Stephen Parsons

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

eIF4E Activation Is Commonly Elevated in Advanced Human Prostate Cancers and Significantly Related to Reduced Patient Survival

Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.

Therapeutic Inhibition of MAP Kinase Interacting Kinase Blocks Eukaryotic Initiation Factor 4E Phosphorylation and Suppresses Outgrowth of Experimental Lung Metastases

Activation of the translation initiation factor 4E (eIF4E) promotes malignant transformation and metastasis. Signaling through the AKT-mTOR pathway activates eIF4E by phosphorylating the inhibitory 4E binding proteins (4E-BP). This liberates eIF4E and allows binding to eIF4G. eIF4E can then be phosphorylated at serine 209 by the MAPK-interacting kinases (Mnk), which also interact with eIF4G. Although dispensable for normal development, Mnk function and eIF4E phosphorylation promote cellular proliferation and survival and are critical for malignant transformation. Accordingly, Mnk inhibition may serve as an attractive cancer therapy. We now report the identification of a potent, selective and orally bioavailable Mnk inhibitor that effectively blocks 4E phosphorylation both in vitro and in vivo. In cultured cancer cell lines, Mnk inhibitor treatment induces apoptosis and suppresses proliferation and soft agar colonization. Importantly, a single, orally administered dose of this Mnk inhibitor substantially suppresses eIF4E phosphorylation for at least 4 hours in human xenograft tumor tissue and mouse liver tissue. Moreover, oral dosing with the Mnk inhibitor significantly suppresses outgrowth of experimental B16 melanoma pulmonary metastases as well as growth of subcutaneous HCT116 colon carcinoma xenograft tumors, without affecting body weight. These findings offer the first description of a novel, orally bioavailable MNK inhibitor and the first preclinical proof-of-concept that MNK inhibition may provide a tractable cancer therapeutic approach.

Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis

ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

Characterization of LY2228820 Dimesylate, a Potent and Selective Inhibitor of p38 MAPK with Antitumor Activity

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

Modulation of 4E-BP1 function as a critical determinant of enzastaurin-induced apoptosis

Enzastaurin (LY317615.HCl) is currently in a phase III registration trial for diffuse large B-Cell lymphoma and numerous phase II clinical trials. Enzastaurin suppresses angiogenesis and induces apoptosis in multiple human tumor cell lines by inhibiting protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K)/AKT pathway signaling. PI3K/AKT pathway signaling liberates eukaryotic translation initiation factor 4E (eIF4E) through the hierarchical phosphorylation of eIF4E binding proteins (4E-BP). When hypophosphorylated, 4E-BPs associate with eIF4E, preventing eIF4E from binding eIF4G, blocking the formation of the eIF4F translation initiation complex. Herein, we show that enzastaurin treatment impacts signaling throughout the AKT/mTOR pathway leading to hypophosphorylation of 4E-BP1 in cancer cells of diverse lineages (glioblastoma, colon carcinoma, and B-cell lymphoma). Accordingly, enzastaurin treatment increases the amount of eIF4E bound to 4E-BP1 and decreases association of eIF4E with eIF4G, thereby reducing eIF4F translation initiation complex levels. We therefore chose to evaluate whether this effect on 4E-BP1 was involved in enzastaurin-induced apoptosis. Remarkably, enzastaurin-induced apoptosis was blocked in cancer cells depleted of 4E-BP1 by siRNAs, or in 4EBP1/2 knockout murine embryonic fibroblasts cells. Furthermore, eIF4E expression was increased and 4E-BP1 expression was decreased in cancer cells selected for reduced sensitivity to enzastaurin-induced apoptosis. These data highlight the importance of modulating 4E-BP1 function, and eIF4F complex levels, in the direct antitumor effect of enzastaurin and suggest that 4E-BP1 function may serve as a promising determinant of enzastaurin activity. Mol Cancer Ther; 9(12); 3158–63. ©2010 AACR.

Abstract C201: The combination of the small molecule TGFβR1 inhibitor LY2157299 monohydrate with CCNU substantially blocks SMAD phosphorylation and significantly suppresses human glioblastoma xenograft growth.

TGF-β signals through the type I and type II TGF-β receptors (TGFβRI and TGFβRII) to phosphorylate the SMAD proteins. In normal tissues, this signaling axis provides a growth inhibitory signal. However, in some cancers, TGF-β is often secreted at high levels and has been implicated in tumor invasion, angiogenesis and decreased immune surveillance. Recent work in GBM indicates that TGFβ may promote aggressive proliferation if the tumor cells are able to express Platelet Derived Growth Factor in response to TGFβ. Indeed, in glioblastoma patient samples, high pSmad2 levels are related to increased proliferation as well as reduced progression-free and overall patient survival. These data strongly implicate aberrant TGFβ signaling in human glioblastoma (GBM) and provide strong rationale to target this pathway for therapeutic intervention. Indeed, phase 1 and 2 clinical trials in GBM patients have begun using the recently developed small molecule inhibitor of TGFβR1 (LY2157299 monohydrate) alone and in combination with lomustine (CCNU). Herein, we show, in GBM cell lines, that LY2157299 monohydrate blocks signaling through the heteromeric TGFβ receptor complex to reduce levels of active, phosphorylated SMAD. Similarly, CCNU treatment of these same GBM cell lines also blocks Smad phosphorylation. In combination, CCNU and LY2157299 monohydrate more effectively block Smad phosphorylation than either agent alone. Furthermore, in U87MG and CRL-2611 human glioblastoma xenografts, oral dosing of LY2157299 monohydrate significantly enhanced the efficacy of CCNU (lomustine). Taken together, these preclinical data support the exploration of LY2157299 monohydrate in combination with CCNU for glioblastoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C201.

Abstract LB-235: Sensitivity of diffuse large B cell lymphoma cells to Enzastaurin (LY317615.HCl) and its primary metabolite (LY326020) is critically dependent upon disruption of the eIF4F translation initiation complex

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Enzastaurin (enza) is currently in phase 3 registration trials for Diffuse Large B Cell Lymphoma (DLBCL) patients at high risk of relapse following R-CHOP therapy. In a phase 2 DLBCL study, 4 of 55 treated patients were progression- free after prolonged, continuous oral enza therapy with 3 of these 4 confirmed as complete responders (Robertson et al., JCO, 2007). The nature of this differential response is unclear, prompting further investigation into the molecular mechanism(s) of enza sensitivity. Enza inhibits PKCβ and related AGC family kinases blocking PKC and PI3K/AKT pathway signaling and inducing apoptosis. In clinical trials, total circulating drug typically reaches steady-state concentrations of 2-4 µM (∼50% enza, ∼50% primary metabolite, LY326020). To identify the critical determinants of sensitivity, we profiled the pro-apoptotic activity of enza and LY326020 in a DLBCL cell panel from both Activated B Cell (ABC) and Germinal Center (GC) subtypes. We show resistant and sensitive DLBCLs from both ABC and GC subtypes. Cells sensitive to enza were also sensitive to LY326020 though, for the first time, we now show that 326020 is decidedly more potent in blocking PI3K-AKT signaling and in inducing apoptosis. In both sensitive and resistant cells, enza and LY326020 reduced phosphorylation of numerous proteins in the PI3K-AKT-TOR pathway (e.g. pGSK3βser9) in a dose and time-dependent manner. However, only sensitive DLBCL cells showed reduced 4EBP1ser65 phosphorylation. By preventing interaction of eIF4E (which binds the 7-mGpppX cap structure of mRNAs) with the scaffolding protein eIF4G, hypophosphorylated 4EBP1 prevents assembly of the translation initiation complex eIF4F (the complex typically elevated in malignancy that preferentially drives translation of potent growth and survival factor mRNAs such as myc, survivin and VEGF). Accordingly, using 7m-GTP co-capture assays to pull down proteins bound to eIF4E, we show a dose and time-dependent increase in 4EBP1: eIF4E binding with concomitant reduction of eIF4E: eIF4G binding. This increase is most pronounced by LY326020. These data indicate that the disruption of the eIF4F complex may be a critical determinant of DLBCL sensitivity to enza and LY326020. Indeed, cells lacking 4EBP1 are insensitive to the pro-apoptotic effects of enza and LY326020, further highlighting the importance of 4EBP1 modulation. Similarly, cells selected for resistance to enza show reduced 4EBP1 expression. These data demonstrate that sensitivity of DLBCL to both enza and LY326020 is critically dependent upon disruption of the eIF4F translation complex. Moreover, these data are the first to show the potent biologic activity of LY326020, the primary metabolite of enza that accounts for ∼50% of total circulating drug in patients and in preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-235. doi:1538-7445.AM2012-LB-235