Leslie Hall

Discovery and Pharmacological Characterization of a Novel Rodent-Active CCR2 Antagonist, INCB3344

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC50 = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.

A Novel Kinase Inhibitor, INCB28060, Blocks c-MET–Dependent Signaling, Neoplastic Activities, and Cross-Talk with EGFR and HER-3

Purpose: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase. Experimental Design: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments. Results: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET–dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET–dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET–driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers. Conclusions: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment. Clin Cancer Res; 17(22); 7127–38. ©2011 AACR.

INCB040093 is a novel PI3Kδinhibitor for the treatment of B cell lymphoid malignancies

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin’s lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1β (MIP-1beta) and tumor necrosis factor-β (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin’s lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

Abstract 4904: The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models

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.

Abstract 5416: Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia

The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) was initially identified as preferential integration sites of the Moloney murine leukemia virus in Eμ-myc mice, resulting in perinatal lymphomagenesis. Molecular characterization has revealed that PIM kinases drive cell proliferation and survival in a number of hematological malignancies beyond lymphomas by mediating responses to cytokines, growth factors and cellular stress. Overexpression of various PIM kinase family members in these malignancies has been associated with poor overall survival and with resistance to chemotherapeutic agents. Therefore, development of a pan-PIM inhibitor may be useful in the treatment of hematological malignancies, both as a single agent and in combination with chemotherapy or targeted agents. The in vitro and in vivo activity of INCB053914, a pan-PIM kinase inhibitor, was determined in a panel of acute myelogenous leukemia (AML) cell lines. Greater than half of all AML cell lines tested were sensitive to single agent INCB053914, with anti-proliferative IC50 potencies Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Sybil O9Connor, Qian Wang, Kathy Wang, Lynn Leffet, Maryanne Covington, Krista Burke, Jason Boer, Kevin Bowman, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia. [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 5416. doi:10.1158/1538-7445.AM2015-5416

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies

The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase–signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. In vitro, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated ex vivo and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. In vivo, single-agent INCB053914 inhibited Bcl-2–associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.

Abstract 143: Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL

The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in “double-hit” DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL. Citation Format: Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL [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 143. doi:10.1158/1538-7445.AM2017-143

Abstract 4635: The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model

Immune checkpoint blockade has shown considerable therapeutic promise in the clinic. However, single agent activity is compromised by the presence of suppressive myeloid cells, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAM) and polymorphonuclear (PMN) cells, in the tumor microenvironment. Epigenetic alterations can significantly contribute to the development of the immunosuppressive tumor microenvironment and recent data have suggested that combining epigenetic-based therapies with immunotherapeutic agents can lead to improved efficacy in preclinical models. Since Lysine Specific Demethylase 1 (LSD1) has been shown to play a critical role in hematopoiesis, we hypothesized that inhibition of LSD1 could have a direct effect on myeloid cell differentiation and potentially restore normal myelopoiesis in cancer patients. To test this hypothesis, we evaluated INCB059872, a potent, selective and orally available FAD-directed covalent inhibitor of LSD1 in several experimental models. In an in vitro differentiation assay, the majority of CD34+ progenitor cells were driven to a monocytic phenotype in the presence of INCB059872, while control treated cells differentiated toward granulocytic PMN cells. Similar results were observed in vivo. Using the orthotopic 4T1 mammary cancer model, the myeloid compartment was characterized in tumor tissues following treatment with INCB059872. Notably, the population of PMN-MDSC was significantly decreased in tumor tissues following oral administration of INCB059872, whereas the macrophage population was increased. These data suggest that INCB059872 can redirect myeloid differentiation toward monocyte/macrophages and inhibit the differentiation of PMN-MDSC in this syngeneic tumor microenvironment. Consistently, intratumoral T lymphocyte infiltration was increased following INCB059872 treatment. The combination of INCB059872 and α-PD-L1 antibody enhanced anti-tumor efficacy in the 4T1 orthotopic tumor model. Collectively, these data suggest that inhibition of LSD1 with INCB059872 can directly affect myeloid differentiation to reduce the accumulation of myeloid suppressive cells, restoring the tumor microenvironment to be more responsive to PD-1/PD-L1 axis blockade. This study supports the therapeutic potential for the combination of an LSD1 inhibitor with immuno-therapeutic agents to improve overall clinical response in cancer patients. Citation Format: Thomas Condamine, Steve Wang, Melody Diamond, Leslie Hall, Huiqing Liu, Antony Chadderton, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle, Holly Koblish, Sang Hyun Lee. The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model [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 4635. doi:10.1158/1538-7445.AM2017-4635

Abstract B191: Preclinical assessment of targeting the PI3K/AKT pathway in combination with other signal transduction pathway inhibitors

Inhibitors targeting the phosphoinositide 3-kinase (PI3K)/serine-threonine protein kinase B (AKT) pathway have been developed and have shown efficacy in inhibiting tumor growth both in preclinical models and in the clinic. The effectiveness of PI3K/AKT inhibitors, however, is attenuated by the activation of alternative signaling pathways. Conversely, the PI3K/AKT pathway is considered to be a resistance mechanism for other therapeutic or cytotoxic agents. Therefore, understanding these mechanisms may help to inform the most optimal combinations for advancement into clinical studies. Here, we explored the potential of targeting the JAK/STAT or PIM signaling pathways in combination with a novel, potent and selective AKT inhibitor, INCB047775. Consistent with published data, pharmacological inhibition of AKT by INCB047775 caused the upregulation of PIM kinases. The combined therapies of PIM inhibition and AKT inhibition exhibited significant synergistic anti-tumor effects. Combination of PIM inhibition with AKT inhibition resulted in synergistic induction of apoptosis in cancer cells. Similarly, the combination of JAK tyrosine kinase inhibition with INCB047775 exerted synergistic inhibition of the proliferation of multiple myeloma cell lines. Similar data were observed with an inhibitor of PI3Kδ in combination with both PIM and JAK inhibition, demonstrating significant crosstalk between the PI3K/AKT pathway and other critical signaling pathways. These data provide the rationale for combinatorial therapies with inhibitors targeting JAK or PIM kinases with PI3K/AKT pathway inhibitors in the clinic. Citation Format: Sang Hyun Lee, Holly Koblish, Xiangdong Liu, Haifen Ye, Jun Li, Xuesong Liu, Melody Diamond, Yue Zhang, Leslie Hall, Valerie Dostalik, Margaret Favata, Elham Behshad, Richard Wynn, Yun-long Li, Andrew Combs, Chu-Bio Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle. Preclinical assessment of targeting the PI3K/AKT pathway in combination with other signal transduction pathway inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B191.