Objective Forkhead box protein O1 (FOXO1) plays a key role in regulating hepatic glucose production, but investigations of FOXO1 inhibition as a potential therapeutic approach have been hampered by a lack of selective chemical inhibitors. By profiling structurally diverse FOXO1 inhibitors, the current study validates FOXO1 as a viable target for the treatment of diabetes. Methods Using reporter gene assays, hepatocyte gene expression studies, and in vivo studies in mice, we profiled our leading tool compound 10 and a previously characterized FOXO1 inhibitor, AS1842856 (AS). Results We show that AS has significant FOXO1-independent effects, as demonstrated by testing in FOXO1-deficient cell lines and animals, while compound 10 is highly selective for FOXO1 both in vitro and in vivo and fails to elicit any effect in genetic models of FOXO1 ablation. Chronic administration of compound 10 improved insulin sensitivity and glucose control in db/db mice without causing weight gain. Furthermore, chronic compound 10 treatment combined with FGF21 led to synergistic glucose lowering in lean, streptozotocin-induced diabetic mice. Conclusions We show that the widely used AS compound has substantial off-target activities and that compound 10 is a superior tool molecule for the investigation of FOXO1 function. In addition, we provide preclinical evidence that selective FOXO1 inhibition has potential therapeutic benefits for diabetes as a monotherapy or in combination with FGF21.
Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [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 3632.
The approval of abemaciclib and additional cyclin dependent kinases 4 & 6 (CDK4/6) inhibitors for the treatment of HR+ breast cancer has provided new therapeutic options to patients. As CDK4/6 inhibitors become part of the standard of care, combination strategies leveraging abemaciclib together with immunotherapy may represent an opportunity to extend benefit to more patients and additional cancers. Accordingly, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with PD-L1 blockade in immunocompetent murine syngeneic tumor models, and directly evaluated the tumor cell and immune cell intrinsic immunologic effect of abemaciclib in vitro. In vivo abemaciclib treatment of murine tumors (CT26, EMT6 and MC38) caused a dose-dependent delay in tumor growth and demonstrated the potential to induce complete tumor regression (CR ~10%). Combination with an anti-PD-L1 antibody after abemaciclib pretreatment greatly enhanced the anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy resulted in 50-60% CRs of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice maintaining CRs after cessation of combination therapy or abemaciclib monotherapy resisted later CT26 rechallenge, demonstrating the ability to generate immunological memory during abemaciclib therapy. Analysis of intra-tumor gene expression showed that abemaciclib monotherapy induced T cell activation and inflammation signatures. Combination therapy substantially enhanced this effect and was additionally associated with DC maturation, antigen presentation, cytokine signaling and helper T cell phenotype. Suppression of cell cycle genes indicative of inhibition of CDK4/6 was also more prominent during the combination therapy. In Jurkat and primary human T cells, treatment with abemaciclib in vitro resulted in a dose-dependent increase in NFAT activity upon TCR stimulation. This correlated with upregulation of both cell surface markers and genes associated with an enhanced T cell activation phenotype, while only modestly affecting T cell expansion. Abemaciclib also amplified expression of antigen presentation and other immune-related genes in human breast cancer cells. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results provide a strong rationale for combining abemaciclib with checkpoint immunotherapy to improve the anti-tumor efficacy. The T cell and tumor cell intrinsic effects, synergistic anti-tumor responses and intra-tumor immune activation, justify clinical investigation of this combination. Citation Format: David Schaer, Richard Beckmann, Jack Dempsey, Lysiane Huber, Amelie Forest, Nelusha Amaladas, Ying Cindy Wang, Erik Rasmussen, Darin Chin, Yanxia Li, Andrew Capen, Marianne Deroose, Carmine Carpenito, Xueqian Gong, Kirk Staschke, Linda Chung, Farhana Merzoug, Trent Stewart, Lacey Litchfield, Philip Iversen, Sean Buchanan, Alfonso de Dios, Ruslan Novosiadly, Michael Kalos. The CDK4/6 inhibitor abemaciclib synergizes with PD-L1 blockade to induce an immune inflamed tumor microenvironment through T cell and tumor cell intrinsic effects [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 4569.
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