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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. [Cancer Res 2009;69(9):3866–73]

Supplementary Table 1 from eIF4E Activation Is Commonly Elevated in Advanced Human Prostate Cancers and Significantly Related to Reduced Patient Survival

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. Cancer Res; 71(5); 1849–57. ©2011 AACR.

In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.

Breast cancer survival rates vary according to stage, tumor grade, and receptor status. While some cancer patients respond well to targeted therapy many patients respond poorly. Critical for advancing new therapies is developing a better understanding of the molecular drivers of breast cancer tumorigenesis and malignant progression. The synthesis of new proteins (translation) is important for cancer cell survival, growth, and proliferation. eIF4E is a rate limiting step in translation. Targeting translation has been suggested as a novel strategy for therapy of malignancies originating in the epithelium since eIF4E regulates the translation of multiple malignancy associated mRNAs. eIF4E is activated by phosphorylation downstream of the Mnk 1 and 2 kinases. We hypothesized that phosphorylation of eIF4E plays a critical role in driving breast cancer and is elevated in advanced cancers and metastases. Using immunochemistry and semi-quantitative cellular pathology approaches we analyzed expression of eIF4E and phospho-eIF4E in 168 FFPE archived surgical specimens of normal breast, benign breast disease and pre-invasive, invasive, and metastatic breast cancer. Immunohistochemical stain in the cytoplasm and nuclei of breast epithelial cells was evaluated microscopically by the area of epithelium stained and the intensity of stain (Histoscore = Area X Intensity). We found that expression of eIF4E in the cytoplasm and nuclei of breast epithelial cells was highly correlated with tumor progression (p < 0.0001 and p = 0.0025, respectively). Similarly phospho-eIF4E expression increased in concert with tumor progression (p < 0.0001) in both the cytoplasm and nuclei of breast epithelial cells. Interestingly, while expression of both eIF4E and phospho-eIF4E was not significantly increased in benign breast disease, expression of both eIF4E and phospho-eIF4E was significantly increased in atypical duct hyperplasia (p = 0.01) and in carcinoma in situ (p < 0.0001) as well as in invasive ductal carcinoma (p < 0.0001) relative to normal duct epithelium. Expression of eIF4E and phospho-eIF4E was also increased in lobular carcinoma in situ relative to expression in normal lobules. Breast cancers of all receptor types (ER+/-, PR +/-, Her2+/-, and triple negative) had increased expression of eIF4E and phospho-eIF4E relative to normal duct epithelium. These data are consistent with the conclusion that eIF4E and phospho-eIF4E are critical factors in breast cancer development and progression and that targeting either eIF4E or phosphorylation of eIF4E may be an effective strategy for therapy of all subtypes of breast cancer. Citation Format: Chad A. Dumstorf, Larry E. Douglass, James A. Deddens, Thomas G. Lewis, Kyle Darpel, Christian Gausvik, Jeremy R. Graff, Julia H. Carter. Phosphorylation of eIF4E is a critical factor in development and progression of breast cancer in women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3919.

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.

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. [Cancer Res 2009;69(9):3866–73]

The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced by >90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a nonviral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen-induced lung tumors from 6.4 to 3.7 tumors per mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention. The somatic mutation hypothesis of cancer predicts that the frequency of cancers will also be reduced. (Mol Cancer Res 2009;7(2):247–54)

DNA polymerase ι (pol ι) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol ι performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol η, pol ι, or both. A deficiency in mouse DNA polymerase η (pol η) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol ι, indicating that pol ι participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol η- and pol ι-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol ι status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol η, pol ι, or both. Although pol ι deficiency alone had no effect, UV-induced skin tumors in pol η-deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol ι. Collectively, these data reveal functions for pol ι in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.

In numerous experimental cancer models, overexpression of eukaryotic translation initiation factor 4E (eIF4E) can drive oncogenic transformation, enable invasiveness and facilitate metastasis. eIF4E binds the 7-methylguanosine cap structure at the 5’ terminus of cellular mRNAs, recruiting these mRNAs into the multi-protein translation complex known as eIF4F. The eIF4F complex is comprised of eIF4E, the eIF4G scaffolding protein and the ATP-dependent RNA helicase, eIF4A. In addition, accessory factors (e.g. the Mnk kinases) can interact with this complex, further refining and regulating the function of the complex and the translation of mRNAs. A wealth of literature has accumulated over the past 20 years demonstrating that enhanced eIF4E function, a common event in many human cancers and experimental cancer models, selectively and disproportionately upregulates the translation of potent growth and survival factors implicated in all aspects of malignancy- dysregulated cellular growth control (c-myc, cyclinD1), enhanced cellular survival (survivin, BCL-2, MCL-1), angiogenesis (VEGF, FGF-2), invasiveness and metastasis (MMP9, osteopontin). The oncogenic function of eIF4E is critically dependent upon the phosphorylation of eIF4E at serine 209- an event exclusively controlled by the Mnk1/2 kinases and dependent upon the binding of both eIF4E and the Mnk kinases to the eIF4G scaffolding protein. We sought to understand better the dynamics of eIF4F complex assembly and eIF4E phosphorylation. Using pharmacologic inhibitors of eIF4F assembly (rapamycin, 4EGI-1, Torin, AZD8055), we now show that eIF4E phosphorylation can be induced- independent of the assembly of the eIF4F complex. This increase in eIF4E phosphorylation was blocked by addition of the Mnk inhibitor cercosporamide, suggesting the event remains Mnk-dependent. Further supporting this notion, eIF4E phosphorylation could not be induced by any treatment in Mnk double knockout mouse embryo fibroblasts (DKO-MEFs). To delineate definitively whether eIF4E could be phosphorylated independent of the eIF4F complex, we transfected glioma cells and H293T cells with eIF4E mutants that cannot bind eIF4G (W73F, G139D and the W73F + G139D double mutants). Phosphorylation of these mutants was evident in all cells tested. In DKO-MEFs, these eIF4E mutants were also phosphorylated when co-transfected with wild-type Mnk1 or Mnk2. Importantly, these eIF4E mutants that cannot bind eIF4G could also be phosphorylated in the DKO-MEFS when co-transfected with Mnk 1 or 2 mutants that also fail to bind eIF4G. These data indicate that the eIF4E interaction with Mnk does not require binding to the eIF4G scaffolding protein and, collectively, suggest a revised model for eIF4E phosphorylation- one dependent upon the Mnk kinases but not necessarily dependent on eIF4F complex assembly. Citation Format: Chad Hall, Chad Dumstorf, Bruce Konicek, Nathaniel Robichaud, Ann McNulty, Steve Parsons, Jerry Pelltier, Nahum Sonenberg, Jeremy R. Graff. eIF4E phosphorylation is Mnk-dependent but does not require assembly of the eIF4F translation initiation complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2014-LB-302

Abstract 1336 Enzastaurin (Enza) is an ATP-competitive inhibitor of PKCβ and related AGC family kinases and blocks signaling through the PI3K/AKT/TOR pathway. Accordingly, in preclinical tumor models, clinically achievable Enza doses suppress phosphorylation of AKT, GSK3β, ribosomal protein S6, mTOR, p70S6 Kinase and 4EBP1. Enza has shown significant activity in Diffuse Large B Cell Lymphoma (DLBCL) patients under clinical study. 4/55 DLBCL patients treated with Enza were progression- free after prolonged, continuous oral enza therapy, with 3/4 confirmed as complete responders (Robertson et al., JCO, 2007). Furthermore, preliminary analyses of intermediate or high-risk DLBCL patients treated with R-CHOP vs. Enza + R-CHOP suggest that Enza improves both progression-free survival and complete response rates (Hainsworth et al., JCO, 29: 2011, suppl; abstract 8016). Enza is currently being investigated in a phase 3 registration trial for DLBCL patients at high risk of relapse following R-CHOP therapy. Gene expression profiling studies have revealed molecular alterations involved in DLBCL. For example, overexpression of PKCβ is particularly evident in recurrent DLBCL and has been associated with reduced patient survival. Similarly, BCL-6, a transcriptional repressor likely involved in the corruption of appropriate B Cell differentiation, is overexpressed in ∼ 70% of DLBCL and the target of translocation in nearly 40% of DLBCL. From these studies, distinct molecular subtypes of DLBCL have emerged. The Activated B Cell (ABC) subtype, which carries the poorest prognosis, is defined by constitutive activation of NFκB whereas the Germinal Center (GC) subtype, which carries a better prognosis, is not. 10% of ABC DLBCLs harbor CARD11 mutations leading to constitutive NFκB activation, while another 20% have mutations in CD79A/B, enabling chronic B Cell receptor signaling. In both ABC and GC subtypes, chronic activation of the B Cell receptor complex signals through PKCβ and the PI3K/AKT pathway. Given its activity in DLBCL patients and its molecular mechanism of action, we chose to investigate the molecular basis for the direct anti-cancer activity of Enza, specifically in DLBCL, and to discern whether sensitivity to Enza is associated with distinct DLBCL subtypes. In multiple DLBCL lines, we show that Enza treatment at clinically achievable concentrations significantly reduces BCL-6 expression. We also show that Enza robustly induces apoptosis in both GC and ABC models, blocking signaling throughout the PI3K/AKT pathway. Moreover, we show in the same DLBCL models that the primary metabolite of enza (LY326020.HCl), which comprises ∼50% of circulating drug levels in patients, more potently inhibits these same signaling pathways and more robustly induces apoptosis. Importantly, we have also identified cell lines that are resistant to both enza and its metabolite. Profiling differences between these resistant and sensitive DLBCL lines, we now show that the pro-apoptotic activity of Enza and LY326020 requires control of the eIF4F translation initiation complex- the complex responsible for enabling translation of critical, malignancy-related mRNAs (e.g. VEGF, BCL-2, c-myc). In insensitive DLBCL cells, 4EBP1 expression levels are low to non-existent, even prior to treatment. In sensitive DLBCL cells, Enza and LY326020 reduce 4EBP1ser65 phosphorylation in a dose and time-dependent manner, increasing 4EBP1: eIF4E binding thereby precluding eIF4E:eIF4G binding and preventing eIF4F complex assembly. Furthermore, selection of DLBCL cells for resistance yields cells with reduced 4EBP1 expression, increased eIF4G expression, or both, highlighting the critical importance of this complex for Enza activity. Collectively, these data show that: 1) Enza and LY326020 have direct anti-cancer activity in DLBCL cells regardless of ABC or GC subtype; 2) Enza and LY326020 directly impact numerous molecular alterations driving DLBCL (BCL-6 expression, PKCβ and PI3K-AKT pathway signaling); 3) the ability to control eIF4F complex assembly is critical for enza and LY326020 –induced apoptosis; and 4) LY326020 is more potent than Enza at blocking signaling and inducing apoptosis. Moreover, these data suggest that baseline expression levels of 4EBP1 and/or levels of phosphorylated eIF4E (an indicator of active eIF4F complex) may be particularly informative in delineating patients most sensitive to treatment. Disclosures: Graff: Eli Lilly and Company: Employment. McNulty:Eli Lilly and Company: Employment. Dumstorf:Eli Lilly and Company: Employment. Konicek:Eli Lilly and Company: Employment. Hall:Eli Lilly and Company: Employment. Parsons:Eli Lilly and Company: Employment.

8082 Background: Enzastaurin (enza) is in ph 3 registration trials for 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 molecular mechanism for this differential response is unclear. Methods: In clinical trials, Enza yields 2-4 µM total circulating drug, comprised of ~50% enza, ~50% primary metabolite, LY326020. We therefore evaluated the sensitivity of a DLBCL cell panel representing both Activated B Cell (ABC) and Germinal Center (GC) subtypes to enza and LY326020. Gene expression analyses, western blotting to explore intracellular signaling and mRNA cap analogue co-capture assays were used to identify the critical effectors of drug sensitivity. Results: For the first time, we show the profound biological activity of LY326020, the primary metabolite that accounts for ~ 50% of circulating drug in patients. Like Enza, though more potently, LY326020 inhibits PKC and PI3K-AKT-TOR pathway signaling and robustly induces apoptosis in both ABC and GC DLBCL cells. 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. Accordingly, we show a dose and time-dependent increase in 4EBP1: eIF4E binding. This increase is most pronounced by LY326020. Moreover, cells selected for resistance to enza show reduced 4EBP1 expression and cells lacking 4EBP1 are insensitive to the pro-apoptotic effects of enza and LY326020. Conclusions: These data demonstrate that sensitivity of DLBCL to both enza and LY326020 is critically dependent upon 4EBP1 modulation and subsequent 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.

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

In a wide variety of malignancies, progressive disease and reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enhances the translation of potent growth and survival factors and oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1, MMP-9, etc.). Recent published reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of the MNK kinases (Map kinase interacting kinase 1 and 2), which phosphorylate eIF4E at S209 (p4ES209). Increased p4ES209 has been linked to a variety of advanced cancers, notably head and neck and non-small cell lung cancers. Herein, we report that p4ES209 is significantly elevated in human prostate cancer (CaP) tissues (n=133) and associated with reduced patient survival. p4ES209 is elevated In both low grade (Gleason score ≤ 6) and high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p < 0.02 and p < 0.0001, respectively). Elevated p4ES209 in invasive CaP is related to significantly reduced patient survival (p = 0.0216) and increased risk of death from CaP (hazard ratio = 2.729). Moreover, p4ES209 is evident in the LNCaP, LNAI, CWR22Rv1 CaP cell lines, with levels being highest in the castrate-resistant cells (LNAI and CWR22Rv1). shRNA-mediated knock-down of MNK 1 and 2 reduced p4ES209 levels and specifically triggered apoptosis. Similarly, treatment of these cell lines with the novel, orally-bioavailable MNK inhibitor, cercosporamide, reduced p4ES209 in a dose-dependent manner and induced apoptosis coincident with reduced expression of key translationally controlled proteins (e.g. Mcl-1). Further, oral administration of this inhibitor can suppress p4ES209 in normal mouse tissues and in xenografted human cancers within 30 minutes, lasting at least 4 hours in tumor tissue. These data demonstrate the utility of cercosporamide in probing MNK function in vitro and in vivo and highlight the potential utility of targeting MNK for prostate cancer 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 A237.

Malignant progression & reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enables translation of potent growth/survival factors & oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1). Recent reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of MNK kinases (Map kinase interacting kinases 1 and 2). Furthermore, increased eIF4E phosphorylation, a consequence of Mnk activity, has been linked to advanced cancers, including head & neck & non-small cell lung cancers. Herein, by immunohistochemical analysis of 133 prostatic tissues from 76 men, we report that eIF4E phosphorylation is significantly elevated in human prostate cancers (CaP) in relation to disease progression. eIF4E phosphorylation is associated with advanced disease (trend analysis, p < 0.0001) & is increased significantly in both low grade (Gleason score ≤ 6) & high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p < 0.02 and p < 0.0001, respectively). Elevated eIF4E phosphorylation in invasive prostate cancer CaP is related to significantly reduced patient survival (p = 0.0216) & increased risk of death from CaP (hazard ratio = 2.729). These data, along with data in experimental models implicating MNK activity in cellular transformation, indicate that therapeutic targeting of MNK activity may provide an attractive anti-cancer therapeutic strategy. Indeed, we now report the identification of a novel, small molecule Mnk inhibitor with IC50 values of 116 nm & 11nm against Mnk 1 & Mnk 2, respectively. This Mnk inhibitor suppresses eIF4E phosphorylation in a dose & time-dependent manner in a wide array of cancer cell lines, including melanomas, colorectal cancers, lymphomas, leukemias & prostate cancers. In HCT116 colon cancer, treatment with the Mnk inhibitor suppresses eIF4E phosphorylation and diminishes expression of the pro-survival factor Mcl-1. Mnk inhibitor treatment also suppresses cellular proliferation, induces apoptosis & dramatically diminishes soft agar colonization. Further, oral administration of this inhibitor suppresses eIF4E phosphorylation in normal mouse tissues and in xenografted human cancers within 30 minutes, lasting at least 4 hours in tumor tissue. Once daily (20mg/kg) or twice daily (10mg/kg) oral dosing significantly suppresses subcutaneous xenograft tumor growth. Moreover, oral dosing reduces dramatically the number and size of pulmonary metastases formed in mice injected intravenously with B16 melanoma cells & treated 12 consecutive days starting 1 day after cell injection. These data demonstrate the utility of this novel Mnk inhibitor in probing Mnk function in vitro & in vivo and highlight the potential anti-cancer therapeutic utility of suppressing Mnk function. 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 LB-384. doi:10.1158/1538-7445.AM2011-LB-384