Min-Jean Yin

PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity

Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC50 of 7.5–47 nmol/L and 3.8–20 nmol/L, respectively) and inhibited cell proliferation (IC50 of 179–313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC50 of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G1 arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non–small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials. Mol Cancer Ther; 10(11); 2189–99. ©2011 AACR.

The Serine/Threonine Kinase Nek6 Is Required for Cell Cycle Progression through Mitosis

The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.

miR-221 Promotes Tumorigenesis in Human Triple Negative Breast Cancer Cells

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis. TNBCs are characterized by their resistance to apoptosis, aggressive cellular proliferation, migration and invasion, and currently lack molecular markers and effective targeted therapy. Recently, miR-221/miR-222 have been shown to regulate ERα expression and ERα-mediated signaling in luminal breast cancer cells, and also to promote EMT in TNBCs. In this study, we characterized the role of miR-221 in a panel of TNBCs as compared to other breast cancer types. miR-221 knockdown not only blocked cell cycle progression, induced cell apoptosis, and inhibited cell proliferation in-vitro but it also inhibited in-vivo tumor growth by targeting p27kip1. Furthermore, miR-221 knockdown inhibited cell migration and invasion by altering E-cadherin expression, and its regulatory transcription factors Snail and Slug in human TNBC cell lines. Therefore, miR-221 functions as an oncogene and is essential in regulating tumorigenesis in TNBCs both in vitro as well as in vivo.

Clusterin Inhibition Using OGX-011 Synergistically Enhances Hsp90 Inhibitor Activity by Suppressing the Heat Shock Response in Castrate-Resistant Prostate Cancer

Small-molecule inhibitors of Hsp90 show promise in the treatment of castrate-resistant prostate cancer (CRPC); however, these inhibitors trigger a heat shock response that attenuates drug effectiveness. Attenuation is associated with increased expression of Hsp90, Hsp70, Hsp27, and clusterin (CLU) that mediate tumor cell survival and treatment resistance. We hypothesized that preventing CLU induction in this response would enhance Hsp90 inhibitor-induced CRPC cell death in vitro and in vivo. To test this hypothesis, we treated CRPC with the Hsp90 inhibitor PF-04929113 or 17-AAG in the absence or presence of OGX-011, an antisense drug that targets CLU. Treatment with either Hsp90 inhibitor alone increased nuclear translocation and transcriptional activity of the heat shock factor HSF-1, which stimulated dose- and time-dependent increases in HSP expression, especially CLU expression. Treatment-induced increases in CLU were blocked by OGX-011, which synergistically enhanced the activity of Hsp90 inhibition on CRPC cell growth and apoptosis. Accompanying these effects was a decrease in HSF-1 transcriptional activity as well as expression of HSPs, Akt, prostate-specific antigen, and androgen receptor. In vivo evaluation of the Hsp90 inhibitors with OGX-011 in xenograft models of human CRPC showed that OGX-011 markedly potentiated antitumor efficacy, leading to an 80% inhibition of tumor growth with prolonged survival compared with Hsp90 inhibitor monotherapy. Together, our findings indicate that Hsp90 inhibitor-induced activation of the heat shock response and CLU is attenuated by OGX-011, with synergistic effects on delaying CRPC progression.

Dihydroxyphenylisoindoline amides as orally bioavailable inhibitors of the heat shock protein 90 (hsp90) molecular chaperone.

The discovery and optimization of potency and metabolic stability of a novel class of dihyroxyphenylisoindoline amides as Hsp90 inhibitors are presented. Optimization of a screening hit using structure-based design and modification of log D and chemical structural features led to the identification of a class of orally bioavailable non-quinone-containing Hsp90 inhibitors. This class is exemplified by 14 and 15, which possess improved cell potency and pharmacokinetic profiles compared with the original screening hit.

miR-122 Regulates Tumorigenesis in Hepatocellular Carcinoma by Targeting AKT3

MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer.

A Novel HSP90 Inhibitor Delays Castrate-Resistant Prostate Cancer without Altering Serum PSA Levels and Inhibits Osteoclastogenesis

Purpose: Prostate cancer responds initially to antiandrogen therapies; however, progression to castration-resistant disease frequently occurs. Therefore, there is an urgent need for novel therapeutic agents that can prevent the emergence of castrate-resistant prostate cancer (CRPC). HSP90 is a molecular chaperone involved in the stability of many client proteins including Akt and androgen receptor (AR). 17-Allylamino-17-demethoxy-geldanamycin (17-AAG) has been reported to inhibit tumor growth in various cancers; however, it induces tumor progression in the bone microenvironment. Methods: Cell growth, apoptosis, and AR transactivation were examined by crystal violet assay, flow cytometric, and luciferase assays, respectively. The consequence of HSP90 therapy in vivo was evaluated in LNCaP xenograft model. The consequence of PF-04928473 therapy on bone metastasis was studied using an osteoclastogenesis in vitro assay. Results: PF-04928473 inhibits cell growth in a panel of prostate cancer cells, induces cell-cycle arrest at sub-G1, and leads to apoptosis and increased caspase-3 activity. These biological events were accompanied by decreased activation of Akt and Erk as well as decreased expression of Her2, and decreased AR expression and activation in vitro. In contrast to 17-AAG, PF-04928473 abrogates RANKL-induced osteoclast differentiation by affecting NF-κB activation and Src phosphorylation. Finally, PF-04929113 inhibited tumor growth and prolonged survival compared with controls. Surprisingly, PF-04929113 did not reduce serum prostate-specific antigen (PSA) levels in vivo; in parallel, these decrease in tumor volume. Conclusion: These data identify significant anticancer activity of PF-04929113 in CRPC but suggest that serum PSA may not prove useful as pharmacodynamic tool for this drug. Clin Cancer Res; 17(8); 2301–13. ©2011 AACR.

Suppression of Heat Shock Protein 27 Using OGX-427 Induces Endoplasmic Reticulum Stress and Potentiates Heat Shock Protein 90 Inhibitors to Delay Castrate-resistant Prostate Cancer

BACKGROUND Although prostate cancer responds initially to androgen ablation therapies, progression to castration-resistant prostate cancer (CRPC) frequently occurs. Heat shock protein (Hsp) 90 inhibition is a rational therapeutic strategy for CRPC that targets key proteins such as androgen receptor (AR) and protein kinase B (Akt); however, most Hsp90 inhibitors trigger elevation of stress proteins like Hsp27 that confer tumor cell survival and treatment resistance. OBJECTIVE We hypothesized that cotargeting the cytoprotective chaperone Hsp27 and Hsp90 would amplify endoplasmic reticulum (ER) stress and treatment-induced cell death in cancer. DESIGN, SETTING, AND PARTICIPANTS Inducible and constitutive Hsp27 and other HSPs were measured by real-time reverse transcription-polymerase chain reaction and immunoblot assays. The combinations of OGX-427 with Hsp90 inhibitors were evaluated in vitro for LNCaP cell growth and apoptosis and in vivo in CRPC LNCaP xenograft models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Tumor volumes were compared using the Kruskal-Wallis test. Overall survival was analyzed using Kaplan-Meier curves, and statistical significance was assessed with the log-rank test. RESULTS AND LIMITATIONS Hsp90 inhibitors induced expression of HSPs in tumor cells and tissues in a dose- and time-dependent manner; in particular, Hsp27 mRNA and protein levels increased threefold. In vitro, OGX-427 synergistically enhanced Hsp90 inhibitor-induced suppression of cell growth and induced apoptosis by 60% as measured by increased sub-G1 fraction and poly(ADP-ribose) polymerase cleavage. These biologic events were accompanied by decreased expression of HSPs, Akt, AR, and prostate-specific antigen, and induction of ER stress markers (cleaved activating transcription factor 6, glucose-regulated protein 78, and DNA-damage-inducible transcript 3). In vivo, OGX-427 potentiated the anticancer effects of Hsp90 inhibitor PF-04929113 (orally, 25mg/kg) to inhibit tumor growth and prolong survival in CRPC LNCaP xenografts. CONCLUSIONS HSP90 inhibitor-mediated induction of Hsp27 expression can be attenuated by OGX-427, resulting in increased ER stress and apoptosis, and synergistic inhibition of CRPC tumor growth. PATIENT SUMMARY This study supports the development of targeted strategies using OGX-427 in combination with Hsp90 inhibitors to improve patient outcome in CRPC.

Discovery of the Highly Potent PI3K/mTOR Dual Inhibitor PF-04979064 through Structure-Based Drug Design.

PI3K, AKT, and mTOR are key kinases from PI3K signaling pathway being extensively pursued to treat a variety of cancers in oncology. To search for a structurally differentiated back-up candidate to PF-04691502, which is currently in phase I/II clinical trials for treating solid tumors, a lead optimization effort was carried out with a tricyclic imidazo[1,5]naphthyridine series. Integration of structure-based drug design and physical properties-based optimization yielded a potent and selective PI3K/mTOR dual kinase inhibitor PF-04979064. This manuscript discusses the lead optimization for the tricyclic series, which both improved the in vitro potency and addressed a number of ADMET issues including high metabolic clearance mediated by both P450 and aldehyde oxidase (AO), poor permeability, and poor solubility. An empirical scaling tool was developed to predict human clearance from in vitro human liver S9 assay data for tricyclic derivatives that were AO substrates.

Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.