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TTK is an essential spindle assembly checkpoint enzyme in many organisms. It plays a central role in tumor cell proliferation and is aberrantly overexpressed in a wide range of tumor types. We recently reported on a series of potent and selective TTK inhibitors with strong antiproliferative activity in triple negative breast cancer (TNBC) cell lines (8: TTK IC50 = 3.0 nM; CAL-51 IC50 = 84.0 nM). Inspired by previously described potent tricyclic TTK inhibitor 6 (TTK IC50 = 0.9 nM), we embarked on a structure-enabled design and optimization campaign to identify an improved series with excellent potency, TTK selectivity, solubility, CYP inhibition profile, and in vivo efficacy in a TNBC xenograft model. These efforts culminated in the discovery of 25 (TTK IC50 = 3.0 nM; CAL-51 IC50 = 16.0 nM), which showed significant single-agent efficacy when dosed iv in a TNBC xenograft model without body weight loss.

Acute myeloid leukemia (AML) is marked by significant unmet clinical need due to both poor survival and high relapse rates where long-term disease control for most patients with relapsed or refractory AML remain dismal. Inspired to bring novel therapeutic options to these patients, we envisioned protein degradation as a potential therapeutic approach for the treatment of AML. Following this course, we discovered and pioneered a novel mechanism of action which culminated in the discovery of CC-90009. CC-90009 represents a novel protein degrader and the first cereblon E3 ligase modulating drug to enter clinical development that specifically targets GSPT1 (G1 to S phase transition 1) for proteasomal degradation. This manuscript briefly summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and efficacy data for CC-90009, which is currently in phase 1 clinical development.

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.

Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) triggers a “don’t eat me” signal to the macrophage, inhibiting phagocytosis. Thus, overexpression of CD47 enables tumor cells to escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the development and characterization of CC-90002, a humanized anti-CD47 antibody. CC-90002 is unique among previously reported anti-CD47 bivalent antibodies that it does not promote hemagglutination while maintaining high-affinity binding to CD47 and inhibition of the CD47–SIRPα interaction. Studies in a panel of hematological cancer cell lines showed concentration-dependent CC-90002-mediated phagocytosis in acute lymphoblastic leukemia, acute myeloid leukemia (AML), lenalidomide-resistant multiple myeloma (MM) cell lines and AML cells from patients. In vivo studies with MM cell line-derived xenograft models established in immunodeficient mice demonstrated significant dose-dependent antitumor activity of CC-90002. Treatment with CC-90002 significantly prolonged survival in an HL-60-disseminated AML model. Mechanistic studies confirmed the binding of CC-90002 to tumor cells and concomitant recruitment of F4-80 positive macrophages into the tumor and an increase in expression of select chemokines and cytokines of murine origin. Furthermore, the role of macrophages in the CC-90002-mediated antitumor activity was demonstrated by transient depletion of macrophages with liposome-clodronate treatment. In non-human primates, CC-90002 displayed acceptable pharmacokinetic properties and a favorable toxicity profile. These data demonstrate the potential activity of CC-90002 across hematological malignancies and provided basis for clinical studies CC-90002-ST-001 (NCT02367196) and CC-90002-AML-001 (NCT02641002).

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.

Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev. Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. Figure 1 Figure 1. Disclosures Nakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL). Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms. The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals. In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. Figure 1 Figure 1. Disclosures Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Triple negative breast cancer (TNBC) is an aggressive disease with high relapse rates and few treatment options. Outlined in previous publications, we identified a series of potent, dual TTK/CLK2 inhibitors with strong efficacy in TNBC xenograft models. Pharmacokinetic properties and kinome selectivity were optimized, resulting in the identification of a new series of potent, selective, and orally bioavailable TTK inhibitors. We describe here the structure-activity relationship of the 2,4-disubstituted-7 H-pyrrolo[2,3- d]pyrimidine series, leading to significant single agent efficacy in a TNBC xenograft model without body weight loss. The design effort evolving an iv-dosed TTK/CLK2 inhibitor to an orally bioavailable TTK inhibitor is described.

Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.

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.

Introduction: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) with enhanced autonomous cell-killing and immunomodulatory activity against multiple myeloma (MM) cells. CC-92480 is currently in phase 1 development in a late-line myeloma patient population (NCT03374085). Here, we sought to characterize the antitumor activity of CC-92480 in combination with dexamethasone (DEX), bortezomib (BORT), or daratumumab (DARA) in MM cell lines in vitro and xenograft mouse models in vivo. Methods: CC-92480 activity in combination with DEX was evaluated in MM cell lines. Apoptosis was measured by quantification of caspase-3 activation. The effect of BORT on CC-92480-induced Ikaros and Aiolos degradation was determined by concurrent treatment of MM cells with BORT and CC-92480. β5-site proteasome activity was also determined in the same experiment. The in vitro activity of CC-92480 in combination with BORT was characterized using washout experiments to more faithfully model the short in vivo exposure but more prolonged, gradually diminishing proteasome inhibitory activity of BORT. Apoptosis and cell viability of CC-92480 with BORT were analyzed by flow cytometry. The effect of CC-92480 on CD38 expression was also evaluated across a panel of MM cell lines. The effect of CC-92480 in combination with DARA was characterized with antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. CC-92480 in combination with DEX or BORT was tested in a lenalidomide-resistant (H929-1051) xenograft mouse model. Female SCID mice were inoculated with H929-1051 cells in the right hind leg. For the DEX combination, groups of tumor-bearing mice (n = 9-10) were dosed with vehicle, DEX, or CC-92480 once daily (QD), or CC-92480 in combination with DEX throughout the study, starting when the tumor volumes reached approximately 115 mm3. For combination with BORT, mice (n = 9-10/group) were dosed with vehicle, CC-92480, or BORT, or the CC-92480 and BORT combination starting when the tumor volumes reached approximately 500 mm3. CC-92480 was administered orally QD for 3 days and BORT as a single intravenous dose. Tumor volumes were measured twice a week for the duration of the studies. Results: CC-92480 synergized with DEX in reducing cell viability and potentiated DEX-induced apoptosis in a concentration-dependent manner in MM cell lines. Of note, the combination showed activity at concentrations of both DEX and CC-92480 that had minimal activity as single agents. In the xenograft model with H929-1051 cells, the combination of CC-92480 and DEX significantly inhibited tumor growth (−84%) when compared with either agent alone (−34% and −20% for CC-92480 and DEX, respectively) and was classified as a synergistic effect using the fractional product method. Although proteasome activity is required for CC-92480-induced degradation of Ikaros and Aiolos, CC-92480 nevertheless maintained its ability to efficiently degrade Ikaros and Aiolos in the presence of doses of BORT that cause clinically relevant levels of proteasome inhibition. The in vitro combination of CC-92480 with BORT resulted in greater cytotoxic activity on MM cells than either single agent alone. The in vivo efficacy of CC-92480 and BORT, administered concurrently, showed a strongly synergistic effect with a near complete or complete tumor regression in every animal, and 6 of 9 animals remained tumor-free through an observation period extending 157 days after the control group was terminated. Anti-CD38 therapies, including DARA and isatuxumab, target CD38-expressing MM cells for killing by immune cells through cytotoxic and phagocytic mechanisms. In a panel of MM cell lines, CC-92480 treatment caused increased cell surface expression of CD38 (2-3 times that of control). Pretreatment of MM cells with CC-92480 resulted in increased DARA-mediated ADCC and ADCP compared with DMSO-treated controls. Conclusions: The strong preclinical synergy in MM cell killing exhibited by CC-92480 in combination with DEX, BORT, and with an anti-CD38 antibody (DARA), highlights its potential to bring clinical benefit to patients with MM in combination with these agents and supports the rationale for testing these combinations in clinical studies. Disclosures Wong: Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment. Bauer:Celgene Corporation: Employment, Equity Ownership. Groza:Celgene Corporation: Employment, Equity Ownership. Gaffney:Celgene: Employment. Havens:Celgene: Equity Ownership; Pfizer: Employment, Equity Ownership. Choi:AnaptysBio Inc: Employment, Equity Ownership; Celgene Corporation: Equity Ownership, Other: Formerly Employed. Lopez-Girona:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment. Cathers:Celgene Corporation: Equity Ownership; Global Blood Therapeutics (GBT): Employment. Carmichael:Celgene plc: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

A series of 1,1-diarylalkene derivatives were prepared to optimize the properties of CC-5079 (1), a dual inhibitor of tubulin polymerization and phosphodiesterase 4 (PDE4). By using the 3-ethoxy-4-methoxyphenyl PDE4 pharmacophore as one of the aromatic rings, a significant improvement in PDE4 inhibition was achieved. Compound 28 was identified as a dual inhibitor with potent PDE4 (IC(50)=54 nM) and antitubulin activity (HCT-116 IC(50)=34 nM and tubulin polymerization IC(50) ∼1 μM). While the nitrile group at the alkene terminus was generally required for potent antiproliferative activity, its replacement was tolerated if there was a hydroxyl or amino group on one of the aryl rings. Conveniently, this group could also serve as a handle for amino acid derivatization to improve the compounds' solubility. The glycinamide analog 45 showed significant efficacy in the HCT-116 xenograft model, with 64% inhibition of tumor growth upon dosing at 20 mg/kg qd.

Historically, synthetic lethal-based drug discovery has yielded a high percentage of novel, first-in-class drugs while uncovering new tumor biology. Using a synthetic lethal approach, we identified one compound that preferentially induced apoptosis in triple-negative breast cancer (TNBC) cell lines while sparing luminal breast cancer cell lines. The synthetic lethal approach evolved into a targeted approach following the identification of the two kinases that were selectively inhibited by CC-671 in enzyme assays: human protein kinase monopolar spindle 1 (hMps1), also known as TTK, and CDC2-like kinase (CLK2). Detailed CC-671 cellular mechanism of action studies showed phosphorylation inhibition of the direct substrates of TTK and CLK2. The CC-671 IC50 concentration that inhibited phosphorylation of the CLK2 substrate SR protein ranged between 530 and 553 nM in CAL51 cells. Gene Set Enrichment Analyses (GSEA) of RNAseq data suggest that CC-671 CLK2 inhibition induces apoptosis by reducing expression of antiapoptotic isoforms via splicing modification. Cellular CC-671 TTK inhibition was demonstrated by significant reduction of TTK autophosphorylation at T686 and phosphorylation of KNL1, one of the well-characterized TTK substrates. CC-671 treatment also led to a decrease of cyclin B and securin protein level. As a result, the percentage of mitotic cells in nocodazole-arrested CAL51 cells was substantially reduced by CC-671. Furthermore, data from time-lapse video microscopy experiments confirmed the mitotic phase length was reduced by approximately half upon CC-671 treatment. Inhibition of phospho-Histone H3 (Ser 10) following a single CC-671 administration was observed in TNBC in vivo tumor model. More importantly, significant CC-671 in vivo efficacy was demonstrated in a cell line-derived TNBC xenograft model with weekly dosing. The data described here suggest that dual inhibition of TTK and CLK2 represents an attractive therapeutic approach for triple-negative breast cancer. CC-671 presents a unique profile for an anticancer kinase inhibitor. Citation Format: Dan Zhu, Gordafaried Deyanat-Yazdi, David Mikolon, Rama K. Narla, Sophie X. Peng, Yuhong Ning, Jennifer R. Riggs, John F. Boylan. Synthetic lethal approach identifies potent and selective TTK and CLK inhibitor with preclinical anticancer activity in triple-negative breast cancer model [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 B177.

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -β, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) expressed on macrophages results in the inhibition of phagocytosis. Thus, tumor cell overexpression of CD47 enables escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the characterization of CC-90002, a humanized anti-CD47 monoclonal antibody (mAb) that is being developed as a potential therapeutic for hematologic malignancies and solid tumors. CC-90002 has a high affinity for binding to CD47 with a subnanomolar dissociation constant (Kd) value. The IC50 required for the blockade of the CD47-SIRPα interaction was also in the subnanomolar range. CC-90002 is unique among previously reported anti-CD47 antibodies for its inability to promote hemagglutination while maintaining high affinity binding to CD47 and inhibition of the CD47-SIRPα interaction. CC-90002 enabled antibody-mediated phagocytosis of a panel of hematological cancer cell lines in vitro, including ALL, multiple myeloma (MM) and acute myeloid leukemia (AML) cells, and primary AML patient samples. The phagocytosis index for this panel (at 1 μg/mL) ranged from approximately 20% to 60% for all the cell lines tested. Antibody concentration-response studies indicated that the CC-90002 effect was concentration-dependent in the ALL and AML cell lines. CC-90002 treatment significantly enabled the phagocytosis of two lenalidomide-resistant MM cell lines and primary AML cells from three patients. Additionally, CC-90002 treatment elicited the phagocytosis of solid tumor lines, including those from ovarian, breast, head and neck, lung, and pancreatic cancers. The in vivo efficacy of CC-90002 was evaluated across cell line-derived and patient-derived xenograft models. Significant dose-dependent antitumor activity was observed with CC-90002 treatment in the MM cell line-derived xenograft models, RPMI 8226 and the parental NCI-H929 and lenalidomide-resistant NCI-H929 (H929/R1). Treatment with CC-90002 demonstrated significant tumor regression in solid tumor xenografts including a cell line-derived model of triple negative breast cancer (TNBC), MDA-MB-231, and a patient-derived TNBC model, AA1126 and significantly prolonged survival in the HL-60-disseminated AML tumor model. Mechanistic studies in the RPMI 8226 xenograft model confirmed binding of CC-90002 to tumor cells and recruitment of F4-80-positive macrophages into the tumor. RPMI 8226 tumor lysates from CC-90002-treated animals demonstrated an increase in select chemokines and cytokines of murine origin. Taken together, the in vitro and in vivo data demonstrate the potential for activity of CC-90002 across both hematological malignancies and solid tumors. CC-90002 is currently in early clinical development. Citation Format: Rama Krishna Narla, Hardik Modi, Lilly Wong, Mahan Abassian, Daniel Bauer, Pragnya Desai, Bonny Gaffney, Pilgrim Jackson, Jim Leisten, Jing Liu, Antonia Lopez-Girona, Maria Romero, WenQing Yang, Brendan P. Eckelman, Quinn Deveraux, Laurie Phillips, Heather K. Raymon, Laure Escoubet, John Boylan, Kandasamy Hariharan. The humanized anti-CD47 monclonal antibody, CC-90002, has antitumor activity in vitro and in vivo [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 4694. doi:10.1158/1538-7445.AM2017-4694

CD47, also known as integrin-associated protein, is over-expressed in several tumor types, including AML, NHL, breast cancer and multiple myeloma. Elevated expression of CD47 on the cell surface protects tumors from phagocytosis by macrophages through binding to signal regulatory protein alpha (SIRPα) on the surface of macrophages. The CD47-SIRPα interaction triggers events that culminate in the inhibition of the phagocytic process. Macrophages are one of the immune cell types frequently found in the tumor microenvironment and exist as a heterogeneous population that includes both M1 and M2 macrophages. While the spectrum of macrophage subpopulations are likely quite diverse, traditionally, it is thought that M1 macrophages are pro-inflammatory, enhancing immune responses against tumor cells, while M2 macrophages are pro-tumor, since they express a wide array of anti-inflammatory molecules, cytokines and growth factors that promote tumor growth, angiogenesis and an immunosuppressive microenvironment. We evaluated CC-90002 efficacy in the RPMI-8226 multiple myeloma and MDA-MB-231 breast cancer xenograft models and enumerated mouse M1/M2-like macrophage populations within in the tumor before and after C-90002 treatment. In both RPMI-8226 and MDA-MB-231 models, M2 macrophages were the primary resident macrophage. Our studies show that when mice bearing RPMI-8226 multiple myeloma xenografts were treated with CC-90002 (a humanized anti-CD47 antibody), tumor regression was preceded by infiltration of macrophages into the xenograft. In the MDA-MB-231 breast cancer model, resident macrophages appeared to mediate the CC-90002 anti-tumor efficacy in vivo without additional macrophage trafficking. Interestingly, in both models, M1 macrophages appear to mediate CC-90002 efficacy regardless of whether macrophages infiltrate the tumor or are tumor-resident. In the MDA-MB-231 breast cancer model where CC-90002 did not induce infiltration, it is possible that resident tumor macrophages were re-educated to an M1 phenotype. In in vitro experiments using human monocyte-derived macrophages, both M1 and M2 macrophages are able to promote phagocytosis and additionally, we observed that CC-90002 selectively inhibited migration of M2 macrophages toward tumor cell conditioned media. This would presumably shift the overall balance of tumor-associated macrophages toward the M1 phenotype and suggests that inhibiting CD47 can both promote tumor phagocytosis and skew tumor macrophage subpopulations toward an anti-tumor phenotype. CC-90002 is currently being tested in two ongoing Phase I clinical studies in subjects with advanced solid and hematologic cancers (NCT02367196, NCT02641002). Citation Format: Bing Zheng, Piu Wong, WenQing Yang, Rama Narla, Michael Burgess, Laure Escoubet, Heather Raymon, Kandasamy Hariharan, John Boylan, Kristen Hege, Victoria Sung. CC-90002 (anti-CD47 antibody) in vivo anti-tumor activity is associated with an increase in M1-polarized macrophages [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 2009. doi:10.1158/1538-7445.AM2017-2009

The structure of [V(eta5-C5H5)2(CH3C(O)CHC(O)CH3)](O3SCF3) (1) (=[VCp2(acac)](O3SCF3)), a dual-function anti-cancer agent with anti-angiogenic and anti-mitotic properties, was determined by single-crystal X-ray diffraction. The geometry is well described as a pseudo-tetrahedral like structure with the centroids of the cyclopentadienyl rings and the two oxygen atoms of the acetylacetonate ring in the ancillary positions of the central vanadium (IV) atom. The bisector of the V(acac) fragment deviates from the C2 axis of the ligand framework by only 4 degrees, compared to a deviation of 7 degrees for the V(acac) fragment in the tetramethylethano-bridged vanadocene acetyl acetonate complex. Crystal data for 1: space group, P2(1)/c; a=7.5544(9) A, b=14.936(2) A, c=16.193(2) A, beta=102.901(2) degrees, V= 1781.0(4) A3; Z=4; R=0.0506 for 2310 reflections with I> 2sigma(I). This report also details the electron paramagnetic resonance, UV/Vis spectroscopy, electrochemical properties and the biological activity profile of this potent anti-cancer agent.

We present experimental data which establish the organometallic compounds vanadocene dichloride (VDC) and vanadocene acetylacetonate (VDacac) as potent anti-proliferative agents. We first examined the effects of VDC and VDacac on the rapid embryonic cell division and development of Zebrafish. Both compounds were capable of causing cell division block at the 8-16 cell stage of embryonic development followed by total cell fusion and developmental arrest. We next examined the effect of VDC and VDacac on proliferation of human breast cancer and glioblastoma cell lines using MTT assays. VDC inhibited the proliferation of the breast cancer cell line BT-20 as well as the glioblastoma cell line U373 in a concentration-dependent fashion with IC50 values of 11.0, 14.9 and 18.6 microM, respectively. VDacac inhibited cellular proliferation with IC50 values of 9.1, 26.9 and 35.5 microM, respectively. Whereas in vehicle-treated control cancer cells mitotic spindles were organized as a bipolar microtubule array and the DNA was organized on a metaphase plate, vanadocene-treated cancer cells had aberrant monopolar mitotic structures where microtubules were detected only on one side of the chromosomes and the chromosomes were arranged in a circular pattern. In contrast to control cells which showed a single focus of gamma-tubulin at each pole of the bipolar mitotic spindle, VDC- or VDacac-treated cells had two foci of gamma-tubulin on the same side of the chromosomes resulting in a broad centrosome at one pole. All monopolar spindles examined had two foci of gamma-tubulin labeling consistent with a mechanism in which the centrosomes duplicate but do not separate properly to form a bipolar spindle. These results provide unprecedented evidence that organometallic compounds can block cell division in human cancer cells by disrupting bipolar spindle formation. In accordance with these results vanadocene treatment caused an arrest at the G2/M phase of the cell cycle. This unique mechanism of anti-mitotic function warrants further development of vanadocene complexes as anti-cancer drugs.

A novel mono-THF containing synthetic anticancer drug (WHI-261) was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of tubulin. The anti-cancer activity of WHI-261 was confirmed using MTT assays. The structure-based design, synthesis, and biological activity of WHI-261 are reported.

CD74, also known as HLA-DR-associated invariant chain, is a type II transmembrane glycoprotein highly expressed in many B-cell malignancies. The limited expression of CD74 in normal tissues suggests it may be a suitable ADC target for these tumor types. Accordingly, we engineered an anti-CD74 human IgG1 antibody (SP7219) using novel Fab-based ribosome display methods. The selected Fabs were readily reformatted and directly screened as IgGs using Sutro's unique high-throughput, cell-free protein synthesis platform, Xpress CFTM. We then developed novel, potent ADCs, SP7676 and SP7675 (STRO-001), comprised of our lead antibody (SP7219) conjugated to non-cleavable DBCO-maytansinoid linker-warheads with an average drug-antibody ratios (DAR) of 2. We used site-specific conjugation technology which results in a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The sites for conjugation were selected based on highest cell killing activity and stablity in vitro and in vivo. Both ADCs demonstrate potent cell killing activity across multiple B-cell tumor lines in vitro, and anti-tumor activity in preclinical multiple myeloma xenograft models. In vitro cytotoxicity assays show nanomolar potency of STRO-001 in four MM cell lines: Mc/CAR (IC50 0.8 nM), MM.1S (IC50 10-11 nM), U266B1 (IC50 8.5 -9.3 nM), and ARP-1 (IC50 4.3-22 nM). CD74 cell surface expression is required for ADC anti-proliferative effect but the expression level does not seem to correlate with in vitro potency. SP7676 elicited a robust anti-tumor response in the ANBL-6 multiple myeloma xenograft model. Durable regressions were observed in all mice at ≥ 3 mg/kg, with equivalent efficacy (regression) at 3 mg/kg (every 3 days x5) and 10 mg/kg (every 3 days x5 or weekly x3). SP7676 also elicited a clear survival benefit in a disseminated multiple myeloma CAG xenograft model starting at 1mg/kg every 3 days x 5 doses. Similarly, both SP7676 and STRO-001 inhibited the formation of internal visceral tumors in the ARP-1 xenograft model after 3 weekly doses of 3 mg/kg. Evaluation of our lead candidate, STRO-001 in additional MM cell lines and primary patient samples is planned. The tolerability of STRO-001 in non-human primates is under evaluation. STRO-001 was administered to cynomolgous monkeys in an exploratory dose-escalating study up to 30 mg/kg x 2 doses on Day 1 and 15. STRO-001 reduces normal B-cell populations at ≥1 mg/kg after a single dose, providing pharmacodynamic evidence of B-cell targeting while other hematopoietic lineages are mostly affected only at the highest dose studied. Anticipated hematologic toxicities were readily reversible at 1, 3 and 10 mg/kg and target organs of interest were identified. Based on these encouraging data, STRO-001 is advancing to IND-enabling studies for the treatment of CD74 expressing multiple myeloma and other B-cell malignancies. Disclosures Abrahams: Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Narla:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Hoffmann:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. High expression of CD74 has been confirmed in follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and other types of NHL with immunohistochemistry (IHC) using the LL1 antibody (Stein et al. Clin Cancer Res 2007). We employed site-specific conjugation technology to generate novel CD74-targeting ADCs, SP7676 and SP7675 (STRO-001) that exhibit a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The human anti-CD74 IgG1 antibody (SP7219) used for ADCs SP7676 and STRO-001 was engineered using novel Fab-based ribosome display methods enabling selection from ~1012 different antibody variants. Hundreds of unique Fabs from this selection were converted to IgGs and expressed directly in Sutro's proprietary cell-free protein synthesis platform, Xpress CFTM, for extensive screening. The top antibody lead derived from this screen is further tested to identify the best sites for conjugation of linker-warheads. Sutro's SP7219 emerged as the top performing antibody and was conjugated to noncleavable DBCO-maytansinoid linker-warheads to form the ADCs SP7676 and STRO-001. Since conjugation sites were selected based on highest stability both in vitro and in vivo, these novel ADCs lose little drug moiety in circulation and have potential for improved PK, safety and activity profiles. In vitro cell proliferation/cytotoxicity assays show potent activity in 1) DLBCL (germinal center B-cell-like [GCB] and "double-hit") lines: SU-DHL-4, IC50 - 1nM; SU-DHL-6, IC50 - 0.4 nM; WSU-NHL, IC50 - 1.6 nM; Pfeiffer, IC50 - .09 nM; NUDUL-1, IC50 - 0.4 nM; HT, IC50 - 0.7 nM; OCI-LY-19, IC50 - 0.7 nM; WSU-DLBCL2, IC50 - 0.3 nM; 2) mantle cell lymphoma (MCL) cell lines: Mino, IC50 - 0.4-0.7 nM; JVM-2, IC50 - 1.7-2.9 nM; Jeko-1, IC50 - 0.4 - 0.6 nM; 3) Ph+ acute lymphoblastic leukemia (ALL): SUP-B15, IC50 - 3.9-4.6 nM; and 4) CLL (EBV-transformed): JVM-13, IC50 - 3.0-3.4 nM. SP7676 elicited strong anti-tumor response in the OCI-LY-10 lymphoma xenograft model with 100% of animals achieving complete regression of tumors at 3mg/kg every 3 days x 5 doses and 10 mg/kg weekly x 3 doses. In the WSU-DLCL2 "double-hit" lymphoma xenograft model, administration of SP7676 (with re-dosing at time of re-growth) produced tumor regressions at 10 mg/kg every 3 days x 5 (6/8 mice tumor free, remaining 2 with small tumors) and 10 mg/kg weekly x 3 (tumor regression in most animals, 4/8 tumor free). Additionally, STRO-001 exhibits dose-dependent tumor growth inhibition in SUDHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. Exploratory testing of our lead candidate, STRO-001 in cynomologous monkeys showed dose-dependent B-cell depletion at 1 - 30 mg/kg doses on Day 1 and 15, confirming the intended pharmacodynamic effect. Our preliminary data demonstrate that SP7676 and STRO-001 generate potent cell killing activity across multiple B-cell lymphoma/leukemia cell lines in vitro, and anti-tumor activity in preclinical B-cell NHL xenografts. Evaluation of STRO-001 in other cell lines and xenograft models and in combination studies is ongoing. GLP toxicology and other IND-enabling studies are planned. Disclosures Li: Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Brown:Celgene: Employment. Narla:Celgene: Employment. Barnes:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Hoffmann:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

A series of 12 organic arsonic acid compounds has been synthesized and evaluated against human B-lineage (NALM-6) and T-lineage (MOLT-3) acute lymphoblastic leukemia (ALL) cell lines. The lead compounds 2-trichloromethyl-4-[4'-(4"-phenylazo)phenylarsonic acid]aminoquinazoline (compound 19, PHI-P518; IC(50)=1.1+/-0.5 microM against NALM-6 and 2.0+/-0.8 microM against MOLT-3) and 2-methylthio-4-(2'-phenylarsonic acid)aminopyrimidine (compound 15, PHI-P381; IC(50)=1.5+/-0.3 microM against NALM-6 and 2.3+/-0.5 microM against MOLT-3) exhibited potent antileukemic activity at low micromolar concentrations.

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.

A novel mono-THF containing synthetic anticancer drug (WHI-261) was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of tubulin. The anti-cancer activity of WHI-261 was confirmed using MTT assays. The structure-based design, synthesis, and biological activity of WHI-261 are reported.

A series of cysteine diazomethyl- and chloromethyl ketone derivatives has been synthesized and evaluated against human B-lineage (Nalm-6) and T-lineage (Molt-3) acute lymphoblastic leukemia cell lines. The chloromethyl ketone compounds showed potent cytotoxicity against these cell lines, with IC50 values in the low micromolar range. The best compounds were N-acetyl-S-dodecyl-Cys chloromethyl ketone (IC50 = 2.0 microM against Nalm-6, 2.3 microM against Molt-3) and N-acetyl-S-trans,trans-farnesyl-Cys chloromethyl ketone (IC50 = 3.0 microM against Nalm-6 and 1.4 microM against Molt-3).

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC50 value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G2-M phase, increased phosphorylation of G2-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [3H]colchicine for binding to tubulin; however, it does not compete with [3H]paclitaxel (Taxol) or [3H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-α (TNF-α) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC50, 270 nmol/L). This inhibitory effect on TNF-α production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy. (Cancer Res 2006; 66(2): 951-9)

The in vitro cytotoxic activity profile of nine novel phenylarsonic acid (CAS 98-05-5, PAA) compounds against 17 human cancer cell lines including (a) ovarian cancer cell lines ES-2, PA-1, CAOV-3, OVCAR-3, (b) testicular cancer cell lines Ntera-2, Tera-2, N2NICP, 833K, and 64CP, (c) multiple myeloma cell lines ARH77, HS-Sultan, RPMI-8226, and U266, and (d) acute lymphoblastic leukemia (ALL) cell lines NALM-6, MOLT-3, ALL-1, and RS4; 11, was determined by the MTT assay. The lead compounds, 2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine (PHI-370) and 2-methylthio-4-(4'-phenylarsonic acid)-aminopyrimidine (PHI-380) caused apoptotic death in all 17 cancer cell lines at low micromolar concentrations, as documented by TUNEL assays and confocal laser scanning microscopy. PHI-380 was also tested and found to be very active against primary tumor cells isolated from surgical biopsy specimens of 14 patients with therapy-refractory non-small cell lung cancer, breast cancer, colon cancer, lymphoma, hepatoblastoma, or Wilm's tumor as well. Because of their broad-spectrum and potent anticancer activity and ability to induce apoptosis in primary tumor cells from therapy-refractory cancer patients, PAA compounds such as PHI-370 and PHI-380 may provide the basis for effective salvage regimens for patients with recurrent cancer.

Background: IMiDs® Immunomodulatory agent lenalidomide (Len) is active in a range of hematologic cancers including diffuse large B-cell lymphoma (DLBCL). The molecular target of Len is cereblon (CRBN), a substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex. In a CRBN-dependent manner, Len promotes ubiquitination of lymphoid transcription factors Ikaros and Aiolos leading to their subsequent degradation. This was shown to contribute to Len’s anti-Multiple Myeloma and T-cell activation properties. CC-122 is a non-phthalimide analog of thalidomide and has potent anti-proliferative, immunomodulatory and anti-angiogenic activities giving it a potentially broader range of activity than Len. CC-122, a first-in-class pleiotropic pathway modifier (PPM®), is currently in a Phase I clinical trial for DLBCL, multiple myeloma, and solid tumors (ClinicalTrials.gov NCT01421524) and showing encouraging clinical activity. The present study investigated the mechanism of action of CC-122 in pre-clinical models of DLBCL and in clinical samples from the ongoing Phase I study. Results: CC-122 inhibited proliferation (IC50=0.01-1.5μΜ) and induced apoptosis (6.5-12 fold) in 4 ABC and 3 GCB DLBCL cell lines in a dose-dependent manner. CC-122 induced rapid degradation of Aiolos and Ikaros in a CRBN-dependent manner; an effect blocked by MLN4924, a small molecule inhibitor of Cullin-RING E3 ligase complexes. In vivo anti-tumor activity of CC-122 was demonstrated in 2 xenograft models of human DLBCL, and was associated with significant reduction in Aiolos and Ikaros expression (94% and 69%, respectively, p The relationship between Aiolos and Ikaros protein expression and DLBCL cell of origin (COO) was further explored in primary DLBCL tissues. A tissue microarray containing 90 DLBCL patient cases immunohistochemically stained for Aiolos, Ikaros and COO markers (Hans) demonstrated Aiolos and Ikaros expression across GCB and non-GCB subtypes, suggesting that CC-122 may have broad-spectrum activity independent of COO. In fact, Aiolos and Ikaros were degraded in lymph node biopsies from both GCB and non-GCB DLBCL patients administered CC-122. The predictive value of Aiolos and Ikaros protein levels for clinical response will be assessed in the Phase I study. Conclusion: Aiolos degradation-dependent upregulation of ISGs represents a novel mechanism for the activity of CC-122. This mechanism bypasses the interferon-signaling pathway and occurs across cell of origin subtypes, suggesting this is a common mechanism accounting for the broad-spectrum activity of CC-122. Understanding of this mechanism has potential to greatly enhance the clinical development of CC-122 in DLBCL by guiding patient selection strategies and rational combination therapies. Disclosures Hagner: Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Breider:Celgene Corp: Employment. Fontanillo:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Raymon:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Barnes:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Daniel:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Pourdehnad:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership.

Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2014-4517

Bis(4,7-dimethyl-1,10 phenanthroline) sulfatooxovanadium(IV) [VO(SO(4) )(Me(2)-Phen)(2)] induces apoptosis in human NALM-6 leukemia cells. In the present report, we demonstrate that VO(SO(4) )(Me(2)-Phen)(2)-induced apoptosis is mediated through the generation of reactive oxygen species (ROS), depletion of glutathione and depolarization of mitochondrial membrane potential (DeltaPsim). Using multilaser flow cytometry methods, we further mapped out the death sequence that occurs in VO(SO(4))(Me(2)-Phen)(2)-treated leukemic cells. Triple labeling method to measure ROS, DeltaPsim and glutathione coupled with multilaser excitation flow cytometry showed that induction of ROS took place before the loss of mitochondrial permeability transition and depletion of glutathione. Correlated two parameter plots of glutathione content versus DeltaPsim showed that loss of DeltaPsim and depletion of glutathione closely follows each other. Translocation of phosphatidylserine to the outer leaflet of the cell membrane was the final step in the process before the cells became apoptotic. These results demonstrate that the mitochondrial permeability transition takes place during VO(SO(4))(Me(2)-Phen)(2)-induced apoptosis and is mediated through induction of ROS and depletion of glutathione.

A murine model of vascular injury-induced neointimal hyperplasia was developed by using a photoactive dye, rose bengal. Photoactivation of rose bengal induced vascular injury to the femoral arteries of C57B1/6 mice and resulted in an occlusive neointimal hyperplasia after 4 weeks. The cellular elements of the hyperplastic neointima were found to be alpha-actin-positive vascular smooth muscle cells expressing epidermal growth factor (EGF) receptor at high levels. EGF-Gen, an EGF-R-specific inhibitor with potent anticancer activity, suppressed the formation of hyperplastic neointima. Morphometric analysis of serial tissue sections at 4 weeks after vascular injury showed that in 75% of the EGF-Gen-treated mice, the maximal stenosis index was only 0.44 +/- 0.13, whereas in 75% of phosphate-buffered saline (PBS)-treated mice, the maximal stenosis index was 1.20 +/- 0.25. The mean neointima/media ratios for areas of maximum neointimal hyperplasia were 0.59 +/- 0.16 (n = 24) for the EGF-Gen-treated group, 0.99 +/- 16 (n = 45) for the PBS group (EGF-Gen vs. PBS, p = 0.0017), and 1.03 +/- 18 (n = 8) for group treated with unconjugated genistein (EGF-Gen vs. Gen, p = 0.0088). EGF-Gen treatment of mice with vascular injury to the left femoral artery was not associated with any clinical signs of toxicity or histopathologic lesions in any of the organs, including the uninjured right femoral artery. EGF-Gen also inhibited VSMC migration in vitro, without affecting VSMC proliferation and viability, suggesting that EGF-Gen is blocking neointima formation by inhibiting cellular migration to vascular injury sites. In conclusion, EGF-Gen may be useful as a nontoxic prophylactic agent for prevention of restenosis in clinical settings.

A series of cysteine chloromethyl ketone compounds with a systematic variation of the S-alkyl chain length have been synthesized in order to gauge the effect of the alkyl chain length on the cytotoxicity of these compounds against human acute lymphoblastic leukemia cells. Comparable activities were observed for compounds with S-alkyl chains ranging from pentyl to dodecyl, with the best being undecyl (IC50= 1.7 microM) and dodecyl (IC50=2.0 microM) against B-lineage leukemia cells and hexyl (IC50 = 0.7 microM) against T-lineage leukemia cells.

In a systematic search for novel dual function antioxidants with potent anti-HIV activity, we evaluated 9 rationally designed non-nucleoside inhibitors (NNI) of HIV-1 RT for antioxidant and anti-HIV activities. Our lead phenethyl-5-bromopyridyl thiourea (PEPT) compounds, N-(2-(2-methoxyphenylethyl))-N 0 -(2-(5-bromopyridyl))-thiourea (2) and N-(2-(2-chlorophenylethyl))- N 0 -(2-(5-bromopyridyl))-thiourea (9), inhibited the oxidation of ABTS to ABTS + by metmyoglobin in the presence of hydrogen peroxide with EC50 values of 79 and 75 mM, respectively. Both compounds eAectively inhibited the oxidation-induced green fluo- rescence emission from the free radical-sensitive indicator dye 2 0 ,7 0 -dichlorodihydrofluorescein diacetate in CEM human T-cells and Nalm-6 human B-cells exposed to hydrogen peroxide. To our knowledge, compounds 2 and 9 are the first NNI of HIV-1 RT with potent anti-oxidant activity. Furthermore, the activity center was defined as the sulfhydryl group since alkylated PEPT derivatives were inactive. The presence of a free thiourea group was also essential for the anti-HIV activity of the PEPT compounds. # 1999 Published by Elsevier Science Ltd. All rights reserved.

The fusion toxin DT ct GMCSF has been constructed by genetically replacing the native receptor-binding domain of diphtheria toxin (DT) with human granulocyte–macrophage colony stimulating factor (GMCSF). This recombinant fusion toxin preserves the catalytic (c) and membrane translocation (t) domains of DT and includes a sterically neutral peptide linker separating the toxin and growth factor domains. Previous work using DT ct GMCSF produced in Escherichia coli has shown that this chimeric toxin is selectively cytotoxic to GMCSF receptor (R)-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. Its clinical development has been hampered due to very low expression levels, requirements for solubilization with guanidine hydrochloride and subsequent refolding, and concerns about bacterial endotoxin contamination. These difficulties prompted us to investigate the utility of a baculovirus/insect cell expression system for the production of DT ct GMCSF. Here, we report that a soluble form of DT ct GMCSF can be produced in the baculovirus expression vector system (BEVS) and purified to homogeneity by column chromatography. The BEVS-derived DT ct GMCSF fusion toxin caused apoptotic death in GMCSF-R-positive human AML cells at nanomolar concentrations. In contrast to the 100 μg/L yields of purified DT ct GMCSF obtained from E. coli, the BEVS allows us to routinely generate 8–10 mg/L of purified DT ct GMCSF. This increased capacity provided by the BEVS for the production of DT ct GMCSF makes it now possible to obtain sufficient quantities to carry out preclinical and clinical trials. To our knowledge, this is the first report of the successful utilization of the BEVS for producing a therapeutic fusion toxin.

Over expression of the PI3 kinase/mTOR/AKT pathway has been well documented in MM patient biopsies and human MM cell lines, suggesting this pathway plays a key role in the survival and proliferation of malignant plasma cells. Rapamycin and the rapalogs are allosteric inhibitors of the mTORC1 complex (consisting of mTOR, raptor, mLST8 and PRAS40), inducing mainly cytostatic effects but not cell death. Inhibition of mTORC1 prevents a negative feedback loop to the mTORC2 complex (consisting of mTOR, Rictor, mLST8 and Sin 1) leading to the phosphorylation of AKT. Phosphorylated AKT is a key inducer of anti-apoptosis mechanisms and cell cycle progression, which may explain the limited results of the rapalogs in the clinic. Recently developed mTOR kinase inhibitors (i.e., CC-223) target both mTORC1 and mTORC2 complexes in order to inhibit tumor growth and importantly, induce cell death. Here we evaluate the effects of CC-223 on a panel of MM cell lines, in combination with current standard of care agents in MM (the corticosteroid, dexamethasone [DEX] and the IMiD® immunomodulatory drugs, lenalidomide [LEN] and pomalidomide [POM]), as well as in the context of LEN resistance. Single agent CC-223 was shown to inhibit cell proliferation in a panel of 10 MM cell lines achieving IC50 values between 0.1-1 µM following 5 days of treatment. CC-223 also reduced cell viability reaching IC50 values between 0.4-1 µM in 5 out of 10 MM cell lines tested. CC-223 induced concentration-dependent G1 phase arrest within 24h of treatment followed by an induction of cell death by 48h. The anti-MM tumor activity of CC-223 (0.3-10 mg/kg) was further tested in SCID mice with xenotransplants of NCI-H929 grown to approximately 100-150 mm3 in size. A dose-dependent tumor growth inhibition and tumor growth delay was seen with once daily dosing of CC-223. Combination of CC-223 with standard of care therapy compounds was also evaluated in vitro. The combination of CC-223 and DEX demonstrated synergistic effects on the inhibition of cell proliferation in 6 MM cell lines (combination index: 0.0002-0.38) tested over 5 days. CC-223 also had synergistic effects on the same panel of MM cell lines when combined with LEN (combination index: 0.05-0.8). Acquisition of drug resistance in patients receiving standard of care therapies is still one of the major clinical problems in MM. POM, the next generation of IMiD® immunomodulatory agents, has shown clinically meaningful results in patients that are resistant or have relapsed to their drug regimens, including LEN. We have recently developed in vitro cellular models of LEN-resistance using the H929 MM cell line. H929 cells with acquired resistance to LEN (H929 R10-1, R10-2, R10-3 and R10-4) were shown to have one copy number loss of cereblon compared to their matched LEN-sensitive control (H929 D1). In addition to this, protein expression analysis identified that these resistant cell lines also gained the activation of signaling pathways such as PI3K/AKT/mTOR, MEK/MAPK as well as anti-apoptotic factors. For example, S473 AKT phosphorylation was highly elevated in LEN-resistant cell lines which correlated with loss of PTEN protein expression (H929 R10-3 and R10-4). Interestingly, regardless of PI3K/AKT/mTOR pathway status, all LEN-sensitive and resistant H929 cells responded to CC-223 treatment with a strong inhibition of cell proliferation (H929 D1 IC50 0.2 µM, and H929 R10 1-4 IC50 0.2-0.35 µM) and to a lesser effect, induction of cell death, over a 5 day period. Similar to the panel of MM cell lines, G1 arrest occurred after 24h treatment and cell death (Sub-G1) was increased by 72h of treatment. CC-223 treatment reduced S473 pAKT and p-4EBP1 after 1h while total AKT and 4EBP1 remained unchanged in both the sensitive and resistant MM cell lines. Combination treatment of LEN-sensitive and resistant H929 cells with CC-223 and POM had synergistic inhibitory effects on cell proliferation (combination index: 0.35-0.7) and cell viability (combination index: 0.15-0.42). In conclusion, the mTOR kinase inhibitor, CC-223 potently inhibited MM cell proliferation by inducing G1 arrest and cell death in a panel of MM cell lines and reduction of tumor volume in vivo. The combination of LEN, POM or DEX with CC-223 had synergistic effects on MM cell proliferation and viability. Therefore, CC-223 in combination with other standard of care agents could become an important clinical tool for the treatment of MM in the future. Disclosures: Rychak: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Miller:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Raymon:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership.

CC-223 is an orally active selective mammalian target of rapamycin (mTOR) kinase inhibitor which is currently in clinical development. In biochemical assays CC-223 was highly selective to mTOR over 249 kinases. In cellular assays CC-223 inhibited both mTORC1 and mTORC2 as evidenced by inhibition of pS6RP(Ser 235/236) and pAkt(Ser 473). The current study was aimed to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship, antitumor activity, and mechanism of action of CC-223 in mice with PC3 human prostate cancer tumors. In PK/PD studies with PC3 tumor-bearing mice, CC-223 exhibited a dose-dependent inhibition mTOR pathway biomarkers, pS6RP, and pAkt, which correlated with plasma exposure. Antitumor activity of CC-223 was tested in mice with PC3 tumors of approximately 100-150 mm3 in size. CC-223 consistently exhibited dose-dependent tumor growth inhibition. The minimum dose required to obtain >65% tumor volume reduction compared with vehicle control was 5 mg/kg BID. In addition to showing antitumor activity in smaller tumors, CC-223 also caused the regression or growth delay of larger PC3 tumors. At 10 mg/kg BID, CC-223 caused the regression of tumors, whereas 5 and 1 mg/kg BID slowed tumor growth compared to the vehicle control. The mechanism of action of CC-223 on PC3 tumors was characterized by analyzing tumors for functional markers (proliferation, apoptosis, and blood vessel markers). The number of proliferating cells in CC-223-treated tumors was significantly reduced compared to vehicle control. A significant increase in the number of apoptotic cells and a significant decrease in blood vessel density was observed in CC-223-treated tumors. In conclusion, treatment with the mTOR kinase inhibitor CC-223 significantly inhibited PC3 prostate tumor growth. 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. Maintenance of >80% and >60% biomarker inhibition for pS6 and pAkt, respectively, and plasma levels greater than 0.2 μM through 8 hours twice daily confers good antitumor efficacy in PC3 tumors. Further immunohistochemical data demonstrated that the antitumor activity of CC-223 was not only due to the inhibition of tumor cell proliferation but also due to the increase in apoptosis and antiangiogenic activity of CC-223. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A165. Citation Format: Rama Krishna Narla, Sophie Peng, Jim Gamez, Jason Katz, Julius Apuy, Mehran Moghaddam, Kimberly E. Fultz, Sabita Sankar, Deborah S. Mortensen, Heather K. Raymon. Antitumor activity of mTOR kinase inhibitor CC-223 in a mouse model of prostate cancer. [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 A165.