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Background Dermatologists would benefit from an easy to use psoriasis severity assessment tool in the clinic. Objective To develop psoriasis assessment tools to predict PASI and Dermatology Life Quality Index (DLQI) using simple measures typically collected in clinical practice. Methods Data included 33 605 dermatology visits among plaque psoriasis patients enrolled in the CorEvitas Psoriasis Registry (4/15/15-7/11/20). Performance (adjusted coefficient of determination [R2adj], root mean square error [RMSE]) in predicting PASI and DLQI was assessed for 16 different linear regression models (specified a priori based on combinations of BSA, Investigator’s Global Assessment [IGA], itch, skin pain, patient global assessment, age, sex, BMI, comorbidity index, prior biologic use), and 2 stepwise selection models and 1 elastic net model based on 56 available variables. For each prediction model, concordance (sensitivity, specificity) of predicted PASI75, PASI90 and DLQI 0/1 with observed values was evaluated. Results Mean (SD) age, BSA, and PASI were 51 (14) years, 6 (11), and 4 (6), respectively; 46% were women, and 87% were biologic experienced. A model predicting PASI using BSA plus IGA performed best among a priori specified models (R2adj = .72, RMSE = 2.93) and only marginally worse than models including additional variables (R2adj range .64-.74, RMSE range 2.82-3.36). Models including IGA had the best concordance between predicted and observed PASI75 (sensitivity range 83-85%, specificity range 88-91%) and PASI90 (sensitivity range 76-82%, specificity range 94-98%). DLQI prediction was limited. Conclusion An assessment tool for psoriasis including BSA and IGA may be an ideal option to predict PASI in a clinic setting.

The aim of this work is to describe real-world biologic-experienced psoriasis patients initiating ixekizumab by prior biologic therapy status and compare the effectiveness of ixekizumab between patients who previously failed secukinumab and those who failed other biologics. We hypothesized that (1) clinical outcomes and patient-reported outcomes would improve following a switch to IXE, and (2) there would be no differences in responses between patients who previously failed secukinumab and those who failed other biologics.Participants (n = 419) included adult psoriasis patients enrolled in the CorEvitas Psoriasis Registry through 9/10/20 who switched to ixekizumab after discontinuing another biologic. Patients were classified by the biologic used immediately prior to ixekizumab and reason for discontinuation: prior secukinumab failure; prior secukinumab non-failure; prior other biologic failure; and prior other biologic non-failure. Discontinuations for efficacy reasons were considered failures; all others were considered non-failures. Baseline descriptive statistics were calculated. Multivariable Poisson regression models estimated the likelihood of response of other failure relative to secukinumab failure.Mean age was 51 years; 48% were women. Psoriasis disease characteristics were similar across prior biologic groups. At 6-month follow-up, disease severity improved for all who initiated ixekizumab after discontinuing another biologic. Secukinumab failure patients who switched to ixekizumab achieved BSA ≤ 1 (49%), BSA ≤ 3 (59%), PASI75 (46%), PASI ≤ 3 (64%), and IGA ≤ 1 (40%). Other failure patients achieved BSA ≤ 1 (55%), BSA ≤ 3 (72%), PASI75 (59%), PASI ≤ 3 (74%), and IGA ≤ 1 (54%). In regression modeling, we observed patients in the other biologics failure group had an increased likelihood of achieving response for BSA ≤ 3, PASI75, PASI90, PASI100, and IGA ≤ 1 compared to patients who failed secukinumab.These findings suggest that patients with psoriasis who switch to ixekizumab after discontinuing another biologic demonstrate improvement in disease severity after six months. Patients who discontinued biologics other than secukinumab may be more likely to respond to ixekiziumab compared to those who switched from secukinumab.

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

Background Long-term efficacy, safety, and quality of life with ixekizumab (IXE) through 5 years in UNCOVER-1 and UNCOVER-2 patients with baseline scalp, nail, or palmoplantar psoriasis were assessed. Methods Patients included in this intent-to-treat subanalysis had baseline involvement in at least one of the three anatomic areas (scalp, fingernail, or palmoplantar locations) and 1) received IXE through week 60, with a 160-mg starting dose 80 mg Q2W through week 12 and Q4W thereafter, 2) achieved a static Physicianrs Global Assessment score of 0 or 1 at week 12, and 3) completed week 60 and continued treatment with IXE Q4W or were escalated to Q2W during the long-term extension. Efficacy outcomes (e.g., percent improvement in Psoriasis Scalp Severity Index [PSSI], Nail Psoriasis Severity Index [NAPSI], Palmoplantar Psoriasis Area and Severity [PPASI], and Dermatology Life Quality Index [DLQI]) were summarized by descriptive statistics through week 264. Results Patients rapidly achieved and sustained improvements in scalp, nail, and palmoplantar psoriasis for up to 5 years with IXE. Patients achieved complete clearance at year 5: observed (scalp, 82%; nail, 73%; palmoplantar, 96%) and mNRI (scalp, 77%; nail, 67%; palmoplantar, 85%). Up to 80% of patients reported DLQI 0,1 responses at week 12, which were sustained through week 264. No increases in the number of annual treatment-emergent adverse events were observed from years 1n5. Conclusion Patients receiving IXE for 5 years sustained high rates of improvement in scalp, nail, and palmoplantar psoriasis, with a long-term quality of life benefit with no unexpected safety signals. J Drugs Dermatol. 2021;20(8):880-887. doi:10.36849/JDD.6101.

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

Summary Purpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.

Summary: Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1−/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1−/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. : Fragile X syndrome (FXS) is caused by dysregulation of translation in the brain. Gkogkas et al. show that phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) is increased in FXS postmortem brains and Fmr1−/y mice. Downregulation of eIF4E phosphorylation in Fmr1−/y mice rescues defects in dendritic spine morphology, synaptic plasticity, and social interaction via normalization of MMP-9 expression.

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.

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.

The combination of tumor targeted therapeutics with PD-L1 checkpoint blockade is being explored as a method to increase the clinical benefits of immunotherapy, and expand response to additional cancer types. Merestinib (Mer) is a kinase inhibitor targeting several oncokinases1 (including MET, MST1R, AXL, MERTK, and MKNK1/2) that can potentially modulate immune function, angiogenesis, as well as target the tumor 1-5. To determine the combinatorial potential with immunotherapy, the effects of Mer were evaluated in vitro on human T cells, PBMCs and murine tumor lines CT26 colon carcinoma (harbors KRASmt G12D expresses low Met/no p-Met/high Axl/p-Axl) and B16F10 melanoma (expressing high Met/pMet/peIF4E). Additionally, the anti-tumor effect of Mer was tested in vivo on established CT26 and B16F10 tumors compared to MET specific TKIs (savolitinib, PF4217903) alone or in combination with PD-L1 antibody (Ab) blockade. In vitro, Mer showed no significant effects on either T cells or PBMCs, but was able to inhibit downstream signaling in both CT26 and B16F10 showing activity on murine tumor cell lines. In vivo, daily Mer monotherapy (6, 12 or 24 mg/kg) showed significant anti-tumor effect at all doses in both CT26 and B16F10, that was not seen with either savolitinib or PF4217903. Concurrent combination of Mer (12 mg/kg) and anti-PD-L1 Ab (0.5 mg qw) in CT26 was found to have anti-tumor activity that was synergistic as compared to each single agent alone. While the effect of Mer monotherapy was lost when treatment ended, tumors continued to regress in the combination group even upon cessation of therapy. The combination was well tolerated and resulted in 90% complete responders compared to 30% with anti-PD-L1 Ab alone, 35 days after completing dosing. To test the ability to generate immunologic memory, complete responders were re-challenged with CT26 cells on the contralateral side. All mice in the combination group resisted re-challenge, showing that Mer/PD-L1 Ab combination was triggering immunologic memory. Although there was no significant change in intra-tumor immune cell populations between groups, combination therapy showed an enhanced and unique intra-tumor immune activation/inflammation gene expression signature compared to PD-L1 Ab monotherapy. The enhanced immune activation of the combination therapy, leading to synergistic anti-tumor efficacy, demonstrates that merestinib has the potential to augment immunotherapy while targeting the tumor directly. This preclinical data provides the rationale for the clinical investigation of merestinib in combination with checkpoint therapies targeting the PD-L1/PD1 axis (NCT02791334). 1 - Yan et al. Invest New Drugs 2013;31:833-44 2 - Balan et al. J Biol Chem 2015;290:8110-20 3 - Eyob et al. Cancer Discov 2013;3:751-60 4 - Lemke G. CSH Persp Biol 2013;5:a009076 5 - Piccirillo et al. Nat Immunol 2014;15:503-11 Citation Format: Sau-Chi Betty Yan, Victoria L. Peek, Jennifer R. Stephens, Um L. Um, Amaladas Nelusha, Colleen A. Burns, Kelly M. Credille, Thompson N. Doman, Scott W. Eastman, Beverly L. Falcon, Gerald E. Hall, Philip W. Iversen, Bruce W. Konicek, Jason R. Manro, Any T. Pappas, Julie A. Stewart, Michael B. Topper, Swee-Seong Wong, Michael Kalos, Ruslan D. Novosiadly, Richard A. Walgren, David Schaer. Combination of an oncokinase inhibitor merestinib with anti-PD-L1 results in enhanced immune mediated antitumor activity in CT26 murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5590. doi:10.1158/1538-7445.AM2017-5590

Background: MET ex14 skipping, present in ~3% of lung cancer, is a strong oncogenic driver which is further evidenced by case reports of patients response to MET TKI treatment. ~15% of tumors in patients that harbor MET ex14 skipping also have MET amplification (amp). Merestinib is a type II MET kinase inhibitor1. Emibetuzumab, a bivalent MET Ab, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET ex14 skipping and MET amp. Methods: Each agent was evaluated in vitro for inhibition of Hs746t cell proliferation and pMET levels. In vivo study in Hs746t-derived xenograft mouse model (n=7 mice/ arm, 28 day dosing) initiated when tumors were 150-350mm3: merestinib at 6 mg/kg (suboptimal dose - insufficient target coverage for 24 hrs) or 12 mg/kg (optimal dose) qd orally, emibetuzumab at 10 mg/kg qw by IP. Results: Merestinib inhibited Hs746t cell proliferation with IC50=34 nM and totally eliminated pMET at 65-100 nM. Emibetuzumab slightly inhibited Hs746t cell proliferation (IC50>100 nM), reduced 10-20% cell surface MET, and no effect on pMET expression (at 130-650 nM). In the Hs746t xenograft model, merestinib (12 mg/kg) treatment resulted in 91.8% tumor regression after 21 day dosing, while 6 mg/kg merestinib provided transient tumor regression followed by re-growth while on treatment with T/C=18.3% after 21 day dosing. No tumor re-growth was observed in 6/7 mice in the 12 mg/kg merestinib cohort during the 5 weeks post-treatment. Emibetuzumab treatment provided transient tumor regression (37.7%) after 3 doses, but tumors re-grew while on treatment. Combination of 6 mg/kg merestinib and 10 mg/kg emibetuzumab resulted in 85% tumor regression for the duration of the 28 day dosing period and the treatment was well tolerated. Tumors in animals re-grew upon termination of this combination treatment. Conclusion: Merestinib (12 mg/kg) treatment resulted in durable and complete response in 6/7 mice bearing Hs746t tumors with MET ex14 skipping and MET amp. When used singly, merestinib (6 mg/kg) or emibetuzumab (10 mg/kg) resulted in only transient tumor regression in this model, while the combination resulted in substantial tumor regression while on treatment. This combination treatment was however, not as durable as was observed with single agent 12 mg/kg merestinib. Single agents and the combinations were well tolerated. As a type II MET inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or those who have progressed on type I MET inhibitor treatment, whose tumors have MET exon 14 skipping and/or MET amplification 2,3. Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). 1 - Yan et al. Invest New Drugs 2013;31:833-844 2 - Ou et al. J Thorac Oncol. 2016; PMID:27666659 3 - Heist et al. J Thorac Oncol. 2016;11:1242-1245 Citation Format: Sau-Chi Betty Yan, Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Wei Zeng, Bruce W. Konicek, Ling Liu, Volker Wacheck, Richard A. Walgren. Evaluation of single agent merestinib (LY2801653) or emibetuzumab (LY2875358) and the combination in a xenograft tumor model bearing MET exon 14 skipping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 528. doi:10.1158/1538-7445.AM2017-528

NF1 mutant cancers are driven by excessive Ras signaling; however, there are currently no effective therapies for these or other Ras-dependent tumors. While MEK inhibitors promote regression of NF1-deficient premalignant lesions, they are ineffective in advanced cancers. Our lab and others have shown that the addition of a PI3K/AKT/mTORC1 inhibitor can co-operate with MEK inhibition to elicit tumor regression in pre-clinical Ras-driven tumor models; however, one concern is that these combinations might not be tolerated in the clinic due to toxicity. Therefore our goal has been to identify more refined, cancer-specific signaling nodes within these pathways. Here we find that eIF4E, a key downstream effector of mTORC1, is constitutively phosphorylated by the MAP kinase-interacting kinases (MNK1 and 2) in NF1 mutant malignant peripheral nerve sheath tumors (MPNSTs). Moreover, we find that combined genetic or chemical suppression of MNK kinases can cooperate with MEK inhibition to reduce cell viability to a similar extent as broad mTOR inhibition. Surprisingly, we find that the MNK kinases are important, previously unrecognized direct targets of the receptor tyrosine kinase inhibitor cabozantinib. Furthermore, we find that cabozantinib but not a more selective MET inhibitor kills NF1-deficient tumor cells when combined with MEK inhibition, and does so in part via eIF4E phosphorylation inhibition. Finally, we show that combining cabozantinib and MEK inhibitors triggers dramatic tumor regression in an aggressive genetically engineered mouse model of MPNSTs. Together, these results identify MNK/eIF4E as a targetable signaling axis when combined with MEK inhibition in these Ras-driven tumors and highlight the utility of both drug repurposing and developing more specific MNK inhibitors for these and other malignancies. Citation Format: Rebecca Lock, Rachel Ingraham, Ophélia Maertens, Bruce Konicek, Sau-chi Yan, Jeremy Graff, Karen Cichowski. Targeting the MNK kinases for the treatment of NF1-mutant cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A18.

Research in our laboratory is aimed at understanding the cellular and molecular mechanisms that govern colony stimulating factor-1 (CSF-1) gene expression. Our hypothesis is that a basal set of trans-acting factors is bound to the CSF-1 gene during fibroblast proliferation, resulting in constitutive CSF-1 gene expression. Modulation of CSF-1 gene transcription by growth-arrest (decrease) or stimulation of growth-arrested fibroblasts (re-initiate) is mediated by changes in the basal set of factors bound and/or by the addition of stimulus-specific factors. We have extended our hypothesis to include other cell types (monocytes) to determine if mechanisms used to control CSF-1 gene expression in fibroblasts are unique or represent common nontissue-specific regulatory mechanisms. Analysis of CSF-1-CAT reporter constructs in transiently transfected fibroblasts and monocytes was used to identify CSF-1 genomic sequences that affect transcriptional activity. DNase I protection, electrophoretic mobility shift, and methylation interference assays were used to identify the putative cis-acting elements. Results of our study suggest multiple trans-acting factors may regulate CSF-1 gene expression; some may be tissue specific, while others, such as AP1, CTF/NF1, Sp1, and Sp3, are shared in common.

Mnk kinases regulate the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), a protein that plays key roles in the initiation of messenger RNA translation and whose activity is critical for various cellular functions. eIF4E is deregulated in acute myeloid leukemia (AML), and its aberrant activity contributes to leukemogenesis. We determined whether cercosporamide, an antifungal agent that was recently shown to act as a unique Mnk inhibitor, exhibits antileukemic properties. Treatment of AML cells with cercosporamide resulted in a dose-dependent suppression of eIF4E phosphorylation. Such suppression of Mnk kinase activity and eIF4E phosphorylation by cercosporamide resulted in dose-dependent suppressive effects on primitive leukemic progenitors (CFU-L) from AML patients and enhanced the antileukemic properties of cytarabine (Ara-C) or mammalian target of rapamycin (mTOR) complex 1 inhibition. Similarly, the combination of cercosporamide with cytarabine resulted in enhanced antileukemic responses in a xenograft mouse model in vivo. Altogether, this work demonstrates that the unique Mnk inhibitor cercosporamide suppresses phosphorylation of eIF4E and exhibits antileukemic effects, in support of future clinical-translational efforts involving combinations of Mnk inhibitors with cytarabine and/or mTOR inhibitors for the treatment of AML.

In cancer, the formation of chimeric gene fusions by genomic rearrangement causes aberrant receptor tyrosine kinase activation resulting in sustained oncogenic signaling driving tumorigenesis. Neurotrophic tyrosine receptor kinase 1 (NTRK1), the cognate receptor for nerve growth factor (NGF), has been reported in 7 tumor types as a NTRK1 kinase domain fused with several reported partners including the 5’ coiled-coil domain of the tropomysin TPM3 gene. The resultant NTRK1 fusion protein is present in about 1.5% of colorectal cancer (CRC), 3% of lung and 12% of papillary thyroid cancers. In addition, gene fusions involving NTRK2 and NTRK3 are present in about 19 different tumor types. Thus pharmacologically targeting NTRK kinase in cancers bearing NTRK fusions may provide treatment options to patients who otherwise might be resistant to standard oncolytic regimens. Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, ROS1 and MKNK1/2. Merestinib and its two primary metabolites, M1 (LSN2800870) and M2 (LSN2887652) were shown in scanMaxSM kinase binding assays to inhibit all three NTRKs with an IC50 ranging from 15-320 nM, and in the cell-based PathHunter® NTRK1 assay with an IC50 ranging from 12-92 nM. Merestinib, M1 and M2 were evaluated in vitro in TPM3-NTRK1 fusion bearing CRC cells (KM-12). Merestinib, M1 and M2 reduced p-NTRK1 levels, cell proliferation (IC50 of 11 nM, 18 nM and 100 nM respectively) and anchorage independent growth (IC50 of 45 nM, 79 nM and 206 nM respectively). Crizotinib previously reported (Nat Med. 2013;19:1469-72) to have moderate activity against NTRK1, was used to treat a patient with NTRK1 fusion resulted with transient response. Crizotinib was shown here to also reduce p-NTRK1 levels, cell proliferation (IC50 = 88nM) and anchorage independent growth (IC50 = 276nM) in vitro in KM-12 cells. Merestinib treatment at 24 mg/kg once daily arrested tumor growth (T/C = 4%) in KM-12 xenograft tumor bearing mice. Crizotinib administered at 25 mg/kg twice daily in this same model did not result in tumor growth arrest (T/C = 39.5%). Merestinib treatment at 24 mg/kg once daily led to tumor regression in a CRC PDX xenograft model (EL1989) bearing the TPM3-NTRK1 fusion. Crizotinib treatment at 25 mg/kg twice daily in this model did not show tumor regression. Further pre-clinical studies of Merestinib inhibition of NTRK2 and NTRK3 gene fusion are ongoing. These data support the clinical evaluation of Merestinib in patients with tumors harboring NTRK fusion. Merestinib is currently being studied clinically in advanced cancers (NCT01285037). Citation Format: Bruce W. Konicek, Steve M. Bray, Andrew R. Capen, John N. Calley, Kelly M. Credille, Philip J. Ebert, Gary Heady, Bharvin K. Patel, Victoria L. Peek, Jennifer R. Stephens, Suzane L. Um, Melinda D. Willard, Isabella H. Wulur, Yi Zeng, Richard A. Walgren, Sau-Chi Betty Yan. Merestinib (LY2801653), targeting several oncokinases including NTRK1/2/3, shows potent anti-tumor effect in colorectal cell line- and patient-derived xenograft (PDX) model bearing TPM3-NTRK1 fusion. [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 2647.

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 eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer. [Cancer Res 2008;68(3):631–4]

Activation of protein kinase Cβ (PKCβ) has been repeatedly implicated in tumor-induced angiogenesis. The PKCβ-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCβ has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3βser9, ribosomal protein S6S240/244, and AKTThr308. Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3β in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3β phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3β phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3β phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

The progression of human prostate cancer from the initial androgen-dependent phase to androgen independence involves diminished apoptosis and a release from the cell cycle block triggered by androgen ablation therapy. FOXO transcription factors play a central role in promoting expression of proapoptotic and cell cycle regulatory genes (e.g., FasL and p27KIP1). Reduced FOXO function might, therefore, play a role in androgen-independent progression of human prostate cancer. Herein, we show that FOXO function is compromised in androgen-independent prostate cancer cells (LNAI) versus androgen-dependent LNCaP cells. The FOXO3a protein, the most highly expressed FOXO family member in prostate cancer cells, is hyperphosphorylated in LNAI cells. FOXO3a expression is also markedly reduced in these androgen-independent LNAI cells when compared with parental LNCaP cells. Together, reduced FOXO3a expression coupled to FOXO3a hyperphosphorylation would suppress FOXO transcriptional activity. Accordingly, activity of the FOXO-responsive p27KIP1 promoter is reduced 60% in these LNAI cells when compared with LNCaP cells. Moreover, mutation of a conserved FOXO response element suppresses p27KIP1 promoter activity, substantiating a regulatory role for this FOXO response element in p27KIP1 promoter transactivation. Finally, we show that the activity of a distinct FOXO-responsive promoter, the 3X-IRS promoter, is also reduced in LNAI cells. Collectively, these data show that reduced FOXO3a expression coupled to increased FOXO3a phosphorylation coincide with reduced FOXO-responsive promoter activity in androgen-independent LNAI cells when compared with androgen-dependent LNCaP cells. To the extent that this model reflects human disease, these data suggest that FOXO function may be compromised with androgen-independent progression of human prostate cancer.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cholangiocarcinoma (CCA) is an aggressive and locally invasive biliary tract malignancy with a poor prognosis and no approved drug treatment. LY2801653 is an orally bioavailable small molecule oncokinase inhibitor1 of MET, a receptor reported to be dysregulated and correlated with a poor outcome in CCA. We evaluated additional oncotargets of LY2801653 (MET/HGF, pMET, AXL/pAXL, the MKNK1/2 substrate p-eIF4E, and ROS1) in 7 CCA cell lines (EGI-1, TFK-1, SNU245, SNU478, SNU869, SNU1079, and SNU1196) and 5 CCA patient-derived mouse xenograft (PDX) tumors. Cell line expression of MET, EGFR and p-eIF4E were evaluated by western blot. Although HGF expression was not detected in any of the cell lines by ELISA, pMET was detected in 5 cell lines (not in TFK-1 or SNU1079). ROS1 fusion was not detected in the 7 cell lines using break-apart FISH probes. Very high AXL and pAXL were detected in the SNU1196 line by western blot. Despite the high levels of pAXL, further increases in pAXL were noted after addition of its ligand, Gas6. In the SNU1196 cell line, pAXL expression and cell proliferation were completely inhibited by LY2801653, but not by a MET-specific inhibitor PF4217903. HGF-induced SNU1196 cell migration was inhibited equally well by LY2801653 and PF4217903. Unlike CCA cell line derived mouse xenograft tumors, the 5 PDX tumors retained prominent desmoplastic stroma. As analyzed by immunohistochemistry (IHC), MET was highly expressed in 3 of the 5 PDX tumors, and high levels of p-eIF4E were expressed in all 5. Increased AXL expression (IHC) appeared to be associated with more poorly differentiated PDX tumors. LY2801653 demonstrated potent in vivo anti-tumor activity in several CCA xenograft models. In a xenograft mouse model with the extrahepatic CCA derived EGI-1 cell line, treatment with LY2801653 at 24 mg/kg dosed twice daily resulted anti-tumor effect of 38.5% (% treated/control). In the SNU869 extrahepatic CCA cell line-derived xenograft mouse model, combination treatment with LY2801653 (12 mg/kg twice daily) and either cisplatin (5 mg/kg once weekly) or gemcitabine (60 mg/kg once weekly) resulted in an additive anti-tumor effect as compared to LY2801653 alone. Moreover, the combination of LY2801653 with gemcitabine resulted in tumor regression in this model. In vivo studies are ongoing with LY2801653 in the extrahepatic SNU1196 CCA cell line-derived xenograft model as well as two of the PDX models. The preclinical data in this study support the ongoing phase 1 clinical evaluation of LY2801653 in cholangiocarcinoma patients (trial I3O-MC-JSBA, [NCT01285037][1]). (1 - Yan et al. Invest New Drugs 2013;31:833-44). Citation Format: Sau-Chi Betty Yan, Suzane L. Um, Victoria L. Peek, Megan N. Thobe, Kelly M. Credille, Jennifer R. Stephens, Jason R. Manro, Darryl W. Ballard, Jessica A. Baker, Joel D. Cook, Bruce W. Konicek, Jeremy R. Graff, Timothy R. Holzer, Richard A. Walgren. Preclinical evaluation of LY2801653, an orally bioavailable small molecule oncokinase inhibitor, in cholangiocarcinoma models. [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 4403. doi:10.1158/1538-7445.AM2014-4403 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01285037&atom=%2Fcanres%2F74%2F19_Supplement%2F4403.atom

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

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27(Kip1) cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27(Kip1). To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27(Kip1) expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27(Kip1) expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

Macrophage colony-stimulating factor (M-CSF; CSF-1) is a member of a complex network of cytokines that regulate monocytic cell development and activity. It is produced in nearly all organs by cell types commonly found in connective tissue, including fibroblasts and monocytes. Whether different cell types share common or have divergent mechanisms for regulating CSF-1 gene expression is not known. To address this question, the identity of cis-acting elements and cognate trans-acting factors was characterized in a region of the CSF-1 promoter known to be more active in monocytes than in fibroblasts. The results of DNase I protection assays performed with fibroblast- or monocyte-derived nuclear extracts revealed a difference in the pattern of DNA-binding proteins. One protected region, common to both fibroblasts and monocytes, spans a putative phorbol ester-responsive element (TRE), and binding to the TRE by AP1 was verified with antibodies directed against c-fos and c-jun family members. Mutational analysis revealed that the TRE is required for CSF-1 gene expression in proliferating fibroblasts and monocytes. Binding of a second putative trans-acting factor, preferentially expressed in fibroblasts, to the region immediately upstream of the TRE was also detected. Screening a mouse expression library with oligonucleotides spanning the putative cis-acting element identified cellular nucleic acid-binding protein (CNBP) as the cognate binding activity, and antiserum to CNBP disrupted the electromobility shift assay complex. Mutational analysis revealed that loss of CNBP binding leads to a decrease in CSF-1 promoter activity in fibroblasts but has no effect on CSF-1 promoter activity in monocytes. Our results demonstrate that control of CSF-1 gene expression in monocytes and fibroblasts is mediated by common and cell type-specific trans-acting factors.

BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Under 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. Citation 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.AM2013-3042 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01285037&atom=%2Fcanres%2F73%2F8_Supplement%2F3042.atom

Circulating and tissue-specific monocytes/macrophages, through production of hydrolytic enzymes and growth factors, can dramatically affect the local tissue environment. Colony-stimulating factor-1 (CSF-1) is a key regulator of monocyte/macrophage cell activity. CSF-1 is produced by stromal elements, including fibroblasts, which are found in all tissues. To understand at the molecular level how changes in CSF-1 gene transcription are initiated in fibroblasts, we set out to identify the cis-acting elements and cognate trans-acting factor(s) that bind regulatory regions of the mouse CSF-1 gene. Analysis of heterologous reporter constructs containing the mouse CSF-1 promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in transiently transfected fibroblasts identified a cis-acting element located between base pairs -88 and -43 of the CSF-1 gene. Electrophoretic mobility-shift assays (EMSAs) and DNase I protection assays with nuclear extracts isolated from proliferating fibroblasts revealed distinct protein binding to the region spanning base pairs -90 to -68. Results from methylation interference assays suggest CTF/NF1 or a CTF/NF1-like factor is the cognate trans-acting factor. Mutation of the putative CTF/NF1 binding site in the CSF-1 promoter lead to a modest decrease in promoter activity in transiently transfected fibroblasts and monocytes. Therefore, we have demonstrated that CTF/NF1 or a CTF/NF1-like protein binds to the CSF-1 gene promoter; however, binding of the CTF/NF1-like protein alone does not significantly effect changes in CSF-1 gene promoter activity.

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.

CTCs are tumor cells in circulation in the blood and are believed to be shed from both the primary and metastatic tumors. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers. It is postulated that these cells are genetically similar to cells comprising the patient's tumor(s). The presence of these cells in blood provides a minimally invasive source of nucleic acid for serial monitoring of tumors for disease progression and emergence of drug treatment resistance without the risks of traditional solid tumor biopsy. Activating mutations in the KRAS gene are frequently found in human cancers (up to 30% of cancer cases), and are present in a large portion of non-small cell lung cancer (NSCLC), pancreatic, and colorectal cancers. Reported mutations are mostly single point mutations in codons 12, 13 and 61, with the majority of mutations present in codon 12. Importantly, KRAS mutation status can be predictive of response to treatment, and may be used to determine the best therapeutic strategy. Therefore, it is critical to develop a robust method for detecting mutations from a limited number of enriched tumor cells (e.g. CTCs or fine needle aspirate) in a high background of normal cells. Our studies utilize competitive allele-specific PCR (castPCR) for detecting mutant KRAS from human cancer cells spiked into and retrieved from blood, and from fixed cells embedded in paraffin. This technology is reportedly capable of detecting one mutant allele molecule in 10 million wild-type molecules. KRAS is mutated in human NSCLC H441 cells (G12V) and in KP-4 pancreatic cells (G12D). After spiking H441 and KP-4 cells into human blood, cells were captured/enriched and enumerated by the Veridex CellTracks™ system. Both CXC and Profile kits (Veridex) were used. Recovered cells were then lysed overnight with Arcturus® PicoPure® DNA extraction lysis buffer, and the resulting DNA was analyzed for mutant KRAS with the castPCR TaqMan® Mutation Detection Assay (Applied Biosystems) specific for the mutation found in H441 cells (G12V). Under these conditions, we detected KRAS mutation in H441 cells with robust sensitivity (20 spiked cells into 7.5ml of blood; KP-4 cells were negative for the G12V mutation). To further examine if this method could be used for detecting mutations in human tissue, FFPE tumor cells were analyzed. The WaxFreeTM DNA kit (TrimGen) was used to extract DNA from FFPE cells, and castPCR was performed. H441 mutant KRAS was detected from as little as ∼200 pg DNA (100-fold dilution of DNA). Ongoing studies involve correlating mutation status between CTCs and tumor FFPE tissue. These data will aid in clinical biomarker development used in clinical trials and improve methods to determine the most beneficial treatment options for patients. Importantly, this approach may enable patients to be serially monitored for tumor mutation status with blood draws. 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 2087. doi:1538-7445.AM2012-2087

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

Summary The HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min−1 and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). Electronic supplementary material The online version of this article (doi:10.1007/s10637-012-9912-9) contains supplementary material, which is available to authorized users.