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LY3295668 chemical characterization

Supplementary Materials and Methods

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

shRNA suppressor screening data for H446 and MDA-MB-468 cells (tabs 1, 2) and RNA expression level associations with LY3295668 Abs IC50 values from 493 cancer cell lines (tab 3).

Supplementary Tables

Supplementary figures

Supplementary Figures

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1mut cancer cells and leads to durable regression of RB1mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. Significance: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors. See related commentary by Dick and Li, p. 169. This article is highlighted in the In This Issue feature, p. 151

Loss-of-function mutations in the retinoblastoma gene

Summary Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-β- d -glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans . Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated ( r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.

Sulfanilanilides with 3',5'-halogen substitutions had Ki values 6- to 57-fold lower than the Ki of sulfamethoxazole when tested against dihydropteroate synthase from Toxoplasma gondii. The compounds acted as competitive inhibitors. These compounds were also active against dihydropteroate synthase from Pneumocystis carinii, Mycobacterium avium, and Escherichia coli but were not significantly more active than sulfamethoxazole. The compounds were significantly more active in culture than were standard agents. Against T. gondii in culture, 50% inhibitory concentrations were 7- to 30-fold lower than that of sulfadiazine; against P. carinii in culture, a concentration of 100 microM caused 33 to 95% inhibition of growth, compared with 9% inhibition with 100 microM sulfamethoxazole.

We developed a combination screening protocol to look for synergistic interactions with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Abemaciclib (LY2835219), has shown cytostatic effects in some cell lines while inducing senescence and apoptosis in particularly sensitive cell lines. Abemaciclib, combined with various compounds, was screened across panels of genomically characterized tumor cells. These screens identified several synergistic interactions that improved the activity of abemaciclib in cancer cells lines that respond to abemaciclib monotherapy (e.g. mantle cell lymphoma, ER+ breast cancer) but additionally revealed certain combinations with synergy in Rb wild-type cancers that do not respond optimally to single agent abemaciclib treatment. Most interestingly, MEK inhibitors and LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Cancer Cell 28:384-98) were found to potentiate the cytostatic effects of abemaciclib in these cell lines leading to apoptosis in vitro and tumor regression in vivo. Further analysis of the effects of combined inhibition of CDK4 and CDK6 and Raf isoforms on downstream signaling pathways provides mechanistic clues that may help explain the observed synergy. Citation Format: Gong Xueqian, Li-Chun Chio, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Phil Iversen, Alfonso De Dios, Ian Smith, Christoph Reinhard, Sheng-Bin Peng, Jack Dempsey, Teresa Burke, Shih-Hsun Chen, Trent Stewart, Richard Beckmann, Wenjuan Wu, Sean G. Buchanan. The identification of combinations for the CDK4 and CDK6 inhibitor, abemaciclib. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A07.

Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models. Citation Format: Xueqian Gong, Wenjuan Wu, Li-Chun Chio, Susan Pratt, Constance King, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Philip Iversen, Christoph Reinhard, Shih-Hsun Chen, Richard Bechmann, Sheng-Bin Peng, Sean Buchanan. An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2818.

It is well established that phosphorylation of Rb-family pocket proteins by CDK4 and CDK6 is important for the commitment of cancer cells to a new cell cycle and the initiation of the G1-S phase transition. Abemaciclib is a potent inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing clinical testing. To better understand the molecular determinants of response to abemaciclib, we tested its anti-proliferative activity across a panel of over 500 well characterized cancer cell lines. Statistical approaches were employed to uncover genomic features associated with the response. Candidate markers of sensitivity and resistance were further tested by genetic manipulations in vitro. In vivo models representing the candidate molecular marker of sensitivity were identified and drug efficacy examined. Three broad classes of response were identified. The class of tumors cells most resistant to abemaciclib showed enrichment for RB1 mutations. Conversely, cell lines with amplification of CCND2 and CCND3 were among the very most sensitive tumor cells and tumor cells with these markers showed evidence of senescence and apoptosis after either depletion of the cognate D-cyclin or treatment with abemaciclib. In vivo models of tumors harboring CCND2 and CCND3 gene amplification were very sensitive to abemaciclib treatment and showed evidence of tumor regression. Citation Format: Xueqian Gong, Li-Chun Chio, MaryJo Lallena, Farhana Merzoug, Shaoyou Chu, Yue Webster, Jack Dempsey, Xiwen Ma, Alfonso De Dios, Richard Beckman, Sean G. Buchanan. Molecular features that determine the sensitivity of cancer cells to abemaciclib, an inhibitor of CDK4 and CDK6. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2015-3104

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broad-spectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a β-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and β-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

In recent years, mounting evidence has indicated that sphingolipid metabolism is an important cell signaling system. Rapid and transient changes in sphingolipid metabolism are closely associated with a wide range of cellular activities, including the stress response, apoptosis, inflammation, cell cycle regulation, and cancer development (19, 20, 33). For instance, stress signals rapidly and transiently elevate the level of cellular ceramide; conversely, growth factors stimulate the rapid, transient generation of sphingosine-1-phosphate (S-1-P). Importantly, the treatment of cells with short-chain ceramide analogs or the expression of sphingomyelinases reproduces most of the effects of endogenous ceramide, particularly in the stress response and apoptosis (19, 20, 33). Furthermore, S-1-P, as a high-affinity ligand of the edg family of G-protein-coupled receptors, promotes cell survival and proliferation by antagonizing ceramide-mediated apoptosis (52, 61). Thus, ceramide, as an important regulatory component in the stress response and apoptosis, and S-1-P, involved in cell survival and proliferation, have attracted enormous scientific interest in recent years. In particular, a large body of evidence has now accumulated to implicate an important role of ceramide in the stress response and apoptosis. It is proposed that the relative levels of cellular ceramide and S-1-P determine whether cells undergo apoptosis or continue to proliferate (61). The sphingoid long-chain bases dihydrosphingosine (DHS) and sphingosine are central components of the sphingolipid metabolic pathway (20). They are substrates for the synthesis of both ceramide and sphingoid base phosphates (S-1-P and DHS-1-phosphate [DHS-1-P]). Sphingosine is also the product of ceramide and S-1-P metabolism. It has been shown that sphingosine is also a highly bioactive molecule (20). However, compared to the situation for ceramide and S-1-P, very few studies have been directed toward characterizing the cellular functions of sphingosine. Interestingly, a recent study showed that, paralleling the change in ceramide levels, a rapid, transient elevation of sphingosine levels is also observed in the stress response and apoptosis (9). It was further shown that treatment of cells with exogenous C2-ceramide not only increases the level of endogenous ceramide but also leads to a marked increase in the level of cellular sphingosine (9). The biological significance of such an increase in the cellular sphingosine level during the stress response and apoptosis is not well understood. These studies do raise the possibility that, like ceramide, sphingosine may also play a role in the stress response and apoptosis. This possibility is further supported by the fact that exogenous sphingosine is a potent elicitor of apoptosis (9, 10, 30). Most importantly, sphingosine regulates multiple key signal transduction factors, such as protein kinase C, 3-phosphoinositide-dependent kinase 1 (PDK1), and p21-activated kinase 1 (PAK1) (4, 18, 32). The basic chemical structure, biosynthesis, and metabolism of sphingolipids are conserved in mammalian and fungal systems (15). In Saccharomyces cerevisiae, an important role for sphingolipids in the stress response was originally proposed based on the observation that SLC (suppressor of long-chain bases) mutants were unable to grow under many types of stress (55). A role for sphingolipids in the yeast stress response to heat now has been well established (28, 71). Interestingly, a detailed analysis of changes in the sphingolipid composition showed that upon heat stress, the levels of the sphingoid long-chain bases DHS and phytosphingosine (PHS) rapidly and transiently increase severalfold within 10 to 20 min (28). Although the level of cellular ceramide also increases severalfold upon heat stress, the increase occurs much more slowly, within 1 to 2 h (28). Furthermore, it was demonstrated that the rapid elevation of the levels of cellular DHS and PHS but not of ceramide is responsible for transient G1 arrest upon heat stress (27) and that heat resistance requires DHS-1-P and PHS-1-phosphate (PHS-1-P) produced by sphingoid base kinase-phosphatase activities (31, 34, 43, 44). Exogenous PHS has also been shown to inhibit yeast growth in a very specific manner that does not involve ceramide (8) but occurs in part through the regulation of ubiquitin-dependent proteolysis (7) and nutrient uptake (59). Moreover, in S. cerevisiae, PHS plays an important role in endocytosis and proper actin cytoskeleton organization by regulating Pkc1 activity via Pkh1/2 (11, 65). Together, these studies strongly suggest that sphingoid bases play a specific signaling role in growth regulation and the stress response in S. cerevisiae. We are using Aspergillus nidulans, a filamentous fungus, as a model genetic system to study the cellular functions of sphingolipids. Recently, Cheng et al. showed that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that the accumulation of cellular ceramide causes cell cycle arrest in G1 (6). In this study, we investigated the cellular functions of DHS and PHS by analyzing the physiological and cellular effects of treating A. nidulans cells with exogenous DHS and PHS. Both DHS and PHS are able to diffuse readily between cell membranes and, most importantly, are able to fully complement the lack of serine palmitoyltransferase (SPT) activity, the first committed and rate-limiting step of the sphingolipid biosynthesis pathway (6). We found that DHS and PHS have potent antifungal activity with remarkable structural and stereochemical specificity. We demonstrate that the antifungal activity of DHS and PHS does not involve ceramide and intriguingly acts through the rapid induction of metacaspase-independent apoptosis. Furthermore, we show that fungal apoptosis is remarkably similar to that of mammalian cells morphologically.

Background Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol. Results ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1β, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis. Conclusion In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems.

14 C-labeled Prolan or 1,1-bis-( p -chlorophenyl)-2-nitropropane was found to be some-what more biodegradable than DDT. This insecticide, although highly resistant to microsomal metabolism, was degraded by elimination to 1,1-bis-( p -chlorophenyl)-1-propene, and by reduction to 1,1-bis-( p -chlorophenyl)-2-aminopropane. The major degradative pathway, however, was by oxidation to 1,1-bis-( p -chlorophenyl)-2-propanone, to 1,1-bis-( p -chlorophenyl)-pyruvic acid, to bis-( p -chlorophenyl)-acetic acid, and ultimately to p,p′ -dichlorobenzophenone. Therefore the ultimate degradative products of Prolan are identical to those produced from DDT.