Jason Oeh

Tumour-secreted miR-9 promotes endothelial cell migration and angiogenesis by activating the JAK-STAT pathway

Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co‐cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR‐9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR‐9 effectively reduced SOCS5 levels, leading to activated JAK‐STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti‐miR‐9 or JAK inhibitors suppressed MV‐induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour‐secreted miRNAs participate in intercellular communication and function as a novel pro‐angiogenic mechanism.

Targeting Activated Akt with GDC-0068, a Novel Selective Akt Inhibitor That Is Efficacious in Multiple Tumor Models

Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. Clin Cancer Res; 19(7); 1760–72. ©2012 AACR.

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL–selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2High/BCL-XLLow. In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132–44. ©2016 AACR.

Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.

Fragment-Based Identification of Amides Derived from trans-2-(Pyridin-3-yl)cyclopropanecarboxylic Acid as Potent Inhibitors of Human Nicotinamide Phosphoribosyltransferase (NAMPT)

Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 μM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 μM, A2780 cell culture IC50 = 0.000 49 μM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.

Retinal Toxicity, in vivo and in vitro, Associated with Inhibition of Nicotinamide Phosphoribosyltransferase

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein with intra- and extra-cellular functions as an enzyme, cytokine, growth factor, and hormone. NAMPT is of interest for oncology, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), which is considered a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. This manuscript describes NAMPT inhibitor-induced retinal toxicity that was identified in rodent safety studies. This toxicity had a rapid onset and progression and initially targeted the photoreceptor and outer nuclear layers. Using in vivo safety and efficacy rodent studies, human and mouse cell line potency data, human and rat retinal pigmented epithelial cell in vitro systems, and rat mRNA expression data of NAMPT, nicotinic acid phosphoribosyltransferase, and nicotinamide mononucleotide adenylyltransferease (NMNAT) in several tissues from rat including retina, we demonstrate that the retinal toxicity is on-target and likely human relevant. We demonstrate that this toxicity is not mitigated by coadministration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway. Further, modifying the physiochemical properties of NAMPT inhibitors could not sufficiently reduce retinal exposure. Our work highlights opportunities to leverage appropriately designed efficacy studies to identify known and measurable safety findings to screen compounds more rapidly and reduce animal use. It also demonstrates that in vitro systems with the appropriate cell composition and relevant biology and toxicity endpoints can provide tools to investigate mechanism of toxicity and the human translation of nonclinical safety concerns.

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC50 = 11 nM; PC-3 antiproliferative IC50 = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC50 = 10nM; A2780 antiproliferative IC50 = 7 nM) in complex with the NAMPT protein was also determined.

Potent, Selective, and Orally Bioavailable Inhibitors of the Mammalian Target of Rapamycin Kinase Domain Exhibiting Single Agent Antiproliferative Activity

Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.

Prediction of Drug Distribution in Subcutaneous Xenografts of Human Tumor Cell Lines and Healthy Tissues in Mouse: Application of the Tissue Composition-Based Model to Antineoplastic Drugs

Advanced tissue composition-based models can predict the tissue-plasma partition coefficient (Kp ) values of drugs under in vivo conditions on the basis of in vitro and physiological input data. These models, however, focus on healthy tissues and do not incorporate data from tumors. The objective of this study was to apply a tissue composition-based model to six marketed antineoplastic drugs (docetaxel, DOC; doxorubicin, DOX; gemcitabine, GEM; methotrexate, MTX; topotecan, TOP; and fluorouracil, 5-FU) to predict their Kp values in three human tumor xenografts (HCT-116, H2122, and PC3) as well as in healthy tissues (brain, muscle, lung, and liver) under steady-state in vivo conditions in female NCR nude mice. The mechanisms considered in the tissue/tumor composition-based model are the binding to lipids and to plasma proteins, but the transporter effect was also investigated. The method consisted of analyzing tissue composition, performing the pharmacokinetics studies in mice, and calculating the corresponding in vivo Kp values. Analyses of tumor composition indicated that the tumor xenografts contained no or low amounts of common transporters by contrast to lipids. The predicted Kp values were within twofold and threefold of the measured values in 77% and 93% of cases, respectively. However, predictions for brain for each drug, for liver for MTX, and for each tumor xenograft for GEM were disparate from the observed values, and, therefore, not well served by the model. Overall, this study is the first step toward the mechanism-based prediction of Kp values of small molecules in healthy and tumor tissues in mouse when no transporter and permeation limitation effect is evident. This approach will be useful in selecting compounds based on their abilities to penetrate human cancer xenografts with a physiologically based pharmacokinetic (PBPK) model, thereby increasing therapeutic index for chemotherapy in oncology study.

Monitoring and Targeting Anti-VEGF Induced Hypoxia within the Viable Tumor by 19F–MRI and Multispectral Analysis

The effect of anti-angiogenic agents on tumor oxygenation has been in question for a number of years, where both increases and decreases in tumor pO2 have been observed. This dichotomy in results may be explained by the role of vessel normalization in the response of tumors to anti-angiogenic therapy, where anti-angiogenic therapies may initially improve both the structure and the function of tumor vessels, but more sustained or potent anti-angiogenic treatments will produce an anti-vascular response, producing a more hypoxic environment. The first goal of this study was to employ multispectral (MS) 19F–MRI to noninvasively quantify viable tumor pO2 and evaluate the ability of a high dose of an antibody to vascular endothelial growth factor (VEGF) to produce a strong and prolonged anti-vascular response that results in significant tumor hypoxia. The second goal of this study was to target the anti-VEGF induced hypoxic tumor micro-environment with an agent, tirapazamine (TPZ), which has been designed to target hypoxic regions of tumors. These goals have been successfully met, where an antibody that blocks both murine and human VEGF-A (B20.4.1.1) was found by MS 19F–MRI to produce a strong anti-vascular response and reduce viable tumor pO2 in an HM-7 xenograft model. TPZ was then employed to target the anti-VEGF-induced hypoxic region. The combination of anti-VEGF and TPZ strongly suppressed HM-7 tumor growth and was superior to control and both monotherapies. This study provides evidence that clinical trials combining anti-vascular agents with hypoxia-activated prodrugs should be considered to improved efficacy in cancer patients.