An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.

Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

A phase I dose-escalation study to assess safety, tolerability, pharmacokinetics, and preliminary efficacy of the dual mTORC1/mTORC2 kinase inhibitor CC-223 in patients with advanced solid tumors or multiple myeloma.

BACKGROUND: The mammalian target of rapamycin (mTOR) pathway is essential for tumor development, yet mTOR inhibitors have yielded modest results. This phase 1 study investigated the mTORC1/mTORC2 inhibitor CC-223 in patients with advanced cancer. METHODS: Patients with advanced solid tumors or multiple myeloma received an initial dose of 7.5-60 mg of CC-223, followed by oral daily dosing in 28-day cycles until disease progression. The primary objective was to determine the safety, tolerability, nontolerated dosage, maximum tolerated dosage (MTD), and preliminary pharmacokinetic profile. Secondary objectives were to evaluate pharmacodynamic effects and to describe preliminary efficacy. RESULTS: Twenty-eight patients were enrolled and received ≥1 dose of CC-223. The most common treatment-related grade 3 adverse events were hyperglycemia, fatigue, and rash. Four patients had dose-limiting toxicities, including hyperglycemia, rash, fatigue, and mucositis. Therefore, 45 mg/d was determined to be the MTD. The pharmacokinetics of CC-223 demonstrated a mean terminal half-life ranging from 4.86 to 5.64 hours and maximum observed plasma concentration ranging from 269 to 480 ng/mL in patients who received CC-223 ≥45 mg/d. Phosphorylation of mTORC1/mTORC2 pathway biomarkers in blood cells was inhibited by CC-223 ≥30 mg/d with an exposure-response relationship. Best responses included 1 partial response (breast cancer; response duration 220 days; 30-mg/d cohort), stable disease (8 patients across ≥15 mg/d cohorts; response duration range, 36-168 days), and progressive disease (12 patients). The disease control rate was 32%. CONCLUSIONS: CC-223 was tolerable, with manageable toxicities. Preliminary antitumor activity, including tumor regression, and evidence of mTORC1/mTORC2 pathway inhibition were observed.

Design and Synthesis of Novel Cereblon Binders for Use in Targeted Protein Degradation.

Modulating the chemical composition of cereblon (CRBN) binders is a critical step in the optimization process of protein degraders that seek to hijack the function of this E3 ligase. Small structural changes can have profound impacts on the overall profile of these compounds, including depth of on-target degradation, neosubstrate degradation selectivity, as well as other drug-like properties. Herein, we report the design and synthesis of a series of novel CRBN binding moieties. These CRBN binders were evaluated for CRBN binding and degradation of common neosubstrates Aiolos and GSPT1. A selection of these binders was employed for an exploratory matrix of heterobifunctional molecules, targeting CRBN-mediated degradation of the androgen receptor.

MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress.

c-Jun, a major transcription factor in the activating protein 1 family of regulatory proteins, is activated by many physiologic and pathological stimuli. We show here that c-Jun was downregulated in response to osmotic stress via ubiquitination-dependent degradation by the PHD/RING finger domain of MEKK1, which exhibited E3 ubiquitin ligase activity toward c-Jun in vitro and in vivo. The reduced c-Jun protein level resulting from exogenous expression of wild-type MEKK1 and the opposite effect induced by expression of a MEKK1 PHD/RING finger domain mutant were consistent with a higher level of c-Jun protein in MEKK1(-/-) cells than in corresponding wild-type cells. The deficiency of MEKK1 blocked posttranslational downregulation of c-Jun in response to osmotic stress. Furthermore, apoptosis induced by osmotic stress was suppressed by overexpression of c-Jun, indicating that the downregulation of c-Jun promotes apoptosis.

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1-S Checkpoint.

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1-S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1-S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1-S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727-38. ©2018 AACR.

CC-115, a dual inhibitor of mTOR kinase and DNA-PK, blocks DNA damage repair pathways and selectively inhibits ATM-deficient cell growth in vitro.

CC-115, a selective dual inhibitor of the mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase (DNA-PK), is undergoing Phase 1 clinical studies. Here we report the characterization of DNA-PK inhibitory activity of CC-115 in cancer cell lines. CC-115 inhibits auto-phosphorylation of the catalytic subunit of DNA-PK (DNA-PKcs) at the S2056 site (pDNA-PK S2056), leading to blockade of DNA-PK-mediated non-homologous end joining (NHEJ). CC-115 also indirectly reduces the phosphorylation of ataxia-telangiectasia mutated kinase (ATM) at S1981 and its substrates as well as homologous recombination (HR). The mTOR kinase and DNA-PK inhibitory activity of CC-115 leads to not only potent anti-tumor activity against a large panel of hematopoietic and solid cancer cell lines but also strong induction of apoptosis in a subset of cancer lines. Mechanistically, CC-115 prevents NHEJ by inhibiting the dissociation of DNA-PKcs, X-ray repair cross-complementing protein 4 (XRCC4), and DNA ligase IV from DNA ends. CC-115 inhibits colony formation of ATM-deficient cells more potently than ATM-proficient cells, indicating that inhibition of DNA-PK is synthetically lethal with the loss of functional ATM. In conclusion, CC-115 inhibits both mTOR signaling and NHEJ and HR by direct inhibition of DNA-PK. The mechanistic data not only provide selection of potential pharmacodynamic (PD) markers but also support CC-115 clinical development in patients with ATM-deficient tumors.

In vitro SCFbeta-Trcp1-mediated IkappaBalpha ubiquitination assay for high-throughput screen.

An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas.

PURPOSE: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. EXPERIMENTAL DESIGN: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance. RESULTS: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death. CONCLUSIONS: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it.

Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase.

p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.

ERK1/2 MAP kinases in cell survival and apoptosis.

ERK1/2 is an important subfamily of mitogen-activated protein kinases that control a broad range of cellular activities and physiological processes. ERK1/2 can be activated transiently or persistently by MEK1/2 and upstream MAP3Ks in conjunction with regulation and involvement of scaffolding proteins and phosphatases. Activation of ERK1/2 generally promotes cell survival; but under certain conditions, ERK1/2 can have pro-apoptotic functions.

The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2.

ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation were resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity.

Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation.

We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 phosphorylates the amino terminus of MEKK1 on serine 67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH(2)-terminal kinase/stress-activated protein kinases (JNK/SAPK). Serine 67 is constitutively phosphorylated in resting 293 cells, but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is negatively regulated by PAK through phosphorylation of serine 67.