BackgroundPreclinical evaluation of drugs targeting the human immune system has posed challenges for oncology researchers. Since the commercial introduction of humanized mice, antitumor efficacy and pharmacodynamic studies can now be performed with human cancer cells within mice bearing components of a human immune system. However, development and characterization of these models is necessary to understand which model may be best suited for different agents.MethodsWe characterized A375, A549, Caki-1, H1299, H1975, HCC827, HCT116, KU-19–19, MDA-MB-231, and RKO human cancer cell xenografts in CD34+humanized non-obese diabetic-scid gamma mice for tumor growth rate, immune cell profiling, programmed death ligand 1 (PD-L1) expression and response to anti-PD-L1 therapy. Immune cell profiling was performed using flow cytometry and immunohistochemistry. Antitumor response of humanized xenograft models to PD-L1 therapy was performed using atezolizumab.ResultsWe found that CD4+and CD8+T-cell composition in both the spleen and tumor varied among models, with A375, Caki-1, MDA-MB-231, and HCC827 containing higher intratumoral frequencies of CD4+and CD8+T cells of CD45+cells compared with other models. We demonstrate that levels of immune cell infiltrate within each model are strongly influenced by the tumor and not the stem cell donor. Many of the tumor models showed an abundance of myeloid cells, B cells and dendritic cells. RKO and MDA-MB-231 tumors contained the highest expression of PD-L1+tumor cells. The antitumor response of the models to atezolizumab was positively associated with the level of CD4+and CD8+tumor-infiltrating lymphocytes (TILs).ConclusionsThese data demonstrate that there are tumor-intrinsic factors that influence the immune cell repertoire within tumors and spleen, and that TIL frequencies are a key factor in determining response to anti-PD-L1 in tumor xenografts in humanized mice. These data may also aid in the selection of tumor models to test antitumor activity of novel immuno-oncology or tumor-directed agents.
This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic−pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.
Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.
TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, which play important roles in tumor growth, survival, cell adhesion, as well as innate immunity, phagocytosis, and immune-suppressive activity. Therefore, targeting both AXL and MERTK kinases may directly impact tumor growth and relieve immunosuppression. We describe here the discovery of INCB081776, a potent and selective dual inhibitor of AXL and MERTK that is currently in phase 1 clinical trials. In cellular assays, INCB081776 effectively blocked autophosphorylation of AXL or MERTK with low nanomolar half maximal inhibitory concentration values in tumor cells and Ba/F3 cells transfected with constitutively active AXL or MERTK. INCB081776 inhibited activation of MERTK in primary human macrophages and partially reversed M2 macrophage–mediated suppression of T-cell proliferation, which was associated with increased interferon-γ production. In vivo, the antitumor activity of INCB081776 was enhanced in combination with checkpoint blockade in syngeneic models, and resulted in increased proliferation of intratumoral CD4+ and CD8+ T cells. Finally, antitumor activity of INCB081776 was observed in a subset of sarcoma patient–derived xenograft models, which was linked with inhibition of phospho-AKT. These data support the potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of both enhancing tumor immune surveillance and blocking tumor cell survival mechanisms.
Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic. Mol Cancer Ther; 11(9); 2045-53. Ó2012 AACR.
Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs), which are amplified, translocated, or over-expressed in numerous types of human cancer. These RTKs play important roles in tumor growth, survival, cell adhesion and migration as well as drug resistance. In addition, it has been shown that both AXL and MER are critical regulators of innate immunity, phagocytosis, and immune-suppressive activity. Therefore targeting both AXL and MER kinases may not only impact the growth, survival and malignant progression of neoplastic cells directly, but also has the potential to restore and enhance host immunity against cancers. INCB081776 is a potent inhibitor of AXL and MER that exhibits selective pharmacological activity and enhanced anti-tumor immune activity. In biochemical assays, INCB081776 potently inhibited the kinase activity of recombinant AXL/MER enzymes and was highly selective against a panel of 192 kinases (IC50 = 0.61±0.31 nM and 3.17±1.97 nM against AXL and MER, respectively). INCB081776 is greater than 30 fold selective against TYRO3. Selectivity against TYRO3 is important as retinal toxicity associated with loss of the Mer gene appears to be modulated by TYRO3 in mice. In cellular assays, INCB081776 effectively blocked auto-phosphorylation of AXL or MER including BAF3 cells transfected with constitutively active AXL or MER, AXL in H1299 tumor cells, or MER kinase in G361 tumor cells, with low nanomolar IC50 values. In addition, INCB081776 inhibited activation of MER kinase in primary human macrophages with low nanomolar IC50 potency. More importantly, in an in vitro functional assay, INCB081776 partially reversed M2 macrophage-mediated suppression of T cell proliferation, and increased IFN-γ in co-cultured macrophages and T cells. In vivo, INCB081776 administration to H1299 tumor-bearing mice dose-dependently inhibited the phosphorylation in tumors. Consistent with the proposed mechanism of action, INCB081776 potently inhibited tumor growth in immunocompetent mice, but not in immunodeficient mice, demonstrating that a functional immune system is important for activity. Treatment was associated with dose-related increases in the percent of tumor-infiltrating effector CD4+ and CD8+ T cells, as well as macrophages with the M1 phenotype. In addition, INCB081776 decreased the percentage of intratumoral M2 macrophages and monocytic myeloid-derived suppressor cell (M-MDSC) immune cell populations. In the 4T1 model, combining INCB081776 with anti-PD-L1 resulted in synergistic anti-tumor effects compared to either single agent. Collectively, these preclinical data support the hypothesis and potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of enhancing tumor immune surveillance mechanisms in cancer patients as a single agent and when combined with therapies mediating immune PD-L1 checkpoint blockade. Citation Format: Margaret Favata, Kerri Lasky, Yvonne Lo, Patricia Feldman, Jun Li, Yaoyu Chen, Christina Stevens, Min Ye, Hui Wang, Ke Liu, Richard Wynn, Yanlong Li, Jennifer Harris, Robert Landman, Yu Li, Xiaozhao Wang, Chunhong He, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Jonathan Rios-Doria, Zhenhai Gao, Maryanne Covington, Xuesong M. Liu, Holly Koblish, Peggy Scherle. Characterization of INCB081776, a potent and selective dual AXL/MER kinase inhibitor [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 3759.
3922 Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30 percent of newly diagnosed cases. Current treatment options for this disease are effective, but not always curative; therefore experimental therapies are being investigated. Diffuse large B-cell lymphoma cells typically over-express the serine/threonine mitotic kinase PLK1, which plays a key role in mitotic cell cycle progression, and has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL lines. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in mouse models harboring human DLBCL disease. MLN0905 has drug-like pharmacokinetic properties and an acceptable toxicity profile making it a good clinical candidate. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. The anti-tumor activity of MLN0905 was evaluated in three human subcutaneous xenograft models OCI-LY10, OCI-LY19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant anti-tumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The anti-tumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI-LY19) setting to better mimic DLBCL in humans. In this disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with Rituximab in the disseminated OCI-LY19 model. Combining Rituximab and MLN0905 provided both a synergistic anti-tumor effect and a synergistic survival advantage. Our findings indicate for the first time that PLK1 inhibition leads to pharmacodynamic pH3 modulation and significant anti-tumor activity in multiple models of DLBCL. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anti-cancer agents in the clinic. Disclosures: Shi: Millennium: The Takeda Oncology Company: Employment. Lasky:Millennium: The Takeda Oncology Company: Employment. Shinde:Millennium: The Takeda Oncology Company: Employment. Duffey:Millennium: The Takeda Oncology Company: Employment. Stringer:Millennium: The Takeda Oncology Company: Employment. Qian:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Liu:Millennium: The Takeda Oncology Company: Employment. Rao:Millennium: The Takeda Oncology Company: Employment. Vos:Millennium: The Takeda Oncology Company: Employment. D'Amore:Millennium: The Takeda Oncology Company: Employment. Hyer:Millennium: The Takeda Oncology Company: Employment.
Inhibitors of the BET family of bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate the expression of key survival and cell fate determining genes such as c-myc. In addition to their role in cancer, studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins controls the expression of pro-inflammatory cytokine genes in macrophages and is therapeutic in models of acute inflammation. These data suggest that in addition to their tumor intrinsic effects, BET inhibitors may also regulate the cytokine milieu within the tumor microenvironment and have immunomodulatory activity in cancer. To study this aspect, we evaluated INCB054329, a novel and selective BET inhibitor currently in Phase 1 trials, alone and in combination either with epacadostat, a highly selective IDO1 inhibitor, or with PD-1/PD-L1 axis blockade in syngeneic tumor models using immunocompetent animals. When used alone, INCB054329 suppressed a panel of cytokines and chemokines in a whole blood assay, confirming that INCB054329 can antagonize a pro-inflammatory response. The potency of INCB054329 in reducing the levels of these inflammatory mediators in the whole blood assay was similar to that for inhibition of c-myc, suggesting that the effects were on-target. INCB054329 was capable of inhibiting the growth of multiple syngeneic tumor models in immunocompetent mice, whereas only modest tumor growth inhibition was observed in immunodeficient mice and a lack of activity was observed in vitro, supporting the immunomodulatory activity of the compound. Because maximal in vivo tumor growth inhibition required an intact immune system, we investigated the impact of INCB054329 on various immune cell subsets, both in vitro and in vivo. Of note, increases in effector T cell populations were observed and efforts are ongoing to further characterize the tumor infiltrating immune cells following INCB054329 treatment. The mechanistic complimentarity of this novel BET inhibitor-mediated immunomodulation was also evaluated in combination with other therapeutically relevant mechanisms, including IDO1 inhibition and PD-1 axis blockade. Enhanced efficacy was observed with all INCB054329-containing regimens. These data demonstrate for the first time that BET inhibition can suppress tumor growth through both tumor-intrinsic and immune modulatory mechanisms, and support the potential of epigenetic-based, immunotherapy combinations as a novel approach to cancer therapy. Citation Format: Holly K. Koblish, Michael Hansbury, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Maryanne Covington, Timothy Burn, Mark Rupar, Christine Gardiner, Thomas Condamine, Kerri Lasky, Matthew C. Stubbs, Eddy Yue, Richard Sparks, Richard Sparks, Thomas Maduskuie, Andrew P. Combs, Gregory Hollis, Reid Huber, Phillip CC Liu, Peggy Scherle. The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4904.
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