Blocking the oncoprotein murine double minute 2 (MDM2)–p53 protein–protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2–p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with Ki = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53–MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53. Cancer Res; 74(20); 1–11. ©2014 AACR.

Blocking the MDM2-p53 protein-protein interaction has long been considered by many to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301) that has been advanced into Phase I clinical trials. SAR405838 binds to MDM2 with Ki = 0.88 nM and has high specificity over other proteins. A co-crystal structure of the SAR405838:MDM2 complex shows that in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 osteosarcoma model. Mechanistically, robust transcriptional up-regulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.

RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor α (ERα) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERα, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERα-complex formation, ERα-mediated gene expression, and ERα-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERα-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment. Cancer Res; 74(19); 1–11. ©2014 AACR.

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody–drug conjugates (ADC). For full control over drug–antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled. 131I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ∼1 nmol/L), and their direct covalent conjugation to 89Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of 131I-tubulysin–NHS esters to 89Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM–trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM–trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment. Cancer Res; 74(20); 1–11. ©2014 AACR.

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug-conjugates (ADCs). For full control over drug-antibody-ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 mAb trastuzumab, are radiolabeled. 131I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 >100nM) and the potent TUB-OMOM (IC50 ~1nM), and their direct covalent conjugation to 89Zr-trastuzumab were established. Radioiodination of tubulysins was 92-98% efficient, conversion to NHS-esters >99%, esters were isolated in an overall yield of 68±5% with radiochemical purity of >99.5%. Conjugation of 131I-tubulysin-NHS-esters to 89Zr-trastuzumab was 45-55% efficient, resulting in ADCs with 96-98% radiochemical purity after size-exclusion-chromatography. ADCs were evaluated for their tumor-targeting potential and anti-tumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent anti-tumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.