Mark T. Uhlik

Modern Phenotypic Drug Discovery Is a Viable, Neoclassic Pharma Strategy

Strategy Jonathan A. Lee,*,† Mark T. Uhlik,‡ Christopher M. Moxham, Dirk Tomandl, and Daniel J. Sall Departments of †Quantitative Biology, ‡Cancer-Angiogenesis, Discovery Informatics, and Discovery Chemistry Research and Technologies, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana ImClone Systems, a Wholly Owned Subsidiary of Eli Lilly and Company, New York, New York ■ INTRODUCTION The pharmaceutical industry (Pharma) is currently facing unprecedented challenges. In addition to strategic patent expirations, the rate of drug launches has been essentially constant for 60 years with overall productivity falling since the 1970s. Similarly, the appearance of novel targets to FDA approved drugs, a measure of industry innovation, has not dramatically improved since the 1980s. Although Pharma productivity is a multifaceted problem, detailed analyses of comprehensive, industry-wide data indicate that late stage clinical failures are a major contributor that has been attributed to poor target validation (TV) and the lack of predictive biomarkers that translate to the clinic. In the spirit of “reinventing innovation” Pharma needs to introspectively identify areas for improvement. This communication considers how choices in drug screening strategies may relate to target validation issues and influence the probability of identifying novel medicines. Target-directed drug discovery (TDD) and phenotypic drug discovery (PDD) are Pharma strategies that have roots in distinct but complementary philosophies. Advantages of genespecific, reductionist approaches include the formulation and testing of specific molecular hypotheses. TDD approaches utilize advances in automation, biochemistry, structural biology, and chemistry related technologies to provide efficient and high capacity testing of unprecedented numbers of compounds and molecular targets. In addition, deep mining of cDNA expressed sequence tags (ESTs) and subsequently entire genomes led to the discovery of thousands of unknown genes and the potential for deep insight into novel drug target biology. Taken together, these advances in science and technology, in conjunction with the innate human desire to seek new challenges, contributed to the rapid adoption of molecular-reductionist views of biology. In contrast, PDD tests compounds in complex biological systems and monitors physiological responses with minimal assumptions concerning the participation of specific molecular targets and/or signaling pathways. Analyses of new molecular entities (NMEs) approved by the FDA between 1999 and 2008 indicate that for first in class molecules, 37% resulted from projects that used phenotypic screening whereas target based screening identified 23%; moreover, the discovery rate of PDD NMEs was greater than TDD NMEs and was invariant over the 9-year study period. Since significantly more TDD efforts were conducted during this period, the overall launch rate for first in class drugs underestimates the intrinsic probability of technical success (pTS) of classic PDD. A hybrid of classic phenotypic and target-directed strategies, which blends the use of physiologically relevant biological systems with the high throughput and statistical robustness of modern assay technologies, may have a higher pTS for launching first in class drugs than either classic PDD or TDD. Academia has utilized modern phenotypic approaches to study cell cycle, stem cell renewal, cell migration, metastasis, and induction of pluripotent stem cells. However, such “neoclassic” PDD approaches are not widely used in Pharma because of concerns about assay performance, statistical robustness, perceived difficulties in establishing compound structure−activity relationships (SARs), anticipated limited applicability of chemoinformatics tools, and the difficulty/ requirement for elucidating a molecular drug target. The results outlined in this presentation address commonly perceived issues related to the use of complex biological systems for modern lead generation. Our data, using an angiogenesis assay incorporating a coculture of primary human endothelial and stromal progenitor cells, indicate that phenotypic assays can be statistically robust, can be used to identify novel compound scaffolds by chemoinformatics enabled hit expansion, and can provide evidence of compound structure−activity relationships. Identification of novel molecular targets important to angiogenesis, acetyl Co-A carboxylase (ACC) and a protein related to cellular β-secretase (β-sec) activity, demonstrates that PDD provides a means to test multiple biologically relevant pathways in a target agnostic fashion. These attributes of PDD enabled the discovery of chemical scaffolds that were readily differentiated, structurally and mechanistically, from antiangiogenic agents that constitute the current standard of care (SOC) and demonstrated in vivo activity. We conclude that PDD complements gene-specific target-directed strategies, may mitigate TV risks, and has the potential to enhance innovation in drug discovery.

A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay.

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

Identification of LY2510924, a Novel Cyclic Peptide CXCR4 Antagonist That Exhibits Antitumor Activities in Solid Tumor and Breast Cancer Metastatic Models

Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC50 value of 0.079 nmol/L, and inhibited SDF-1–induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1–induced cell migration with IC50 value of 0.26 nmol/L and inhibited SDF-1/CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1–stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non–Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer. Mol Cancer Ther; 14(2); 480–90. ©2014 AACR.

Antagonist antibodies to vascular endothelial growth factor receptor 2 (VEGFR-2) as anti-angiogenic agents.

Interaction of numerous signaling pathways in endothelial and mesangial cells results in exquisite control of the process of physiological angiogenesis, with a central role played by vascular endothelial growth factor receptor 2 (VEGFR-2) and its cognate ligands. However, deregulated angiogenesis participates in numerous pathological processes. Excessive activation of VEGFR-2 has been found to mediate tissue-damaging vascular changes as well as the induction of blood vessel expansion to support the growth of solid tumors. Consequently, therapeutic intervention aimed at inhibiting the VEGFR-2 pathway has become a mainstay of treatment in cancer and retinal diseases. In this review, we introduce the concepts of physiological and pathological angiogenesis, the crucial role played by the VEGFR-2 pathway in these processes, and the various inhibitors of its activity that have entered the clinical practice. We primarily focus on the development of ramucirumab, the antagonist monoclonal antibody (mAb) that inhibits VEGFR-2 and has recently been approved for use in patients with gastric, colorectal, and lung cancers. We examine in-depth the pre-clinical studies using DC101, the mAb to mouse VEGFR-2, which provided a conceptual foundation for the role of VEGFR-2 in physiological and pathological angiogenesis. Finally, we discuss further clinical development of ramucirumab and the future of targeting the VEGF pathway for the treatment of cancer.

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

High-content multiplexed tissue imaging and quantification for cancer drug discovery.

Targeting multiple hallmarks of cancer with drug combinations may provide unique opportunities for cancer therapeutics; however, phenotypic quantification is necessary to understand in vivo mechanisms of action of each drug alone or in combination. Immunohistochemistry (IHC) can quantify phenotypic changes, but traditional methods are not amenable for high-throughput drug discovery. In this article, we describe a high-content method to quantify changes in tumor angiogenesis, vascular normalization, hypoxia, tumor cell proliferation, and apoptosis using IHC. This method to quantify tumor model phenotypes can be useful for cancer drug discovery by increasing the understanding of: (i) tumor models used in efficacy studies, (ii) changes occurring during the growth of the tumor, and (iii) novel mechanisms of actions of cancer therapeutics.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor. Mol Cancer Res; 10(6); 739–49. ©2012 AACR.

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model

SummaryLY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

Stromal-Based Signatures for the Classification of Gastric Cancer

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

An In Vitro Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.