Kathleen Keegan

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.

PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton’s tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton’s tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton’s tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton’s tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).

Synthesis and structure–activity relationship of trisubstituted thiazoles as Cdc7 kinase inhibitors

The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.

Abstract 2673: Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is the largest lymphoma subtype representing approximately one-third of all cases of non-Hodgkin9s lymphoma. Gene expression profiling showed that DLBCL can be stratified into activated B-cell (ABC) or germinal center B-cell (GCB) subtypes (Alizadeh et al., Nature 2000). The overall five year survival rate is only approximately 50% (Shaffer et al., Annu. Rev. Immunol. 2012), thus a medical need exits for the treatment of this disease, particularly for patients who relapse after the first line chemoimmunotherapy regimen. Idelalisib is an investigational, highly selective oral inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) delta isoform. PI3K delta signaling is required for B lymphocyte activation, proliferation and survival and is dysregulated in several B-cell malignancies. We investigated the activity of idelalisib and ibrutinib, an inhibitor of Bruton9s tyrosine kinase, on the inhibition of proliferation against a panel of 22 DLBCL (9 ABC, 13 GCB) and 3 transformed follicular lymphoma cell lines. Idelalisib has potent activity (EC50 Citation Format: Jia Y. Liu, Tom Kenney, Leslie Butterworth, Adam Kashishian, Sarah Meadows, Peng Yue, Li Li, Kathleen Keegan, Christophe Queva, Stacey Tannheimer. Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2673. doi:10.1158/1538-7445.AM2015-2673

Discovery of a Phosphoinositide 3-Kinase (PI3K) β/δ Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kβ Potency in a PI3Kδ-Selective Template by Targeting Nonconserved Asp856

Phosphoinositide 3-kinase (PI3K) β signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kβ/δ inhibitors in which PI3Kβ potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kβ/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

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