Justin Huard

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG1) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122), and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the anti-tumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

Preclinical Evaluation of AMG 925, a FLT3/CDK4 Dual Kinase Inhibitor for Treating Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D–dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4–RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses. Mol Cancer Ther; 13(4); 880–9. ©2014 AACR.

FLT3 and CDK4/6 inhibitors: Signaling mechanisms and tumor burden in subcutaneous and orthotopic mouse models of acute myeloid leukemia

FLT3ITD subtype acute myeloid leukemia (AML) has a poor prognosis with currently available therapies. A number of small molecule inhibitors of FLT3 and/or CDK4/6 are currently under development. A more complete and quantitative understanding of the mechanisms of action of FLT3 and CDK4/6 inhibitors may better inform the development of current and future compounds that act on one or both of the molecular targets, and thus may lead to improved treatments for AML. In this study, we investigated in both subcutaneous and orthotopic AML mouse models, the mechanisms of action of three FLT3 and/or CDK4/6 inhibitors: AMG925 (Amgen), sorafenib (Bayer and Onyx), and quizartinib (Ambit Biosciences). A composite model was developed to integrate the plasma pharmacokinetics of these three compounds on their respective molecular targets, the coupling between the target pathways, as well as the resulting effects on tumor burden reduction in the subcutaneous xenograft model. A sequential modeling approach was used, wherein model structures and estimated parameters from upstream processes (e.g. PK, cellular signaling) were fixed for modeling subsequent downstream processes (cellular signaling, tumor burden). Pooled data analysis was employed for the plasma PK and cellular signaling modeling, while population modeling was applied to the tumor burden modeling. The resulting model allows the decomposition of the relative contributions of FLT3ITD and CDK4/6 inhibition on downstream signaling and tumor burden. In addition, the action of AMG925 on cellular signaling and tumor burden was further studied in an orthotopic tumor mouse model more closely representing the physiologically relevant environment for AML.

Abstract A279: Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard care treatment, the disease eventually relapses. Activating FLT3 mutations are found in approximately 30% of AML patients. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here preclinical evaluation of AMG 925, a potent, selective and bioavailable FLT3/CDK4 dual kinase inhibitor. The compound inhibited AML xenograft tumor growth by >99% without detectable body weight loss. AMG 925 was also found to inhibit FLT3 mutants (e.g, D835Y) that are resistant to the current FLT3 inhibitors (e.g., quizartinib/AC220, sorafenib). CDK4 is a cyclinD-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-Rb pathway in cancer development has been well established. CDK4 specific inhibitors are being developed for treating Rb positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve clinical response rates. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A279. Citation Format: Kang Dai, Kathleen Keegan, Zhihong Li, Ma Ji, Cong Li, John Eksterowicz, Coberly Suzanne, David Hollenback, Margret Weidner, Justin Huard, Lingming Liang, Grace Alba, Jessica Orf, Mei-Chu Lo, Sharon Zhao, Rachel Ngo, Ada Chen, Lily Liu, Timothy Carlson, Lawrence R. McGee, Julio Medina, Alexander Kamb, Dineli Wickramasinghe. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A279.

Abstract 2351: CDK4/FLT3 dual inhibitors as potential therapeutics for acute myeloid leukemia.

CDK4 is a cyclin D dependent kinase that promotes cell cycle progression in a broad range of tumor types by phosphorylating the tumor suppressor retinoblastoma protein (Rb) and releasing transcription factor E2F. Critical involvement of the cyclin D-CDK4-Rb pathway in carcinogenesis is strongly supported by a large amount of genetic evidence. In addition, promoter methylation with consequent silencing of expression of the CDK4 inhibitor, p15, has been reported in 44-60% of acute myeloid leukemia (AML) patients. It is also well established that constitutive activation of the tyrosine kinase FLT3 via mutation contributes to the development of AML, with 30% of AML carrying such activating mutations. FLT3 tyrosine kinase inhibitors used as single agents reduce peripheral blood and bone marrow blasts in only a minority of AML patients, and the effect tends to be transient. This may be due to insufficient FLT3 inhibition, the selection of drug-resistant clones, or the independence of the cell on FLT3 signaling for proliferation and survival. In preclinical models, a synergistic effect of CDK4 inhibition and FLT3 inhibition resulting in increased apoptosis of AML cell lines was reported (Wang et al., Blood, 2007). From a HTS hit through SAR optimization led to AM-5992, a potent and orally bioavailable dual inhibitor of CDK4 and FLT3 including all FLT3 mutants reported to date. AM-5992 inhibits the proliferation of a panel of human tumor cell lines including MDA-MB-435(Rb+), colo-205(Rb+), U937(FLT3WT) and induced cell death in MOLM13(FLT3ITD), MV4-11(FLT3ITD), and even in MOLM13(FLT3ITD, D835Y) which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. In mouse models of leukemia using cells with the FLT3ITD mutation, AM-5992 treatment at 150 mpk qd on days 6-16 after leukemia cell injection significantly reduced the leukemia burden and prolonged survival 11 days over that of vehicle controls. Collectively, these data support the hypothesis that simultaneously inhibition of CDK4 and FLT3 may improve the durability of clinical response in AML; and consequently that this hypothesis should be tested in the clinic. Citation Format: Zhihong Li, Kang Dai, Kathleen Keegan, Ji Ma, Mark Ragains, Jacob Kaizerman, Dustin McMinn, Jiasheng Fu, Benjamin Fisher, Michael Gribble, Lawrence R. McGee, John Eksterowicz, Cong Li, Lingming Liang, Margaret Weidner, Justin Huard, Robert Cho, Timothy Carlson, Grace M. Alba, David Hollenback, John Hill, Darrin Beaupre, Alexander Kamb, Dineli Wickramasinghe, Julio C. Medina. CDK4/FLT3 dual inhibitors as potential therapeutics for acute myeloid leukemia. [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 2351. doi:10.1158/1538-7445.AM2013-2351