represents one of the most frequent genetic alterations in cancer. Targeting of downstream effectors of RAS, including of MEK and ERK, has limited clinical success in cancer patients with K‐RAS mutations. The reduced sensitivity of K‐RAS‐mutated cells to certain MEK inhibitors (MEKi) is associated with the feedback phosphorylation of MEK by C‐RAF and with the reactivation of mitogen‐activated protein kinase (MAPK) signaling. Here, we report that the RAF dimer inhibitors lifirafenib (BGB‐283) and compound C show a strong synergistic effect with MEKi, including mirdametinib (PD‐0325901) and selumetinib, in suppressing the proliferation of K‐RAS‐mutated non‐small‐cell lung cancer and colorectal cancer (CRC) cell lines. This synergistic effect was not observed with the B‐RAFV600E selective inhibitor vemurafenib. Our mechanistic analysis revealed that RAF dimer inhibition suppresses RAF‐dependent MEK reactivation and leads to the sustained inhibition of MAPK signaling in K‐RAS‐mutated cells. This synergistic effect was also observed in several K‐RAS mutant mouse xenograft models. A pharmacodynamic analysis supported a role for the synergistic phospho‐ERK blockade in enhancing the antitumor activity observed in the K‐RAS mutant models. These findings support a vertical inhibition strategy in which RAF dimer and MEKi are combined to target K‐RAS‐mutated cancers, and have led to a Phase 1b/2 combination therapy study of lifirafenib and mirdametinib in solid tumor patients with K‐RAS mutations and other MAPK pathway aberrations., The reduced sensitivity of K‐RAS‐mutated cancer cells to MEK inhibitors (MEKi), such as selumetinib and mirdametinib, is associated with feedback phosphorylation of MEK by upstream RAF reactivation. RAF dimer inhibitors, such as lifirafenib and compound C, in combination with MEKi potently suppress RAF‐dependent MEK phosphorylation and lead to sustained inhibition of MAPK signaling and tumor growth.

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at + 324 bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

K-RAS mutation represents one of the most frequent genetic alterations in cancers. Monotherapy approaches targeting RAS downstream effectors such as MEK and ERK have had limited clinical success in patients with K-RAS-mutated cancers. The reduced sensitivity of K-RAS-mutated cells to certain MEK inhibitors is associated with feedback phosphorylation of MEK and reactivation of mitogen-activated protein kinase (MAPK) signaling. Herein, we report that RAF dimer inhibitor lifirafenib (BGB-283) shows strong synergistic effect with MEK inhibitor mirdametinib (PD-0325901) in suppressing proliferation of K-RAS-mutated cancer cell lines. This synergistic effect was not observed using vemurafenib, a first-generation B-RAFV600E selective inhibitor. Mechanistic analysis revealed that RAF dimer inhibition could suppress RAF-dependent MEK reactivation and led to sustained inhibition of MAPK signaling in K-RAS-mutated cells. Furthermore, combination synergy was observed in K-RAS mutant xenograft models. Pharmacodynamic analysis supported the role of synergistic phospho-ERK blockade in enhancing the antitumor activity in the K-RAS mutant models. These findings support the rationale to combine a RAF dimer inhibitor and a MEK inhibitor to treat K-RAS-mutated cancers and led to the ongoing a Phase Ib/II clinical trial of lifirafenib and mirdametinib in patients with K-RAS mutations or other MAPK pathway aberrations (Clinical Trial ID: NCT03905148). Citation Format: Xi Yuan, Xinwen Zhang, Rong Du, Shing-Hu Cheung, Min Wei, Changyou Zhou, Lai Wang, Todd Shearer, Badreddin Edris, Mary Smith, Zhan Yao, Claire N. Thant, Neal Rosen, Lusong Luo. RAF dimer inhibitor lifirafenib enhances the antitumor activity of MEK inhibitor mirdametinib in RAS mutant tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6415.

Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between −806 bp and −756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.

Oncogenic BRAF, which drives cell transformation and proliferation, has been detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating BRAFV600E metastatic melanoma, their clinical efficacy in BRAFV600E colorectal cancer is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon BRAF inhibition might contribute to the relative unresponsiveness of colorectal cancer to the first-generation BRAF inhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigation. In vitro, BGB-283 potently inhibits BRAFV600E-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAFV600E and EGFR mutation/amplification. In BRAFV600E colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. In vivo, BGB-283 treatment leads to dose-dependent tumor growth inhibition accompanied by partial and complete tumor regressions in both cell line-derived and primary human colorectal tumor xenografts bearing BRAFV600E mutation. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating colorectal cancer harboring BRAFV600E mutation. Mol Cancer Ther; 14(10); 2187–97. ©2015 AACR.

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. Here, we tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE) located at +324bp from the ADTRP transcription start site. The ARE is required for the transcriptional activation of ADTRP . HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was reduced by ADTRP siRNA and enhanced by overexpression of ADTRP . These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

The Signal Transducer and Activator of Transcription 3 (STAT3) is necessary for ES cell renewal, plays critical roles during vertebrate development, and has oncogenic potential. STAT3 also mediates cytokine responses notably in the induction of acute phase response genes in the liver. Thus STAT3 is a pleiotropic regulator during cell proliferation and a cell-specific mediator of pro-inflammatory responses. How STAT3 fulfils both roles is unclear. To address this question we attempted to characterise pre-initiation complexes (PICs) on STAT3-responsive promoters containing the c-myc P2 promoter element (P2E) or c-fos Serum-Inducible Element (SIE). Although both promoters mediated cytokine responses in HepG2 cells, poor binding of STAT1 and STAT3 in vitro precluded isolation of active promoter complexes on the P2E. The inability of STAT3 to bind the P2E in vitro correlated with failure of the P2E to mediate cytokine-responsive gene expression in several other cell types. Thus the c-myc P2E behaves as a dual-purpose STAT3 element with anomalous characteristics in HepG2 cells.

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.

Reactive oxygen species (ROS) promote tumor cell proliferation and survival by directly modulating growth-regulatory molecules and key transcription factors. The signal transducer and activator of transcription 3 (STAT3) is constitutively active in a variety of tumor cell types, where the effect of ROS on the Janus kinase/STAT pathway has been examined. We report here that STAT3 is directly sensitive to intracellular oxidants. Oxidation of conserved cysteines by peroxide decreased STAT3 binding to consensus serum-inducible elements (SIE) in vitro and in vivo and diminished interleukin (IL)-6–mediated reporter expression. Inhibitory effects produced by cysteine oxidation in STAT3 were negated in redox-insensitive STAT3 mutants. In contrast, ROS had no effect on IL-6–induced STAT3 recruitment to the c-myc P2 promoter. Expression of a redox-insensitive STAT3 in breast carcinoma cells accelerated their proliferation while reducing resistance to oxidative stress. Our results implicate STAT3 in coupling intracellular redox homeostasis to cell proliferation and survival. Cancer Res; 70(20); 8222–32. ©2010 AACR.

Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in approximately 70% of human malignant melanomas and 5-15% of colorectal cancers (CRC). B-RAFV600E mutation, which gives rise to constitutive MAPK signaling, accounts for at least 90% of oncogenic B-RAF mutations. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating B-RAFV600E metastatic melanoma, their clinical efficacy in B-RAFV600E CRC is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon B-RAF inhibition contributed to the intrinsic resistance of CRC to the first generation B-RAF inhibitors. This report represents the first characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigations. BGB-283 is a potent and selective pan-RAF and EGFR inhibitor with IC50 ranging from 5 to 47 nM on RAF and EGFR kinases. In vitro, BGB-283 potently inhibits B-RAFV600E-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harbouring B-RAFV600E and EGFR mutation/amplification. In B-RAFV600E CRC cell lines, BGB-283 effectively inhibits the reactivation of EGFR and achieved sustained inhibition of MAPK pathway. In vivo, BGB-283 is highly efficacious in inhibiting tumor growth accompanied by partial and complete tumor regressions in both BRAFV600E mutant cell derived CRC xenograft models, including HT29, Colo205, WiDr, as well as two primary human CRC xenograft models. In particular, BGB-283 shows compelling efficacy and potent inhibition of EGFR/MAPK signaling in WiDr xenograft model where EGFR reactivation occurs upon B-RAF inhibition. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating CRC harboring B-RAFV600E mutation. Citation Format: Zhiyu Tang, Xi Yuan, Rong Du, Shing-Hu Cheung, Guoliang Zhang, Jing Wei, Yuan Zhao, Yingcai Feng, Yi Zhang, Yunguang Du, Xiaoxia Hu, Wenfeng Gong, Yong Liu, Yajuan Gao, Rui Hao, Jiafu Ji, Lianhai Zhang, Shuangxi Li, David Sutton, Min Wei, Changyou Zhou, Lai Wang, Lusong Luo. BGB-283, a novel RAF kinase and EGFR dual inhibitor, displays potent antitumor activity in B-RAF mutated colorectal cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4692. doi:10.1158/1538-7445.AM2015-4692

Mutation in K-RAS, which drives constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, is one of the most frequent genetic alterations in human cancers. Pharmacological targeting RAS mutants directly is extremely challenging and targeted inhibition of K-RAS downstream effector MEK has shown limited clinical activity in suppressing the progression of K-RAS mutant tumors. The reduced sensitivity of K-RAS-mutated cancer cells to MEK inhibitors (MEKi) is associated with feedback phosphorylation of MEK and rebound of phosphorylated ERK levels after prolonged treatment. A synthetic lethal screen provided the rational of targeting both RAF1 and MEK in K-RAS mutated cancers. In this study, we investigated whether combining BGB-283, a potent inhibitor to RAF dimers, and a series of MEKi could achieve better therapeutic effect of the latter. We confirmed that selumetinib (AZD6244) induced strong MEK phosphorylation in K-RAS mutated cancer cells. Either knockdown of B-RAF/C-RAF or addition of BGB-283 in the presence of selumetinib potently inhibited RAF-dependent MEK phosphorylation and led to sustained inhibition of ERK activity. Furthermore, BGB-283 and selumetinib showed synergistic effect in inhibiting proliferation of several non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines harboring RAS mutations. In vivo, combined dosing of BGB-283 and AZD6244 also exhibited enhanced antitumor activity in K-RAS mutant NSCLC and CRC xenograft models compared to monotherapies. Pharmacodynamic (PD) analysis revealed improved inhibition of ERK phosphorylation in dually treated tumors. Together, these findings suggested that combining RAF dimer inhibitor BGB-283 and MEKi could be a promising strategy in treating RAS mutated cancers. Citation Format: Xi Yuan, Zhiyu Tang, Rong Du, Shing-Hu Cheung, Jing Wei, Yuan Zhao, Yunguang Du, Rui Hao, Xiaoxia Hu, Wenfeng Gong, Yong Liu, Yajuan Gao, Min Wei, Changyou Zhou, Lai Wang, Lusong Luo. BGB-283 effectively enhances MEK inhibitor induced tumor suppression in RAS mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2015-669