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The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

Supplementary Methods

In vivo effect of treatment on body weight loss.

Summary of antitumor activity of LY3214996 and other MAPK pathway inhibitors in cancer models with dominant MAPK pathway alterations

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

Wind assisted propulsion vessels have presented itself as a revolutionary alternative to reduce emissions in a vessel. With the increasing regulations on emission control, many companies have started adopting wind assisted ships into their fleet. With increasing demand so does the need for a deep and better understanding on various aspects of such vessels especially into the operational aspects. This thesis aims at Estimating and analysing the added resistance of a wind assisted vessel and the contribution of drift angle seen in such vessels to added resistance. This is achieved by an experimental study using a wind assisted bulk carrier design, "SOShip" designed by SINTEF Ocean, in various wave conditions simulating a wind assisted vessel with a drift angle of 9 degree and conditions without a drift angle. Followed by a Numerical analysis using state of the art numerical panel codes used in the industry such as WAMIT,VERES3D and VERES (ShipX). The numerical study also aims at creating an outlook on the capabilities of various numerical codes and methods used within the codes to predict first order motions (heave and pitch) and added resistance in various wave conditions. The thesis also includes a brief theory on how to find first order solutions, second order mean drift forces and also covers some particular aspects in the theory used behind each of the numerical code which are of interest for the study. The experimental study concludes that a drift angle increases the added resistance by 5-10% and the numerical study concludes that a drift angle of 9 degree causes less than 5% increase in added resistance. The Experimental estimation of added resistance for shorter waves poses large uncertainty which makes it difficult to make conclusions about added resistance in short wavelengths and also to validate the numerical results. At zero Froude number experimental added resistance estimation and numerical estimation are in good agreement. WAMIT V7 well predicts the effect of tank walls in added resistance for zero speed test conditions. At forward speed the added resistance curve along the wavelength found experimentally has a shift to the direction of smaller wavelengths in comparison to the numerical results. At following sea conditions all the numerical codes predict an added thrust in most of the wavelengths which contradicts the experimental results. The numerical results for added resistance in forward speed is observed to be sensitive to the changes in the panel model of the vessel which can generate misleading estimations.

The combination of BRAF and MEK inhibitors have been clinically studied, and three different combinations (dabrafenib plus trametinib, vemurafenib plus cobimetinib and encorafenib plus binimetinib) are approved by the FDA for treatment of melanoma patients with BRAF V600E mutation. Published reports indicate that each combination has a different efficacy and toxicity profile. It is believed that the toxicity profile of some of these drugs is linked to their pharmacokinetic (PK) profile leading to prolonged pharmacodynamics (PD) or target inhibition. ERK inhibitors have been discovered recently to overcome the acquired resistance to BRAF plus MEK inhibitors in BRAF-mutant melanoma and for combination therapies in RAS/MAPK altered cancers including KRAS-mutant cancers. Several ERK inhibitors have entered clinical development as monotherapy or in combination. However, it is not clear what the minimum time on target is required for efficacy without causing significant toxicity, and whether that would differentiate the therapeutic index of one inhibitor from another. Therefore, we have conducted PK-PD-efficacy relationship studies of different MEK and ERK inhibitors with different PK profiles in mice in BRAF-mutant or KRAS-mutant xenograft models to determine the minimum time on target required for efficacy. We have tested the PK-PD-efficacy of MEK inhibitors trametinib and cobimetinib, as well as ERK inhibitors BVD-523, GDC-0994 and LY3214996 in BRAF V600E or KRAS mutant xenograft models including A375 BRAF V600E melanoma, Colo205 BRAF V600E colorectal carcinoma and HCT116 KRAS G13D colorectal carcinoma. Additionally, an infusion study with LY3214996 was conducted in rats implanted with HCT116 colorectal carcinoma tumors, where drug exposure was controlled for a specified time to get ≥50% pRSK1 inhibition. Our data suggests that ≥50% inhibition of pRSK1 for 8h is required for significant efficacy of ERK inhibitor LY3214996, which is similar to the data presented for GDC-0994 (Robarge K et al, Abstract number DDT02-03, AACR Annual Meeting 2014). Overall assessment of the data in different models suggested that ≥50% target inhibition for 8-16h is enough for significant tumor growth inhibition and regressions by an ERK inhibitor in BRAF-mutant or KRAS-mutant tumors without any obvious toxicity as measured by body weight loss or mortality. LY3214996 is in Phase 1 clinical development and the preliminary PK profile in patients looks as predicted from preclinical data. Therefore, we believe that the PK profile of LY3214996 offers flexibility with dose and schedule to balance efficacy and safety, and to achieve a better therapeutic index in combination therapy for RAS/MAPK pathway altered cancers. Citation Format: Shripad V. Bhagwat, Weihua Shen, Baohui Zhao, Shufen Cai, Lisa Kindler, William T. McMillen, Eunice Yuen, Denis McCann, Jason Manro, Andrew J. Dropsey, Melinda D. Willard, Sajan Joseph, Sheng-Bin Peng. Temporal inhibition of ERK is sufficient for tumor growth inhibition in KRAS-mutant or BRAF-mutant tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5225.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third most frequent cause of cancer deaths in the US and is expected to be the second deadliest cancer by 2030. The 5-year survival rate in PDAC patients is only 6% and the poor prognosis is attributed to several factors including late-stage diagnosis, aggressive disease progression and high resistance to conventional therapies. Therefore, there is an urgent need for more effective systemic treatment strategies in PDAC patients. Nab-paclitaxel (Nab-pac) combined with gemcitabine (Gem) or FOLFIRINOX combination chemotherapy regimen are approved as standard of care treatment for frontline metastatic PDAC. Due to limited clinical efficacy of current cytotoxic chemotherapy regimens for PDAC patients, novel approaches are needed to further improve patient survival outcomes. In PDAC, activating mutations in KRAS occur at a frequency of ~90% rendering this a potential therapeutic target of interest. Developing drugs that directly target mutant KRAS protein remains challenging and alternative strategies focus on inhibition of downstream targets of the RAS-MAPK cascade. The addition of ERK inhibitor LY3214996 to the chemotherapy regimen (Nab-pac+Gem) was tested in MIA PaCa-2 (KRAS G12C), Capan-2 (KRAS G12V) and SW1990 (KRAS G12D) pancreatic cancer xenograft models. In the Capan-2 model, combination of LY3214996 with Gem alone was statistically additive (p Citation Format: Shripad V. Bhagwat, Wenjuan Wu, Baohui Zhao, Weihua Shen, Lisa Kindler, Jennifer Stephens, Jason Manro, William McMillen, Sajan Joseph, Sheng-Bin Peng, Ramon V. Tiu. Inhibition of ERK by LY3214996 augments nab-paclitaxel and gemcitabine combination chemotherapy efficacy in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1307.

Cancer patients with KRAS mutations have poor prognosis and represent an unmet medical need. Mutant KRAS driven MAPK activation is present in ~ 40% and ~ 30% of CRC and NSCLC patients, respectively. ERK1/2, a key downstream effector of KRAS mutations, is involved in the critical signaling network to drives cell proliferation, survival, metastasis and drug resistance. LY3214996, an ERK1/2 inhibitor which is in Phase I clinical trial (NCT02857270), has potent anti-tumor activities in KRAS mutant tumor cells in vitro and in vivo. However, some KRAS mutant cancer cells were less sensitive to LY3214996 single agent, suggesting that combination therapy is needed to maximize the benefit from ERK1/2 inhibition. The combined inhibition of RAS/RAF/MEK/ERK pathway components (such as the inhibition of both BRAF and MEK) has shown the promising anti-cancer activity in melanoma. Based on interaction between activated RAS with RAF which is responsible for cancer progression and resistance in KRAS mutant cancers, we hypothesized that inhibiting both ERK and RAF may augment efficacy of the single agents and delay the resistance. Therefore in this study we have investigated the combination effect of LY3214996 with a pan-RAF inhibitor LY3009120 in KRAS mutant CRC and NSCLC in vitro and in vivo. The combination of LY3214996 and LY3009120 showed synergistic or additive inhibition of cell proliferation in all KRAS mutant CRC (n=8) and NSCLC (n=8) cell lines tested. The LY3214996 and LY3009120 combination significantly decreased the phosphorylation levels of ERK, Rb, S6 and H3, and induced more complete cell death in NCI-H2122 (G-12C), A549 (G-12S), NCI-H441 (G-12V), HCT-116 (G-13D) and in LY3009120-resistant HCT-116 cells compared with either single agent. Subsequent combination efficacy was evaluated in the HCT-116 xenograft model. LY3214996 alone, LY3009120 alone and the combination of LY3214996 and LY3009120 resulted in 52%, 68% and 94% tumor growth inhibition, respectively; and the combination significantly augmented the efficacy (p Citation Format: Wenjuan Wu, Shripad Bhagwat, Lisa Kindler, William McMillen, Sajan Joseph, Sean Buchanan, Christoph Reinhard, Ramon V. Tiu, Sheng-Bin Peng. Combination of an ERK1/2 inhibitor (LY3214996) with pan-RAF inhibitor enhances anti-tumor activity in KRAS mutant colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-083.

3001 Background: LY3214996 is a selective and potent ERK1/2 inhibitor that has demonstrated tumor growth inhibition in several pre-clinical tumor models with BRAF, RAS, or MAP2K1 mutations. This is the first-in-human Phase 1 Study of LY3214996 in adv CA pts. Methods: The goals of this DE study were to determine a recommended Phase 2 dose (RP2D), safety, pharmacokinetic (PK), and preliminary efficacy of LY3214996 (NCT02857270; I8S-MC-JUAB; Eli Lilly & Co.). Pts with adv CA, ≥18 yrs of age, ECOG ≤1, and with adequate organ function were eligible. Pharmacodynamic (PD) biomarkers including pRSK were evaluated in blood and paired tumor tissue. The DE phase evaluated PO doses using the Bayesian model-based toxicity band method. Results: A total of 51 pts with median age of 62 yrs (range: 21-81) received at least 1 dose of LY3214996 with a median of 3 cycles (range: 1-12). Most pts had a mutation in RAS (N = 33) or BRAF (N = 16) and had a median of 4 prior lines of treatment. The DLTs observed in the study include grade (G) 3 cough and fatigue, G3 dehydration, increased creatinine (Cr), G3 increased CPK, G3 rash > 7 days, and 1 pt with renal failure. TRAEs to LY3214996 occurring in ≥10% of pts included nausea, vomiting, diarrhea, dermatitis acneiform, fatigue, pruritus, and blurred vision. LY3214996 exposures increased with dose. Tumor regression was observed in 7 pts with BRAF/non -BRAF mutant CA including 5 pts who failed prior IO/MAPK inhibitors. Four pts achieved stable disease (2 BRAF, 1 RAS and 1 CRAF mutation) that lasted > 4 mos. Up to 100% pRSK decrease from baseline in tumor was observed. Conclusions: LY3214996 had an acceptable safety profile, favorable PK, and potent tumor PD inhibition at RP2D. This supports further exploration of LY3214996 as monotherapy and in combination in CA pts with activating MAPK pathway alterations. Clinical trial information: NCT02857270.

Lung cancer is a leading cause of cancer death worldwide. ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment. KRAS mutation driven MAPK activation is present in ~ 30% of lung cancer patients. Non-small cell lung cancer (NSCLC) patients with KRAS mutation have poor prognosis and represents an unmet medical need. LY3214996, an ERK1/2 inhibitor which is in a phase I clinical trial, has anti-tumor activities in RAS mutant tumor cells in vitro and in vivo (http://cancerres.aacrjournals.org/content/77/13_Supplement/4973). Ramucirumab, a fully-human monoclonal antibody to human VEGFR-2 was approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma including endothelial cells have been shown to be effective in various tumor subtypes. In this study, the combination effect of LY3214996 with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LY3214996 treatment alone resulted in 41%, 91% and 101% tumor growth inhibition in H2122, A549 and H441 xenograft tumors, respectively. DC101 treatment alone resulted in 64%, 75% and 102% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LY3214996 with DC101 led to better tumor growth inhibition 83%, 115% (i.e. 15% regression) and 146% (i.e. 46% regression) for H2122, A549 and H441, respectively when compared with single agent treatment. The molecular mechanism was further investigated in H2122 tumor xenograft tumors in terms of MAPK signaling, MAPK gene signatures, tumor vascularization, cell proliferation and apoptosis. LY3214996 together with DC101 led to greater reduction in tumor blood vessels density compared to DC101 alone. Similarly, the analysis of multiple cell cycle markers (including pRb, pH3 and Ki67) indicated that the combination treatment resulted in greater inhibited of cell proliferation compared to single agent. Moreover, the combination effect of LY3214996 with ramucirumab was also investigated via tumor cell driven cord formation assays in vitro; and the data indicated that the combination enhanced the inhibition of cord formation when compared to single agent. Overall, combined inhibition of ERK1/2 and VEGFR-2 enhanced both anti-angiogenesis and antitumor effects. Taken together, these data support further clinical development of the combination of an ERK1/2 inhibitor, LY3214996 with ramucirumab in the treatment of KRAS mutant NSCLC. Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Bonita D. Jones, Michelle L. Swearingen, Beverly L. Falcon, William T. McMillen, Sajan Joseph, Sean Buchanan, Sheng-Bin Peng, Christoph Reinhard, Ramon V. Tiu. Combination of an ERK1/2 inhibitor (LY3214996) with VEGFR-2 inhibitor enhances anti-tumor activity in KRAS mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-185.

Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies, many of which do not cross-react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections, and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.

The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good antiproliferative agents.

The synthesis and kinase inhibitory activity of a series of novel 1,7-annulated indolocarbazoles 6 and 16 is described. These compounds exhibited potent inhibitory activity against cyclin-dependent kinase 4 and good antiproliferative activity in a human colon carcinoma cell line.

The transforming growth factor β (TGFβ) signaling pathway is a pleiotropic cellular pathway that plays a critical role in cancer. In fact, aggressive tumors are typically associated with high ligand levels and thus associated with poor prognosis in various tumor types. Cancer cells use autocrine and paracrine TGFβ signaling to modulate tumor cells and the tumor microenvironment leading to a highly invasive and metastatic phenotype, inducing and increasing tumor vascularization, modulating the extracellular matrix in the stroma, and inhibiting immune surveillance and antitumor immunity. Clinical studies with galunisertib (aka LY2157299 monohydrate), a small molecule inhibitor targeting the TGFβ pathway, have provided proof of concept data supporting the role of TGFβ in cancer and the utility of targeting the TGFβ pathway. Here we describe the identification of LY3200882, a next generation small molecule inhibitor of TGF-β receptor type 1 (TGFβRI). The molecule is a potent, highly selective inhibitor of TGFβRI embodied in a structural platform with a synthetically scalable route. It is an ATP competitive inhibitor of the serine-threonine kinase domain of TGFβRI. Mechanism of action studies reveal revealed that LY3200882 inhibits various pro-tumorigenic activities. LY3200882 potently inhibits TGFβ mediated SMAD phosphorylation in vitro in tumor and immune cells and in vivo in subcutaneous tumors in a dose dependent fashion. In preclinical tumor models, LY3200882 showed potent anti-tumor activity in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlated with enhanced tumor infiltrating lymphocytes in the tumor microenvironment. Durable tumor regressions in the orthotopic 4T1-LP model were observed and rechallenge of congenic tumors resulted in complete rejection in all mice. In in vitro immune suppression assays, LY3200882 has shown the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naïve T cell activity and restore proliferation. Therefore, LY3200882 shows promising activity as an immune modulatory agent. In addition, LY3200882 has shown anti-metastatic activity in vitro in migration assays as well as in vivo in an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer). Finally, LY3200882 shows combinatorial anti-tumor benefits with checkpoint inhibition (anti-PD-L1) in the syngeneic CT26 model. In conclusion, we have developed a novel potent and highly selective small molecule inhibitor of TGFβRI for the treatment of cancer. Citation Format: Huaxing Pei, Saravanan Parthasarathy, Sajan Joseph, William McMillen, Xiaohong Xu, Stephen Castaneda, Ivan Inigo, Karen Britt, Bryan Anderson, Gaiying Zhao, Scott Sawyer, Douglas Beight, Talbi Kaoudi, Chandrasekar Iyer, Huimin Bian, Amy Pappas, David Surguladze, David Schaer, Karim Benhadji, Michael Kalos, Kyla Driscoll. LY3200882, a novel, highly selective TGFβRI small molecule inhibitor [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 955. doi:10.1158/1538-7445.AM2017-955

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. ERK inhibitors have the potential to address resistance caused by ERK reactivation. Herein, a potent, selective small molecule ERK1/2 inhibitor is described. LY3214996 possesses an optimal balance of potency (hERK1 IC50 5 nM, hERK2 IC50 5nM, pRSK IC50 0.43 µM), solubility (FaSSIF solubility at pH 6.5 0.133 µM), PK properties (dog, AUCoral 23800 nM*hr, CL 12.1 mL/min/kg, bioavailability 75.4%), IVTI (TED50 =16 mg/kg pRSK1) and demonstrated significant in vivo efficacy in several human cancer xenograft models. LY3214996 is currently undergoing early clinical evaluation. Citation Format: Gaiying Zhao, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Lisa Kindler, Robert S. Flack, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jennie Walgren, Eunice Yuen, Jason Manro, Xueqian Gong, Guillermo Cortez, Johnathan McLean, Michael J. Rodriguez, Ramon V. Tiu, Shripad V. Bhagwat, Sajan Joseph. Identifying high quality, potent and selective pyrimidinylthienopyrrolone inhibitors of ERK1/2 kinase: LY3214996 [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 3231. doi:10.1158/1538-7445.AM2017-3231

The RAS/MAPK pathway is dysregulated in approximately 30% of human cancers, and the extracellular-signal-regulated kinases (ERK1 and ERK2) serves as key central nodes within this pathway. The feasibility and clinical impact of targeting the RAS/MAPK pathway has been demonstrated by the therapeutic success of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma. However, resistance develops frequently through reactivation of the pathway. Therefore, simultaneous targeting of multiple effectors such as RAF, MEK and ERK in this pathway, offers a potential for enhanced efficacy while delaying and overcoming resistance. LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. In tumor xenograft models, LY3214996 inhibits PD biomarker phospho-p90RSK1 in tumors and the PD effects are correlated with compound exposures and anti-tumor activities. LY3214996 shows either similar or superior anti-tumor activity as compared to other published ERK inhibitors in BRAF or RAS mutant cell lines and xenograft models. Oral administration of single-agent LY3214996 significantly inhibits tumor growth in vivo and is well tolerated in BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic cancer xenografts or PDX models. Therefore, LY3214996 can be tailored for treatment of cancers with MAPK pathway alteration. In addition, LY3214996 has anti-tumor activity in a vemurafenib-resistant A375 melanoma xenograft model due to MAPK reactivation, may have potential for treatment of melanoma patients who have failed BRAF therapies. More importantly, LY3214996 can be combined with investigational and approved agents in preclinical models, particularly KRAS mutant models. Combination treatment of LY3214996 and CDK4/6 inhibitor abemaciclib was well tolerated and results in potent tumor growth inhibition or regression in multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers. Here, we first report the preclinical characterization of LY3214996, a novel small molecule ERK1/2 inhibitor currently in Phase I clinical trials in patients with advanced and metastatic cancers (NCT02857270). Citation Format: Shripad V. Bhagwat, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Lisa Kindler, Robert S. Flack, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jason Manro, Jennie Walgren, Eunice Yuen, Xueqian Gong, Michael J. Rodriguez, Jianping Huang, Ramon V. Tiu, Sajan Joseph, Sheng-Bin Peng. Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations [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 4973. doi:10.1158/1538-7445.AM2017-4973

ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270). Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC) [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 317. doi:10.1158/1538-7445.AM2017-317

Background LY2584702 tosylate (hereafter referred to as LY2584702) is a potent, highly selective adenosine triphosphate (ATP) competitive inhibitor against p70 S6 kinase, a downstream component of the phosphatidylinositol-3-kinase signalling pathway which regulates cell proliferation and survival. LY2584702 exhibited anti-tumour activity in preclinical analysis. Methods Patients with advanced solid tumours were treated with LY2584702 orally on a 28-day cycle until the criteria for maximum tolerated dose (MTD) were met. Skin biopsies were collected for pharmacodynamic analysis, and levels of phospho-S6 protein were examined. The primary objective was to determine a phase II dose and schedule with secondary objectives of observing safety and tolerability. Dose escalation was based upon Common Terminology Criteria for Adverse Events Version 3.0. Results Thirty-four patients were enrolled onto this phase I study and treated with LY2584702 on a QD (once-daily) or BID (twice-daily) dosing schedule. Part A dose escalation (n = 22) began with 300 mg BID (n = 2). Due to toxicity, this was scaled back to doses of 25 mg (n = 3), 50 mg (n = 8), 100 mg (n = 3), and 200 mg (n = 6) QD. Part B dose escalation (n = 12) included 50 mg (n = 3), 75 mg (n = 3), and 100 mg (n = 6) BID. Seven patients experienced dose-limiting toxicity (DLT). All DLTs were Grade 3 and included vomiting, increased lipase, nausea, hypophosphataemia, fatigue and pancreatitis. Conclusion The MTD was determined to be 75 mg BID or 100 mg QD. No responses were observed at these levels. Pharmacokinetic analysis revealed substantial variability in exposure and determined that LY2584702 treatment was not dose proportional with increasing dose.

Renewable energy source is an echo friendly source of electrical energy. This paper develops a maximum power point tracker (MPPT) for optimizing the power output from any source under varying load condition. Simulations are carried out in MATLAB using Perturb and observe algorithm and incremental conductance algorithm and results compared.

SAR about the B-ring of a series of N 2 -aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o -aminoalkylether and B-ring p -amine probes of the S1′ and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

During the last decade, 2,3-dihydro-4-pyridones have become important intermediates for the synthesis of various alkaloids and biologically active compounds. The versatility of these heterocyclic building blocks has been demonstrated by the synthesis of several complex natural products. This review will cover recent work on the synthesis of substituted dihydropyridones, and their application in the synthesis of complex molecules.

During the last decade, 2,3-dihydro-4-pyridones have become important intermediates for the synthesis of various alkaloids and biologically active compounds. The versatility of these heterocyclic building blocks has been demonstrated by the synthesis of several complex natural products. This review will cover recent work on the synthesis of substituted dihydropyridones, and their application in the synthesis of complex molecules.

PI3K/AKT/mTOR/S6K signaling pathway (AKT pathway) controls cell survival, cell-cycle progression, cell growth and metabolism through a cascade phosphorylation of a number of key substrates. This pathway is regulated by three well characterized tumor suppressors; pten, tsc2, and lkb1. Deletion of these genes results in activation of the AKT pathway and proliferative disorders. Similarly, activating mutations of the receptor tyrosine kinases or PI3 Kinase result in the activation of the pathway. Therefore, multiple nodes of the pathway have become drug targets. As part of a comprehensive drug discovery platform aimed at targeting the PI3K pathway, we have developed a potent small molecule inhibitor of p70 S6 kinase that is a downstream effector in this pathway. LYS6K1 is a potent, highly selective ATP competitive inhibitor against p70 S6 kinase with an IC50 of 0.002 uM. In vitro, LYS6K1 inhibits the phosphorylation of S6 ribosomal protein in HCT116 colon cancer cells with an IC50 of 0.2 uM and similar activity is seen in a broad range of other cell lines. In vivo, LYS6K1.tosylate demonstrates potent phospho-S6 inhibition in nude mice bearing HCT116 colon carcinoma cells, with an ED50 value of 2.2 mg/kg and a ED90 value of 10 mg/kg 4 hours after a single oral dose. In these studies, LYS6K1.tosylate did not show statistically significant elevation of phospho-AKT, in spite of potent p70 S6 kinase inhibition. Proportional dose, exposure, and pharmacodynamic relationships were observed for LYS6K1 in dose- and time- dependent studies. In vivo, LYS6K1.tosylate effectively inhibits the growth of HCT116 colon carcinoma xenografts in mice and the growth of U87MG glioblastoma tumor as a single agent at 2.5 mg/kg given twice daily. Overall, LYS6K1.tosylate has high permeability that resulted in good oral absorption and PK properties. Based on these pre-clinical observations, LYS6K1.tosylate has advanced to Phase I studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 352.