Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively, and less commonly in other cancers. We previously demonstrated that LY3962673 is a highly potent inhibitor of KRAS G12D and is selective against wild-type (WT) KRAS in mutant -cell lines and -in vivo models. Here, we describe the mechanism by which LY3962673 inhibits KRAS G12D and report a more comprehensive evaluation of LY3962673 activity across a panel of genetically and histologically diverse cancer cell lines, as well as in multiple patient-derived xenograft (PDX) models. LY3962673 is a non-covalent KRAS G12D inhibitor with high affinity binding to KRAS G12D-GDP (Kd 0.071 nM) compared to KRAS G12D-GTPγS (Kd 26.7 nM). In a panel of cancer cell lines with KRAS G12D mutations, non-G12D mutations or KRAS WT, LY3962673 selectively suppressed MAPK signaling and inhibited the growth of KRAS G12D mutant cancer cells while sparing KRAS WT and non-G12D mutant cells. Sensitivity to LY3962673 varied among the KRAS G12D-mutant cells tested, suggesting that not all cell lines share the same dependence on KRAS G12D for their growth and survival. Furthermore, in multiple KRAS G12D-mutant PDX models representing diverse tumor types, LY3962673 demonstrated anti-tumor activities, ranging from tumor growth inhibition to robust tumor regression. LY3962673 also showed enhanced efficacy when combined with other anti-cancer agents. Taken together, the findings underscore the potential of LY3962673 as a monotherapy or in combination with other anti-cancer agents, as a promising oral therapeutic option for a range of cancer types with KRAS G12D mutations. Citation Format: Xueqian Gong, Hong Gao, Mark H. Bender, Wenyu Ming, Youyan Zhang, Trent R. Stewart, Chun Ping Yu, Wei Guo Xu, Aurthur Xintian You, Wen Ting Bian, Binghui Li, Tao Wang, Huimin Bian, Manuj Tandon, Andrew Capen, Rachel N. Cavitt, Bryan D. Anderson, Wayne Bocchinfuso, Anke Klippel, Chandrasekar Iyer. LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3316.

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively and less commonly in other cancers. Here, we describe LY3962673, a highly potent inhibitor of KRAS G12D that is selective against wild-type (WT) KRAS and orally bioavailable. Compound potency and selectivity were measured using surface plasmon resonance (SPR) and cell-based assays monitoring the inhibition of pERK (Thr202/Tyr204) and cell growth. In SPR assays, LY3962673 had Kd values of 0.058 and 4.7 nM for GDP-bound KRAS G12D and WT KRAS, respectively (81-fold selective). In KRAS G12D-mutant HPAC, LY3962673 inhibited ERK1/2 phosphorylation with an IC50 of 4.2 nM contrasting 231 nM IC50 in WT KRAS MKN45 cells (55-fold selective). Similarly, LY3962673 inhibited cell growth in KRAS G12D and WT KRAS cell lines with IC50 values of 7.7 nM and 1800 nM, respectively (233-fold selective). LY3962673 shows a clean safety profile in a 133 off-target panel screen. LY3962673 demonstrates favorable in vitro ADME properties. LY3962673 shows oral bioavailability across preclinical species when dosed either as freebase or salt form. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, LY3962673 administered orally demonstrated tumor regressions in KRAS G12D-driven tumor models, without body weight loss. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients. These data demonstrate that LY3962673 potently inhibits KRAS G12D while sparing WT KRAS and other off-targets with its selectivity. We hypothesize that LY3962673’s low nanomolar potency, desirable selectivity profile, and oral bioavailability will provide efficacy and tolerability in patients with KRAS G12D-driven cancers. An IND submission is planned in the first half of 2024. Citation Format: Chandrasekar Iyer, Binghui Li, Trent R Stewart, Tao Wang, Andrew Capen, Rachel Cavitt, Bryan D Anderson, Wayne Bocchinfuso, Gaiying Zhao, Michael J Rodriguez, Santiago Carballares, Andrew Cooke, Robert Bondi, Lee Burns, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, James R Henry, Chong Si. Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B115.

Time-dependent increase in apoptosis induction by LY3214996, pan-RAF inhibitor LY3009120 and MEK inhibitor trametinib in KRAS-mutant HCT116 colorectal cancer cells.

KRAS is altered in ~16% of all cancers and is an oncogenic driver in non-small cell lung, pancreatic, colorectal, and other cancers. Next generation KRAS inhibitors designed to target multiple oncogenic KRAS mutations, while simultaneously sparing wild-type (WT) HRAS and NRAS inhibition, are expected to offer expanded activity and favorable safety. We have discovered a series of highly potent and selective pan-KRAS inhibitors with activity against KRAS G12C, G12D, and G12V mutants, that also display high selectivity over WT HRAS and NRAS, thus providing an expanded therapeutic index. Here, we describe the preclinical profile of these pan-KRAS inhibitors. Compound potency and selectivity were measured using surface plasmon resonance (SPR) assays and cell-based assays measuring inhibition of p-ERK and 3D cell growth of KRAS-mutant tumor cell lines. These pan-KRAS inhibitors have IC50 values ranging from 3-14 nM for KRAS G12C, G12D, G12V, and WT KRAS in phospho-ERK cell-based assays and selectivity of >200-fold over NRAS WT and >100-fold over HRAS WT. These pan-KRAS inhibitors show a clean safety profile in a 133 off-target panel screen. These pan-KRAS inhibitors demonstrate favorable in vitro ADME properties and oral bioavailability in preclinical species. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, the pan-KRAS inhibitors administered orally demonstrated dose-dependent target inhibition in KRAS-mutant xenograft models. These data demonstrate that our pan-KRAS inhibitors potently and selectively inhibit KRAS G12D, G12C, and G12V mutations and WT KRAS, while sparing HRAS, NRAS, and other off-targets. We hypothesize that this potency and selectivity profile, along with high oral bioavailability, will provide efficacy and tolerability for patients with KRAS-mutant-driven cancers. An IND submission is planned in 2024. Citation Format: Lourdes Prieto Vallejo, Chandrasekar Iyer, Noelle Goggin, Binghui Li, Peiyi Yang, Huimin Bian, Jessica Podoll, Stefan Grotegut, Manuj Tandon, Bryan D Anderson, Andrew Capen, Min Xiao, Tao Wang, Trent R Stewart, Sean Aronow, Desta Bume, Isabel Rojo Garcia, Chong Si, Andrew Cooke, Robert Bondi, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, Lauire LeBrun, James R Henry, Tim Kercher. Preclinical characterization of orally bioavailable, highly potent pan-KRAS inhibitors with selectivity over HRAS and NRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B116.

Abs IC50 of LY3214996 and ERK pathway alterations in 60 cancer cell lines profiled for antitumor activity in cell proliferation assay

Biochemical and cellular selectivity profile of LY3214996 (DiscoveRx and ActiveX data)

Effect of LY3214996 on body weight change and calculation of %dT/C in HCT116 xenograft model.

Generation of vemurafenib-resistant A375 model in vivo.

PDF file - 86K, Supplemental Table 1. Kinases where LY2228820 shows >1000-fold selectivity in vitro (p38 MAPK vs. other kinase).

pan-RAF inhibitor can block increase in ERK phosphorylation due to inhibition of negative feedback on CRAF by ERK inhibitor.

In vivo effect of treatment on body weight loss.

In vivo effect of treatment on body weight loss.

Inhibition of downstream signaling by LY3214996 in a BRAF-mutant A375 melanoma cells (A) and MEK1 mutant SW48 colorectal cancer cells (B).

PDF file - 90K, shRNA silencing of p38a MAPK in U-87MG glioma (Westerns and xenograft tumor growth data).

PDF file - 43K, Effect of LY2228820 on MK2 phosphorylation in mouse peripheral blood mononuclear cells (PBMC) and from patients with multiple myeloma.

Abstract Transition metal chalcogenide nanoparticles (NPs) are of interest for energy applications, including batteries, supercapacitors, and electrocatalysis. Many methods have been established for synthesizing Ni NPs, and conversion chemistry to form Ni oxide and phosphides from template Ni NPs is well‐understood. Sulfidation and selenidation of Ni NPs have been much less explored, however. We report a method for the conversion of Ni template NPs into sulfide and selenide product NPs using elemental sulfur, 1‐hexadecanthiol, thiourea, trioctylphosphine sulfide, elemental selenium, and selenourea. While maintaining mole ratios of 2 mmol sulfur/selenium precursor: mmol Ni, products with phases of Ni3S2, Ni9S8, NiS, NiSO4·6H2O, Ni3S4, Ni3Se2, and NiSe have been obtained. The products have voids that form through the Kirkendall effect during interdiffusion. Trends relating the chemical properties of the precursors to the phases of the products have been identified. While some precursors contained phosphorus, there was no significant incorporation of phosphorus in any of the products. An increase of the NP size during sulfidation and selenidation is consistent with ripening. The application of Ni sulfide and selenide NPs as electrocatalysts for the hydrogen evolution reaction is also demonstrated.

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).

The thermal reshaping of gold nanorods in a polymer matrix is an important phenomenon for many potential applications. However, a fundamental understanding of the various mechanisms that govern the nanorod reshaping dynamics is still lacking. Here, we provide evidence for a phenomenological model of the gold nanorod shape transformation based on the measurements and detailed analysis of the time-resolved thermal reshaping for a variety of gold nanorods having different geometries (aspect ratio, volume, diameter) in a cross-linked epoxy matrix at application relevant temperatures (120-220 °C). Our analysis suggests that (a) the nanorod reshaping dynamics consist of two temporal regimes that are governed by different phenomena and (b) the ultimate amount of reshaping at a given temperature depends strongly on the initial particle geometry and the mechanical stiffness of its surroundings. At short times, the shape transformation is dominated by a curvature-induced surface diffusion process, in which the activation energy for diffusion depends on curvature. At long times, however, the surrounding environment plays a key role in slowing the diffusion and stabilizing the nanorod shape. We show that the long-time behavior can be well described using a modified surface diffusion model that takes into account the slowing of atomic diffusivity as a result of external forces arising from mechanical constraints. The ability to tune both the final shape and the reshaping dynamics in nanocomposites opens up new possibilities in tailoring the optical properties of these materials.

Abstract 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