Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Preclinical studies of RA475, a guanidine-substituted spirocyclic candidate RPN13/ADRM1 inhibitor for treatment of ovarian cancer.

There is an urgent unmet need for more targeted and effective treatments for advanced epithelial ovarian cancer (EOC). The emergence of drug resistance is a particular challenge, but small molecule covalent inhibitors have promise for difficult targets and appear less prone to resistance. Michael acceptors are covalent inhibitors that form bonds with cysteines or other nucleophilic residues in the target protein. However, many are categorized as pan-assay interference compounds (PAINS) and considered unsuitable as drugs due to their tendency to react non-specifically. Targeting RPN13/ADRM1-mediated substrate recognition and deubiquitination by the proteasome 19S Regulatory Particle (RP) is a promising treatment strategy. Early candidate RPN13 inhibitors (iRPN13) produced a toxic accumulation of very high molecular weight polyubiquitinated substrates, resulting in therapeutic activity in mice bearing liquid or solid tumor models, including ovarian cancer; however, they were not drug-like (PAINS) because of their central piperidone core. Up284 instead has a central spiro-carbon ring. We hypothesized that adding a guanidine moiety to the central ring nitrogen of Up284 would produce a compound, RA475, with improved drug-like properties and therapeutic activity in murine models of ovarian cancer. RA475 produced a rapid accumulation of high molecular polyubiquitinated proteins in cancer cell lines associated with apoptosis, similar to Up284 although it was 3-fold less cytotoxic. RA475 competed binding of biotinylated Up284 to RPN13. RA475 shows improved solubility and distinct pharmacodynamic properties compared to Up284. Specifically, tetraubiquitin firefly luciferase expressed in leg muscle was stabilized in mice more effectively upon IP treatment with RA475 than with Up284. However, pharmacologic analysis showed that RA475 was more rapidly cleared from the circulation, and less orally available than Up284. RA475 shows reduced ability to cross the blood-brain barrier and in vitro inhibition of HERG. Treatment of mice with RA475 profoundly inhibited the intraperitoneal growth of the ID8-luciferase ovarian tumor model. Likewise, RA475 treatment of immunocompetent mice inhibited the growth of spontaneous genetically-engineered peritoneal tumor, as did weekly cisplatin dosing. The combination of RA475 and cisplatin significantly extended survival compared to individual treatments, consistent with synergistic cytotoxicity in vitro. In sum, RA475 is a promising candidate covalent RPN13i with potential utility for treatment of patients with advanced EOC in combination with cisplatin

Development and anticancer properties of Up284, a spirocyclic candidate ADRM1/RPN13 inhibitor.

Bortezomib has been successful for treatment of multiple myeloma, but not against solid tumors, and toxicities of neuropathy, thrombocytopenia and the emergence of resistance have triggered efforts to find alternative proteasome inhibitors. Bis-benzylidine piperidones such as RA190 covalently bind ADRM1/RPN13, a ubiquitin receptor that supports recognition of polyubiquitinated substrates of the proteasome and their subsequent deububiqutination and degradation. While these candidate RPN13 inhibitors (iRPN13) show promising anticancer activity in mouse models of cancer, they have suboptimal drug-like properties. Here we describe Up284, a novel candidate iRPN13 possessing a central spiro-carbon ring in place of RA190's problematic piperidone core. Cell lines derived from diverse cancer types (ovarian, triple negative breast, colon, cervical and prostate cancers, multiple myeloma and glioblastoma) were sensitive to Up284, including several lines resistant to bortezomib or cisplatin. Up284 and cisplatin showed synergistic cytotoxicity in vitro. Up284-induced cytotoxicity was associated with mitochondrial dysfunction, elevated levels of reactive oxygen species, accumulation of very high molecular weight polyubiquitinated protein aggregates, an unfolded protein response and the early onset of apoptosis. Up284 and RA190, but not bortezomib, enhanced antigen presentation in vitro. Up284 cleared from plasma in a few hours and accumulated in major organs by 24 h. A single dose of Up284, when administered to mice intra peritoneally or orally, inhibited proteasome function in both muscle and tumor for >48 h. Up284 was well tolerated by mice in repeat dose studies. Up284 demonstrated therapeutic activity in xenograft, syngeneic and genetically-engineered murine models of ovarian cancer

Early and consistent overexpression of ADRM1 in ovarian high-grade serous carcinoma

Abstract Background Ovarian carcinoma is highly dependent on the ubiquitin proteasome system (UPS), but its clinical response to treatment with the proteasome inhibitor bortezomib has been disappointing. This has driven exploration of alternate approaches to target the UPS in ovarian cancer. Recently, proteasome inhibitors targeting the 19S regulatory particle-associated RPN13 protein have been described, such as RA190. RPN13, which is encoded by ADRM1, facilitates the recognition by the proteasome of its polyubiquinated substrates. Inhibition of RPN13 produces a rapid, toxic accumulation of polyubiquitinated proteins in ovarian and other cancer cells, triggering apoptosis. Here, we sought to determine if RPN13 is available as a target in precursors of ovarian/fallopian tube cancer as well as all advanced cases, and the impact of increased ADRM1 gene copy number on sensitivity of ovarian cancer to RA190. Methods ADRM1 mRNA was quantified by RNAscope in situ hybridization and RPN13 protein detected by immunohistochemistry in high grade serous carcinoma (HGSC) of the ovary and serous tubal intraepithelial carcinoma (STIC). Amplification of ADRM1 and sensitivity to RA190 were determined in ovarian cancer cell lines. Results Here, we demonstrate that expression of ADRM1mRNA is significantly elevated in STIC and HGSC as compared to normal fallopian tube epithelium. ADRM1 mRNA and RPN13 were ubiquitously and robustly expressed in ovarian carcinoma tissue and cell lines. No correlation was found between ADRM1 amplification and sensitivity of ovarian cancer cell lines to RA190, but all were susceptible. Conclusions RPN13 can potentially be targeted by RA190 in both in situ and metastatic ovarian carcinoma. Ovarian cancer cell lines are sensitive to RA190 regardless of whether the ADRM1 gene is amplified

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma

Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

<p><i>Left panel:</i> immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. <i>Right panel:</i> Quantification of the Ubiquitin/GAPDH ratios.</p