Timothy Paul Burkholder

Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, in Human Cancer Cells METABOLIC BASIS AND POTENTIAL CLINICAL IMPLICATIONS

Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.

Developing and applying a gene functional association network for anti-angiogenic kinase inhibitor activity assessment in an angiogenesis co-culture model

BackgroundTumor angiogenesis is a highly regulated process involving intercellular communication as well as the interactions of multiple downstream signal transduction pathways. Disrupting one or even a few angiogenesis pathways is often insufficient to achieve sustained therapeutic benefits due to the complexity of angiogenesis. Targeting multiple angiogenic pathways has been increasingly recognized as a viable strategy. However, translation of the polypharmacology of a given compound to its antiangiogenic efficacy remains a major technical challenge. Developing a global functional association network among angiogenesis-related genes is much needed to facilitate holistic understanding of angiogenesis and to aid the development of more effective anti-angiogenesis therapeutics.ResultsWe constructed a comprehensive gene functional association network or interactome by transcript profiling an in vitro angiogenesis model, in which human umbilical vein endothelial cells (HUVECs) formed capillary structures when co-cultured with normal human dermal fibroblasts (NHDFs). HUVEC competence and NHDF supportiveness of cord formation were found to be highly cell-passage dependent. An enrichment test of Biological Processes (BP) of differentially expressed genes (DEG) revealed that angiogenesis related BP categories significantly changed with cell passages. Built upon 2012 DEGs identified from two microarray studies, the resulting interactome captured 17226 functional gene associations and displayed characteristics of a scale-free network. The interactome includes the involvement of oncogenes and tumor suppressor genes in angiogenesis. We developed a network walking algorithm to extract connectivity information from the interactome and applied it to simulate the level of network perturbation by three multi-targeted anti-angiogenic kinase inhibitors. Simulated network perturbation correlated with observed anti-angiogenesis activity in a cord formation bioassay.ConclusionWe established a comprehensive gene functional association network to model in vitro angiogenesis regulation. The present study provided a proof-of-concept pilot of applying network perturbation analysis to drug phenotypic activity assessment.

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.

Design and synthesis of a novel series of [1-(4-hydroxy-benzyl)-1H-indol-5-yloxy]-acetic acid compounds as potent, selective, thyroid hormone receptor β agonists

The design, synthesis, and structure activity relationships for a novel series of indoles as potent, selective, thyroid hormone receptor β (TRβ) agonists is described. Compounds with >50× binding selectivity for TRβ over TRα were generated and evaluation of compound 1c from this series in a model of dyslipidemia demonstrated positive effects on plasma lipid endpoints in vivo.

Dose study of the multikinase inhibitor, LY2457546, in patients with relapsed acute myeloid leukemia to assess safety, pharmacokinetics, and pharmacodynamics

Background: Acute myeloid leukemia (AML) is a life-threatening malignancy with limited treatment options in chemotherapy-refractory patients. A first-in-human dose study was designed to investigate a safe and biologically effective dose range for LY2457546, a novel multikinase inhibitor, in patients with relapsed AML. Methods: In this nonrandomized, open-label, dose escalation Phase I study, LY2457546 was administered orally once a day. Safety, pharmacokinetics, changes in phosphorylation of target kinases in AML blasts, and risk of drug–drug interactions (DDI) were assessed. Results: Five patients were treated at the starting and predicted minimal biologically effective dose of 50 mg/day. The most commonly observed adverse events were febrile neutropenia, epistaxis, petechiae, and headache. The majority of adverse events (81%) were Grade 1 or 2. One patient had generalized muscle weakness (Grade 3), which was deemed to be a dose-limiting toxicity. Notably, the pharmacokinetic profile of LY2457546 showed virtually no elimination of LY2457546 within 24 hours, and thus prevented further dose escalation. No significant DDI were observed. Ex vivo flow cytometry studies showed downregulation of the phosphoproteins, pcKIT, pFLT3, and pS6, in AML blasts after LY2457546 administration. No medically relevant responses were observed in the five treated patients. Conclusion: No biologically effective dose could be established for LY2457546 in chemotherapy-resistant AML patients. Lack of drug clearance prevented safe dose escalation, and the study was terminated early. Future efforts should be made to develop derivatives with a more favorable pharmacokinetic profile.

Abstract 2820: LY2784544, a small molecule JAK2 inhibitor, induces apoptosis in inflammatory breast cancer spheres through targeting IL-6-JAK-STAT3 pathway

Inflammatory breast cancer (IBC) is more aggressive and deadly than other breast cancers (K Rowan. JNCI. 2009;101(19):1302-1304). IBC cells secrete angiogenic and vasculogenic growth factors, such as VEGF, bFGF, IL-6 and IL-8, resulting in a cluster of tumor cells, termed anembolus. Through maintaining cell-cell interactions, the IBC emboli can become resistant to “anoikis”, a cell death process following the loss of contact from their neighboring cells. It is hypothesized that this resistance may allow IBC cells to survive the migration through lymphatic vessels and facilitate development of skin and lymph node metastasis. With current therapy, the disease-free survival for IBC patients is only about 35% (SD Merajver et al. J Clin Oncol. 1997;15(8):2873-81). Clearly better therapy is needed for patients with IBC and insights may be gained from exploration of and subsequent interventions in the roles these growth factors play in the biology of IBC. In this study, we investigated the role of IL-6 in activating the signal transducer and activator of transcription 3 (STAT3), conferring resistance to anoikis, and promoting cell proliferation in IBC SUM-149 tumor spheres. Moreover, we targeted JAK2, a transducer of IL-6 signaling to STAT3, by a small molecule JAK2 inhibitor, LY2784544, to inhibit the IL-6-Stat3 pathway and induce apoptosis in SUM-149 tumor spheres. In brief, SUM-149 cells were grown to form tumor spheres under low adherence culture conditions. IL-6 mediated-Stat3 activation and the induction of the cleavage of PARP were evaluated by Western Blotting. Anoikis and cell viability were measured by calcein-AM accumulation and the expression of IL-6 was examined by RT-PCR. We found that exogenous IL-6 activated STAT3, conferred resistance to anoikis and promoted the cell proliferation in SUM-149 cells under the low adherent culture condition. Furthermore, the expression of IL-6 was increased by 50-fold in IBC tumor spheres as measured by RT-PCR. The autocrine production of IL-6 activated STAT3 and inhibited anoikis- induced apoptosis in SUM-149 tumor spheres. Very importantly, we showed that targeting JAK2 by LY2784544 potently inhibited the phosphorylation of STAT3 in a dose dependent manner with an IC50 of 0.25 µM. Blockage of this signal pathway by LY2784544 resulted in potent induction of apoptosis (EC50=0.49 µM). Taken together, these studies suggest the IL-6-JAK-STAT3 pathway may serve as a molecular signature of IBC and as a potential therapeutic target. LY2784544 is currently in clinical studies for the treatment of myeloid proliferative neoplasm indications and the results reported here support clinical investigation for the treatment of IBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2011-2820

Discovery of a Highly Selective NAMPT Inhibitor That Demonstrates Robust Efficacy and Improved Retinal Toxicity with Nicotinic Acid Coadministration

NAMPT, an enzyme essential for NAD+ biosynthesis, has been extensively studied as an anticancer target for developing potential novel therapeutics. Several NAMPT inhibitors have been discovered, some of which have been subjected to clinical investigations. Yet, the on-target hematological and retinal toxicities have hampered their clinical development. In this study, we report the discovery of a unique NAMPT inhibitor, LSN3154567. This molecule is highly selective and has a potent and broad spectrum of anticancer activity. Its inhibitory activity can be rescued with nicotinic acid (NA) against the cell lines proficient, but not those deficient in NAPRT1, essential for converting NA to NAD+. LSN3154567 also exhibits robust efficacy in multiple tumor models deficient in NAPRT1. Importantly, this molecule when coadministered with NA does not cause observable retinal and hematological toxicities in the rodents, yet still retains robust efficacy. Thus, LSN3154567 has the potential to be further developed clinically into a novel cancer therapeutic. Mol Cancer Ther; 16(12); 2677–88. ©2017 AACR.

Abstract 3042: LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnUnder normal conditions MET receptor activation, internalization, and degradation is a highly controlled process regulated by the binding of its ligand, HGF. In many cancers however, MET becomes highly dysregulated, and MET overexpression, with or without gene amplification, is associated with a poorer prognosis. Aberrant activation of MET can also occur through mutation, or increased autocrine or paracrine HGF production. MET activation leads to increased oncogenic activities including cell migration, proliferation, invasion, survival, tumor neovascularization and decreased apoptosis. Several inhibitors of MET, including type I and type II kinase inhibitors, are currently being investigated in clinical trials. LY2801653 is a type II MET kinase inhibitor being investigated in patients with advanced cancer (trial I3O-MC-JSBA, [NCT01285037][1]). In addition to MET, LY2801653 also inhibits ROS1 kinase, and the serine/threonine kinases MKNK1/2, with cell-based IC50 of 23nM and 7nM respectively. ROS1 is an orphan receptor that can be constitutively activated as a fusion protein either by genomic microdeletion or translocation. ROS1 fusion proteins are found in glioblastoma, NSCLC and cholangiocarcinoma. MKNK1/2 are kinases downstream of ERK and p38 and are capable of regulating translation through the direct phosphorylation of eIF4E. Increased MKNK1/2-dependent phosphorylation of the translation factor eIF4E is observed in head and neck squamous cell carcinoma and is correlated with a poor prognosis. This study evaluated the relative inhibitory effects of LY2801653, Crizotinib, XL184, XL880, and PF4217903 in a variety of cell lines against MET, ROS1, MKNK1/2, ERK, and other downstream molecules. In HCC78, a ROS1-fusion protein bearing cell line, and in NIH3T3 cells stably transfected with the FIG-ROS(s) fusion, LY2801653, XL184 and XL880 were more potent inhibitors of ROS1 compared with Crizotinib. Moreover, LY2801653 and Crizotinib were both efficacious in NIH3T3 FIG-ROS(s) xenografts, although LY2801653 was more potent in reducing p-ROS1 in these tumors than Crizotinib. In NCI-H2122 and Hs746T cells (mutated MET), A2780 cells (MET-negative), KP-4 cells (MET autocrine), H441 cells (HGF-independent; MET positive), HCT116 (mutated KRAS), and the cholangiocarcinoma cell lines EGI-1 and TFK-1, LY2801653 demonstrated more potent inhibitory activity against MKNK1/2 versus treatment with XL184 and XL880, while Crizotinib had no observed inhibitory activity against MKNK1/2. LY2801653 also showed inhibitory activity against MKNK1/2 in MET-amplified MKN45 cells. These findings suggests there may be clinical merits to testing LY2801653 in tumor types associated with increased MKNK1/2 activity or ROS1 fusion proteins alone, or in conjunction with aberrant MET activity, such as colorectal carcinoma, head and neck squamous cell carcinoma, and cholangiocarcinoma.nnCitation Format: Sau-Chi B. Yan, Megan N. Thobe, Bruce W. Konicek, Victoria L. Peek, Suzane L. Um, Richard A. Walgren, Huaxing Pei, Timothy P. Burkholder, Jeremy R. Graff. LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2013-3042nn [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01285037&atom=%2Fcanres%2F73%2F8_Supplement%2F3042.atom