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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.

Supplementary Figures S1-S3, Tables S1-S7. Suppl. Figure S1: Prolif. Curves, PC3 Cell Cycle & solid tumor apoptosis Suppl. Figure S2: PK/PD Model Performance and eIF4E In Vivo Biomarker Suppl. Figure S3: HCT-116, MBA MD231 & A549 Efficacy Studies Suppl. Table S1: Kinase Selectivity Suppl. Table S2: Cellular Biomarker with SEM Suppl. Table S3: Cellular Proliferation with SEM Suppl. Table S4: Hematological Panel Suppl. Table S5: IRF4 Gene and Protein Data Suppl. Table S6: PK/PD Data PK Data Suppl. Table S7: In Vivo Efficacy Across Tumor Types

SUPPLEMENTAL METHODS • Correlative analysis of CC-671 sensitivity data with genomic features in the 240 Oncopanel • Correlative analysis of CC-671 sensitivity data with genomic features or tumor subtypes in 49 breast cancer cell lines • Molecular of action studies • Xenograft efficacy studies SUPPLEMENTAL DATA • Table S1. Primer sequences for PCR • Table S2. Invitrogen kinase panel data • Table S3. Growth inhibition (IC50 and AUCR Values) of CC-671 in breast cancer panel • Table S4. CC-671 effects on cancer cell proliferation, apoptosis, and cell cycle • Table S5. Cell lines in Oncopanel with mutations in CTNNB1 exon 3 • Table S6. Correlation between CC-671 IC50 values and mutations in CTNNB1 exon 3 in cancer cell lines • Table S7. Correlation of CDKN2A and TP53 mutations with sensitivity or resistance to CC-671 in lung and melanoma cell lines from the Oncopanel • Figure S1. CLK2 knockdown by siRNA reduced phosphorylation of SRp75 in CAL51 cells • Figure S2. Induction of apoptosis in vivo with a single dose of CC-671 in MDA-MB-231 tumors • Figure S3. Antitumor activity of CC-671 in CAL51 and MDA-MB-231 TNBC xenograft models • Figure S4. CC-671 potently and selectively inhibits proliferation and induces apoptosis in cancer cell lines from different anatomic origins • Figure S5. Cancer cell lines with CTNNB1 exon 3 mutations tend to be more sensitive to CC-671 in Oncopanel, but the correlation does not reach statistical significance • Figure S6. TTK expression and CC-671 potency correlate with the growth rates (t0) of cell lines in both breast cancer panel and Oncopanel

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. Mol Cancer Ther; 14(6); 1295–305. ©2015 AACR.

PDF file - 2080K, Tunable expression of EGFRvIII in genetically modified models correlates with autophagy induction upon CC214-1 treatment.

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PDF file - 5300K, CC214-1 synergizes at low doses with rapamycin. Quantification of the blots shown in Fig. 2B. PTEN detection on DEAL captured GBM39 cells upon CC214-1 treatment.

PDF file - 3457K, Time course dependent CC214-1 treatment in a panel of isogenic GBM cell lines, U87EGFRvIII, U251 and LN229.

PDF file - 1554K, Cap-dependent translation is enhanced by EGFRvIII expression and CC214-1 strongly inhibits P-4E-BP1 phosphorylation.

PDF file - 2561K, Treatment with CC214-1, leads to induction of autophagy in GBM39 cells and genetic inhibition of autophagy sensitizes U87EGFRvIII cells to apoptosis.

PDF file - 5321K, In vitro and in vivo pharmacological inhibition of autophagy sensitizes U87EGFRvIII cells and orthotopic xenografts to CC214-1/CC214-2 mediated apoptosis.

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. Clin Cancer Res; 19(20); 5722–32. ©2013 AACR.

PDF file - 1694K, EGFRvIII expression sensitizes cells to CC214-1 mediated inhibition of proliferation and to autophagy.

CC-91516 (also called CC0776314), a selective and potent inhibitor of ERK1/2 and NLK, was discovered via a phenotypic screen of a kinase-focused library for compounds that synergize with mTOR kinase inhibitor CC-223 to induce apoptosis in combination with CC-223. Broad kinase selectivity profiling identified ERK1/2 and NLK as targets of CC-91516. Crystal structure of CC-91516 in complex with ERK2 reveals that its 2, 4, 6-trichlorophenyl moiety binds to a unique back pocket of the adenosine-5′-triphosphate (ATP) binding site of ERK2, which is not accessible to other ERK inhibitors such as BVD-523 and GDC-0994. This unique binding mode of CC-91516 leads to a slow off-rate for its ERK binding with long residence time disrupting both the active and inactive ERK forms. Consequentially, CC-91516 causes sustained inhibition of the MAPK pathway in BRAF mutant colorectal cancer cells. In addition, CC-91516 also regulates Wnt/β-catenin and YAP pathways in multiple cancer cell lines. CC-91516 shows potent yet selective anti-proliferative activity against a large panel of cancer cell lines. Activating mutations in BRAF or CTNNB1 gene associate with sensitivity to CC-91516-mediated anti-proliferative activity while mutations in RB and PI3K/PTEN pathway associate with resistance. CC-91516 inhibits ex vivo colony formation of PDX models with BRAF and CTNNB1 mutations. In addition, CC-91516 potently induces apoptosis, inhibits survival, and overcomes resistance to MEK inhibitor trametinib of BRAF or CTNNB1 mutant cancer cells in long-term culture assay in vitro. CC-91516 has good oral bioavailability and shows excellent anti-tumor activity in vivo against both BRAF and CTNNB1 mutant xenograft models. DMPK and toxicology studies showed robust oral exposure across preclinical species. In summary, CC-91516 has demonstrated preclinical anti-tumor activities and DMPK and safety profiles in support of its clinical development. Citation Format: Shuichan Xu, Tam Tran, Dan Zhu, Tao Shi, David Mikolon, Jim Leisten, Philip Chamberlain, Laurie LeBrun, Sogole Bahmanyar, Ning Jiang, Jingjing Zhao, Mehnaz Malek, Ellen Filvaroff, Heather Raymon, Robert Hubbard, John Boylan. Discovery of CC-91516, a potent and selective ERK/NLK inhibitor, with anti-tumor activity in preclinical cancer models harboring BRAF or CTNNB1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1180.

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.

// Toshiya Tsuji 1 , Lisa M. Sapinoso 1 , Tam Tran 1 , Bonny Gaffney 1 , Lilly Wong 1 , Sabita Sankar 1 , Heather K. Raymon 2 , Deborah S. Mortensen 3 and Shuichan Xu 1 1 Oncology Research, Celgene Corporation, San Diego, CA 92121, USA 2 Pharmacology, Celgene Corporation, San Diego, CA 92121, USA 3 Medicinal Chemistry, Celgene Corporation, San Diego, CA 92121, USA Correspondence to: Shuichan Xu, email: sxu@celgene.com Toshiya Tsuji, email: ttsuji@celgene.com Keywords: mTOR kinase, DNA-PK, DNA damage repair, ATM Received: February 17, 2017 Accepted: July 25, 2017 Published: August 18, 2017 ABSTRACT 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.

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.

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

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.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.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%.CC-223 was tolerable, with manageable toxicities. Preliminary antitumor activity, including tumor regression, and evidence of mTORC1/mTORC2 pathway inhibition were observed.

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. Mol Cancer Ther; 14(6); 1295–305. ©2015 AACR.

CC-671 is a potent and selective TTK1 (Mps1) and CLK2 inhibitor. In support of a phase I proof of concept, we used an image-based, multiplex cell assay to evaluate the antiproliferative impact of CC-671 across a broad panel of cancer cell lines representing tumors from various anatomic origins. CC-671 potently and selectively inhibited proliferation and induced apoptosis. Cell lines representing leukemia, lymphoma, colorectal cancer (CRC), head and neck (H&N), and bladder cancers were uniformly sensitive to CC-671 while the cell lines from other tumor types, such as breast and lung cancers, were differentially sensitive to CC-671. Cell cycle analysis using phospho-histone H3 (pHH3) as a mitosis biomarker suggested that CC-671 had cell line-specific mitotic effects. CC-671 accelerated mitotic exit in 120 cell lines while causing mitotic block in 37 cell lines at concentrations < 1 μM. Custom gene expression signatures correlating with sensitivity or resistance were generated. Mapping these signatures to a gene expression database of clinical tumor samples identified additional CC-671 sensitive tumor types including Wilms tumor, superficial bladder cancer, triple-negative, and luminal Btype breast cancer, and several squamous cell carcinomas (lung, esophageal, cervical, H&N). To identify a breast cancer patient selection profile, the CC-671 antiproliferative activity was assessed in a panel of 49 breast cancer cell lines. CC-671 potently inhibited the growth of breast cancer cell lines with 50% inhibitory concentrations (IC50 values) < 100 nM in 14 lines and > 10 μM in 21 lines. Triple-negative breast cancer (TNBC) cell lines were significantly more sensitive to CC-671 than non-TNBC (ER+/PR+ and/or HER2+) lines. Within TNBC, the mesenchymal (M) and basal-like 1 (BL1) subtypes were more sensitive to CC-671. In addition, TNBC lines with cancer stem cell (CSC)-like properties were also sensitive, indicating a potential role of TTK1 and CLK2 in cancer stem cells. Interestingly, mutations in several genes (phosphatidylinositol-4,5-bisphosphate 3kinase 110 kDa catalytic subunit alpha [PIK3CA], serine/threonine kinase 11 [STK11], and phosphatase and tensin homolog [PTEN]) in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway tend to associate with CC-671 TNBC sensitivity. These mutational correlates are being confirmed in additional TNBC lines. Citation Format: Shuichan Xu, Tam Tran, Tao Shi, Ning Jiang, Dan Zhu, Jennifer R. Riggs, John Boylan. Identification of a patient enrichment strategy supporting development of a potent and selective dual TTK/CLK2 inhibitor CC-671 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A043.

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 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.

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.

The protein cereblon is part of the CRL4-CRBN E3 ubiquitin ligase complex, and has been shown to be the molecular target for the drugs lenalidomide and pomalidomide. These drugs bind to the surface of cereblon, triggering the recruitment of substrate proteins to the ligase complex where they can be ubiquitinated and subsequently degraded by the proteosome. By this mechanism, lenalidomide and pomalidomide cause the degradation of the zinc finger transcription factors Ikaros and Aiolos, which mediate the antimyeloma activity of these compounds. Here we describe the discovery of CC-885, a novel cereblon modulator with potent and broad-spectrum antiproliferative activity against a panel of tumor cell lines. Acute myeloid leukemia (AML) cell lines and patient-derived AML cells show particular sensitivity to CC-885 treatment. CC-885 achieves this activity by causing the degradation of G1 to S phase transition 1 (GSPT1), a translation termination factor required for the release of nascent peptides from the ribosome. A crystal structure of cereblon in complex with the ligase adapter protein DDB1, as well as CC-885 and GSPT1, reveals that GSPT1 interacts with both CC-885 and the surface of cereblon. The principal molecular feature on GSPT1 that binds to cereblon is a beta-hairpin incorporating a glycine residue that docks against CC-885. Surprisingly, we found evidence that a similar molecular feature mediates Ikaros recruitment, even though there is no common structural fold or sequence homology other than the key glycine residue. We thereby define the common molecular feature, or degron, shared by the known cereblon neomorphic substrates. We further describe a novel therapeutic target, GSPT1, with promise in cancer such as AML. These results further show that cereblon-mediated protein degradation can be directed against new proteins and that this mechanism enables the targeting of multiple protein classes that may be considered undruggable with conventional approaches. Citation Format: Mary Matyskiela, Gang Lu, Takumi Ito, Barbra Pagarigan, Chin-Chu Lu, Karen Miller, Wei Fang, Nai-Yu Wang, Derek Nguyen, Jack Houston, Gilles Carmel, Tam Tran, Mariko Riley, Lyn'Al Nosaka, Gabriel Lander, Svetlana Gaidarova, Shuichan Xu, Alexander Ruchelman, Hiroshi Handa, James Carmichale, Thomas O. Daniel, Brian E. Cathers, Antonia Lopez-Girona, Philip Chamberlain. Ligand-directed degradation of GSPT1 by a novel cereblon modulator drives potent antitumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY37-02. doi:10.1158/1538-7445.AM2017-SY37-02

Mitogen-activated protein (MAP) kinase cascades are involved in transmitting signals that are generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK), and a MEK kinase (MEKK). Protein-protein interactions within these cascades provide a mechanism to control the localization and function of the proteins. MEKK1 is implicated in activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ERK1/2 MAP kinase pathways. We showed previously that MEKK1 binds directly to JNK/SAPK. In this study we demonstrate that endogenous MEKK1 binds to endogenous ERK2, MEK1, and another MEKK level kinase, Raf-1, suggesting that it can assemble all three proteins of the ERK2 MAP kinase module.

Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast [1] [2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3] [4] [5] [6] [7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

Mitogen-activated protein (MAP) kinases mediate responses to a wide array of cellular stimuli. These cascades consist of a MAP kinase or extracellular signal-regulated kinase (ERK), activated by a MAP/ERK kinase (MEK), in turn activated by a MEK kinase (MEKK). MEKK1 has been shown to be a strong activator of the c-Jun N-terminal kinase/stress-actived protein kinase (JNK/SAPK) pathway. We report here that JNK/SAPK binds directly to the N-terminal, noncatalytic domain of MEKK1 in vitro and in transfected cells. Immobilized MEKK1-derived peptides extract JNK/SAPK selectively from cell lysates. MEKK1 coimmunoprecipitates with multiple JNK/SAPK isoforms in transfected cells. Expression of the N terminus of MEKK1 lacking the kinase domain increases activation of endogenous JNK/SAPK by MEKK1. The data are consistent with a model in which MEKK1-JNK/SAPK binding facilitates the receipt of signals from upstream inputs and localizes JNK/SAPK to intracellular targets of the pathway.

Mitogen-activated protein (MAP)/ERK kinase (MEK)1 and MEK2 are the upstream activators of the MAP kinases, ERK1 and ERK2. MEK1 and MEK2 are approximately 85% identical in sequence but have unique inserts in their C-terminal domains. MEK isoform-specific antibodies were used to examine expression and regulation of each enzyme. MEK1 and MEK2 were expressed in approximately equal amounts in several cell lines; in some, MEK1 was present in slight excess. Activation of tyrosine kinase-containing receptors, heterotrimeric G proteins, and protein kinase C enhanced the activities of both MEK isoforms in 293 and PC12 cells. AIF4-stimulated both MEK1 and MEK2 in PC12 cells expressing a dominant interfering Ras mutant that prevents nerve growth factor-dependent activation of the cascade. Carbachol also stimulated the pathway in these cells. Thus, in addition to their ability to activate Ras/Raf and the downstream ERK pathway, heterotrimeric G proteins also appear to trigger a Ras-independent mechanism to regulate this kinase cascade. In U373, Chinese hamster ovary (CHO), and INS-1 cells, MEK1 was activated by regulators of ERKs, while MEK2 was not. These data suggest that, like the MAP kinases ERK1 and ERK2, in some cell settings the two similar MEK isoforms are differentially regulated.

Post-natal growth of cardiac muscle cells occurs by hypertrophy rather than division and is associated with changes in gene expression and muscle fiber morphology. We show here that the protein kinase MEKK1 can induce reporter gene expression from the atrial natriuretic factor (ANF) promoter, a genetic marker that is activated during in vivo hypertrophy. MEKK1 induced both stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) activity; however, while the SAPK cascade stimulated ANF expression, activation of the ERK cascade inhibited expression. C3 transferase, a specific inhibitor of the small GTPase Rho, also inhibited both MEKK- and phenylephrine-induced ANF expression, indicating an additional requirement for Rho-dependent signals. Microinjection or transfection of C3 transferase into the same cells did not disrupt actin muscle fiber morphology, indicating that Rho-dependent pathways do not regulate actin morphology in cardiac muscle cells. While active MEKK1 was a potent activator of hypertrophic gene expression, this kinase did not induce actin organization and prevented phenylephrine-induced organization. These data suggest that multiple signals control hypertrophic phenotypes. Positive and negative signals mediated by parallel MAP kinase cascades interact with Rho-dependent pathways to regulate hypertrophic gene expression while other signals induce muscle fiber morphology in cardiac muscle cells.

Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, α-SAPK, and p38 and their upstream activators, MEK1, −2, −3, and −4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and α-stress-activated protein kinase could also be inactivated by either phosphatase, and α-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 μmol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.

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. Clin Cancer Res; 19(20); 5722–32. ©2013 AACR.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

The prototype mitogen-activated protein (MAP) kinase module is a three-kinase cascade consisting of the MAP kinase, extracellular signal-regulated protein kinase (ERK) 1 or ERK2, the MAP/ERK kinase (MEK) MEK1 or MEK2, and the MEK kinase, Raf-1 or B-Raf. This and other MAP kinase modules are thought to be critical signal transducers in major cellular events including proliferation, differentiation, and stress responses. To identify novel mammalian MAP kinase modules, polymerase chain reaction was used to isolate a new MEK family member, MEK5, from the rat. MEK5 is more closely related to MEK1 and MEK2 than to the other known mammalian MEKs, MKK3 and MKK4. MEK5 is thought to lie in an uncharacterized MAP kinase pathway, because MEK5 does not phosphorylate the ERK/MAP kinase family members ERK1, ERK2, ERK3, JNK/SAPK, or p38/HOG1, nor will Raf-1, c-Mos, or MEKK1 highly phosphorylate it. Alternative splicing results in a 50-kDa alpha and a 40-kDa beta isoform of MEK5. MEK5 beta is ubiquitously distributed and primarily cytosolic. MEK5 alpha is expressed most highly in liver and brain and is particulate. The 23 amino acids encoded by the 5' exon in the larger alpha isoform are similar to a sequence found in certain proteins believed to associate with the actin cytoskeleton; this alternatively spliced modular domain may lead to the differential subcellular localization of MEK5 alpha.

Summary 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.

The extracellular signal-regulated kinases ERK1 and ERK2 are 43- and 41-kd enzymes activated by many extracellular cues. They lie within a protein kinase cascade that is used to achieve many cellular responses. In addition to the wide variety of regulatory contexts in which they are activated, they phosphorylate important regulatory proteins, including receptors, transcription factors, cytoskeletal proteins, and other protein kinases. Thus, the stimulation of this kinase cascade is thought to have a pleiotropic action. ERK1 and ERK2 are controlled by phosphorylation on threonine and tyrosine. To understand the regulatory mechanisms, wild-type and mutant ERKs were expressed in bacteria and phosphorylated with MEK, the enzyme that is upstream of ERKs. Wild-type proteins could be activated 500- to 1,000-fold in vitro by MEK. ERK3, an enzyme of 62 kd and only 50% identical to ERK1 and ERK2 in the catalytic core, was also phosphorylated by MEK in vitro. This suggests that all three of these enzymes are targets of common signaling pathways.

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.

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.

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.

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.

Introduction: PIK3CA and PTEN are among the top five most frequently mutated genes in breast cancer. Activating mutation of PIK3CA or loss of function mutation in PTEN leads to constitutive activation of AKT and mTOR driving multiple downstream metabolic and proliferative pathways important to oncogenesis. There is also cross-talk between the PI3K/AKT/mTOR pathway and ER signaling and activation of the PI3K/AKT/mTOR pathway is associated with resistance to endocrine therapy. CC-223 is a potent and selective ATP-competitive mTOR kinase inhibitor, targeting both TORC1 and TORC2 complexes. Results: Pre-clinically, CC-223 potently inhibits the growth of breast cancer cell lines with GI50 values below 1 uM in 38 of 43 lines. Luminal-derived cell lines are more sensitive to CC-223 than basal-derived lines. Within the luminal subset, ER+, HER2+, PIK3CA mutant, or wild-type TP53 cell lines are more sensitive to CC-223; PTEN loss is not associated with increased CC-223 sensitivity. During the dose escalation part of the phase I clinical trial, 3 pts with breast cancer were enrolled. One pt with HR+/Her2- breast cancer had a durable PR (31 weeks). An expansion cohort of HR+/Her2- breast cancer enrolled 17 pts at a CC-223 dose of 45 mg QD in 28-day cycles and 13 pts were evaluable for tumor response. Deep sequencing of tumors for multiple cancer-related genes was performed. The most common (> 20%) related adverse events (all grades) reported in the breast cancer cohort were nausea, stomatitis, fatigue, anorexia, diarrhea, vomiting, hyperglycemia, rash, and thrombocytopenia. Exposure-dependent TORC1 (p4EBP1) and TORC2 (pAKT) inhibition was observed in blood cells; analysis of paired tumor biopsies is ongoing. Reduction in glucose uptake (> 25% decrease in SUV) on PET imaging at day 15 was observed in 4 of 8 patients with PET imaging data currently available. Three pts demonstrated RECIST PR in target lesions (one categorized as PD due to a new bone lesion); PIK3CA mutations were present in all 3 subjects. Of the two additional pts with PIK3CA mutations, one had SD > 6 months. The one PIK3CA mutated subject with PD at first restaging had a concurrent p53 mutation. Additional genetic abnormalities in mTOR and related pathways in subjects with target lesion PR involved PTEN, Rictor, and IGFR1 genes. Six subjects had SD after 2 cycles, with minor target lesion regression (0 to -30%) in 5 of 6, and with SD > 24 weeks in 1 of 6. Conclusion: The safety profile of CC-223 is typical for drugs targeting the mTOR pathway. Preclinical and clinical data support the activity of CC-223 in HR+ positive breast cancer, particularly in tumors with PIK3CA mutations. Breast Cancer Accrual: Cedars-Sinai (Mita): 4; SCRIL (Arkenau): 4; IGR (Varga): 4; SCRI (Bendell): 3 (all Part A); Moffitt (Mahipal): 2; UCSF (Munster): 1; JSOM (Paz-Ares): 1; ICR (DeLord): 1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A68. Citation Format: Monica M. Mita, Hendrik-Tobias Arkenau, Johanna C. Bendell, Pamela N. Munster, Amit Mahipal, Jean-Pierre Delord, Luis G. Paz-Ares, Jean-Charles Soria, Shuichan Xu, Tam Tran, Tao Shi, Xiaoling Wu, Rajesh Chopra, Kristen Hege, Andrea Varga. Activity of the TORC 1/2 kinase inhibitor, CC-223, in hormone receptor positive (HR+) breast cancer cell lines and patients (pts) with genetically characterized HR+ breast cancer in a Phase I clinical trial. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A68.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.

2606 Background: CC-223 is an ATP-competitive inhibitor of the mTOR kinase, including both TORC1 and TORC2. CC-223 was selected to address resistance of rapamycin analogues mediated by TORC2 activation. Methods: Following establishment of the MTD (reported at ASCO 2012), subjects with select advanced, refractory solid tumors, including NSCLC, HCC, NET, GBM and breast were enrolled in expansion cohorts of up to 20 evaluable subjects. CC-223 was dosed at 45 mg once daily in 28 day cycles until disease progression. Results: As of 09 January, 2013, 101 solid tumor subjects have been treated, including NSCLC (26), HCC (25), NET (23), breast (14), and GBM (13). Results from the NSCLC, HCC, and GBM cohorts are reported here; NET results are reported separately. The most common (> 20%) related adverse events (all grades) were fatigue, rash, stomatitis, hyperglycemia, anorexia, nausea, vomiting and diarrhea. In addition, related serious adverse events included infection (1), pneumonitis (4), renal insufficiency (2) and pancreatitis (2). CC-223 dose reduction was required in > 50% of subjects with NSCLC and HCC, usually during cycle 1 or 2. Exposure-dependent TORC1 (p4EBP1) and TORC2 (pAKT) inhibition was observed across cohorts. mTOR pathway inhibition and/or decreased proliferation was demonstrated in paired tumor biopsies, but results were inconsistent. Reduction in glucose uptake (> 25% decrease in SUV) on PET imaging at day 15 was observed in 78% (14/18) of NSCLC and 69% (11/16) of HCC subjects. Partial tumor responses were observed in evaluable subjects with NSCLC (1/17; confirmed, treatment duration 36 weeks) and HCC (3/15; 1 confirmed, treatment duration 15 – 26 weeks). Disease control rate in the overall NSCLC cohort was 42% (11/26) and in the HCC cohort, 40% (10/25). GBM subjects underwent salvage resections on study and none were progression-free at 6 months. CC-223 was present in all (11/11) resected GBM tumors with plasma:tumor ratios of 16 - 77%, confirming transit across the blood-brain barrier. Conclusions: Encouraging signals of biomarker and clinical activity were observed in HCC and NSCLC. Due to the frequency of dose reductions, select additional cohorts will be enrolled at a starting dose of 30 mg QD. Clinical trial information: NCT01177397.

8522 Background: CC-223 is an ATP-competitive inhibitor of the mTOR kinase, including both TORC1 and TORC2 complexes. CC-223 was selected to address resistance of rapamycin analogues mediated by TORC2 activation. Methods: Following establishment of the MTD (reported at ASCO 2012), subjects with advanced DLBCL, MM and select solid tumors were enrolled in parallel expansion cohorts of up to 20 evaluable subjects. CC-223 was dosed at 45 mg once daily in 28 day cycles until disease progression. Results: As of 09 January, 2013, 35 subjects were enrolled including DLBCL (21) and MM (14). Results in solid tumor cohorts are reported separately. The most common (> 20%) related adverse events (all grades) were fatigue, hyperglycemia, rash, anorexia, nausea, vomiting and diarrhea. In addition, related serious adverse events included infection (2), pneumonitis (1), renal insufficiency (2), pancreatitis (1) and thrombocytopenia (1). CC-223 dose reduction was required in 9 subjects (27%), usually during cycle 1 or 2, and 4 additional subjects with DLBCL dropped out during cycle 1 due to toxicity. Systemic exposure was similar between the two tumor cohorts and was associated with inhibition of TORC1 (p4EBP1) and TORC2 (pAKT) biomarkers in blood cells. Reduction in glucose uptake (32 – 98% decrease) on PET imaging at day 15 was observed in 7/7 DLBCL subjects with results currently available. Three of 17 evaluable subjects with DLBCL had partial responses (PR) after 2 cycles; two with rapid and near complete regression (> 90%) of target lesions by CT and PET with PR confirmed and treatment ongoing at 6 and 8 cycles. Both had failed multiple prior treatment regimens (5 and 3, respectively), including autologous stem cell transplant (ASCT). No responses were observed in 10/14 evaluable subjects with MM although 2 subjects have prolonged stable disease (SD) with treatment ongoing at 12 and 14 cycles. Conclusions: Encouraging signals of biomarker and clinical activity were observed in DLBCL, including two near complete responses, which are ongoing. Due to dose reductions and interruptions, a starting dose of 30 mg QD is recommended for future studies. Clinical trial information: NCT01177397.

CC-223 is a potent and selective mTOR kinase inhibitor currently undergoing Phase 1 clinical evaluation. We evaluated combinatorial effect of CC-223 with 5-Aza and erlotinib at three different ratios of the two combining agents in three different orders of addition in eight NSCLC lines. Combinational indices (CIs) were calculated based upon published method (Chou and Talalay, 1984). CC-223 synergized with azacitidine (CI Citation Format: Tam Tran, Kristen Hege, Deborah Mortensen, Heather Raymon, Shuichan Xu. CC-223, a selective and potent mTOR kinase inhibitor, synergizes with 5-Aza and Erlotinib in eight NSCLC lines in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2013-5252

Three-dimensional (3D) in vitro models have been used in cancer research to overcome limitations of 2D systems. 3D in vitro models recapitulate many aspects of actual tumors including hypoxia, metabolism, proliferation gradients, cell-cell and cell-matrix interactions and closer clinical gene expression profiles than those seen in 2D. Importantly, some cancer drugs showed high potency only in the 3D environment compared to 2D (Muranen, et al, Cancer Cell, 2012). In order to identify improved cancer drugs, we established a simple and versatile 3D spheroid culture approach for high-throughput screening for compounds that selectively kill cancer cells with certain genetic backgrounds. We used HCT116 (parental: HCT116/KRas-mut) and an isogenic counterpart that expresses a genetically modified KRas gene (HCT116/KRas-wt) for 3D cell culture. To validate this approach, HCT116/KRas-mut and HCT116/KRas-wt were cultured separately, treated with 15 selected tool compounds and viability was measured using a caspase 3/7 assay. We found 4 compounds that induced caspase 3/7 activity only in 3D culture of HCT116/KRas-mut and not in 2D. Furthermore, 5 compounds were predominantly sensitive to HCT116/KRas-wt cells in 3D culture, but not in HCT116/KRas-mut 3D culture. These results indicate that the more physiological relevant 3D synthetic lethal screen is an attractive approach to cancer drug discovery and may identify compounds missed in conventional 2D culture model screens. Citation Format: Toshiya Tsuji, Lisa M. Sapinoso, I-Lin Lin, Shuichan Xu, John F. Boylan. Development of a 3-dimensional synthetic lethality screening approach targeting KRas-mut cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3839. doi:10.1158/1538-7445.AM2013-3839