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Immune checkpoint blockade has shown impressive efficacy in patients with inflamed tumors, although minimal activity has been observed in tumors lacking T cells. Myeloid cells are one of the most abundant cell types in both inflamed and non-inflamed tumors, and may contribute to immune checkpoint blockade resistance. The plasticity of macrophages enables them to directly and indirectly modulate T cell responses, and directly kill tumor cells via phagocytosis. This suggests that targeting myeloid cells could be an effective therapeutic approach. Class I PI3Ks are a family of dual specificity lipid and protein kinases. Unlike other class I PI3Ks, PI3Kγ is predominantly expressed in myeloid cells. PI3Kγ has been shown to be a key mediator that drives the immunosuppressive macrophage program by stimulating AKT/mTOR signaling and promote C/EBPβ expression while inhibiting NF-кB activity (Keneda MM. Nature. 2016;17:437-442). Here, we present the discovery and characterization of INCB098377, a potent and selective PI3Kγ inhibitor. Specific inhibition of PI3Kγ with INCB098377 may induce anti-tumor activity by reshaping the tumor immune microenvironment. In cell-based assays, INCB098377 has an IC50 of 1.4 nM and is greater than 100-fold selective over other PI3K isoforms. It also shows a favorable PK profile in several animal species. Treatment of M2 polarized macrophages with INCB098377 resulted in changes towards a more pro-inflammatory phenotype. CD163 and CD206 were decreased, whereas HLA-DR and co-stimulatory CD80/86 molecules were increased. MHC-I expression was unchanged, suggesting a role for these macrophages in MHC-II-mediated antigen presentation. Furthermore, INCB098377 treatment reduced macrophage-mediated immunosuppression and restored T cell proliferation in M2 polarized macrophages co-cultured with allogeneic human T cells. In vivo, significant tumor growth inhibition was observed with once-daily dosing of 10 mg/kg INCB098377 in both syngeneic and humanized mouse tumor models without toxicity. Moreover, efficacy was observed in inflamed and non-inflamed tumor models. Consistent with the proposed mechanism of action, INCB098377 inhibited phospho-AKT levels in vivo and in human PBMCs. Treatment with INCB098377 induced pro-inflammatory responses without macrophage depletion suggests that robust tumor microenvironment changes are responsible for observed anti-tumor efficacy. In addition, INCB098377 inhibited neutrophil migration in the Carrageenan-induced paw inflammation model. INCB098377, a potent and selective inhibitor of PI3Kγ, shows effective anti-tumor activity in a variety of mouse and humanized cancer models through the inhibition of immunosuppressive cells trafficking into the tumor, modulation of myeloid cell function, and enhancement of T cell proliferation. Acknowledgments: Diana Alvarez Arias and Stephen Douglass contributed equally to this study. Citation Format: Diana A. Arias, Stephen Douglass, Lisa Truong, Qian Wang, Kathy H. Wang, Gengjie Yang, Michael Hansbury, Sybil O’Connor, Kevin Bowman, Robert Collins, Matthew Stubbs, Leslie Hall, Christina Stevens, Christopher Maddage, Brent Douty, Maryanne Covington, Lynn Leffet, Eddy Yue, Andrew Combs, Sunkyu Kim, Niu Shin, Holly Koblish, Rodrigo Hess. Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5162.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3Kδ inhibitor that differs in structure from first-generation PI3Kδ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3Kδ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3Kδ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3Kδ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)

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.

[Image: see text] A medicinal chemistry effort focused on identifying a structurally diverse candidate for phosphoinositide 3-kinase delta (PI3Kδ) led to the discovery of clinical candidate INCB050465 (20, parsaclisib). The unique structure of 20 contains a pyrazolopyrimidine hinge-binder in place of a purine motif that is present in other PI3Kδ inhibitors, such as idelalisib (1), duvelisib (2), and INCB040093 (3, dezapelisib). Parsaclisib (20) is a potent and highly selective inhibitor of PI3Kδ with drug-like ADME properties that exhibited an excellent in vivo profile as demonstrated through pharmacokinetic studies in rats, dogs, and monkeys and through pharmacodynamic and efficacy studies in a mouse Pfeiffer xenograft model.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, including the skin, joints and kidneys. Though SLE typically cycles through periods of flares and remission, patients often eventually succumb to end-stage kidney or cardiovascular damage1. SLE is characterized by the presence of autoantibodies that form immune complexes resulting in kidney deposition that significantly contribute to lupus nephritis pathogenesis. Phosphatidylinositol 3-kinases (PI3Ks) are divided into three classes (Class I, II, and III) according to their structure, regulation and substrate specificity. The Class I PI3K delta isoform (PI3Kδ) has been implicated in autoimmune diseases associated with aberrant B cell and antibody responses. INCB050465 is an oral small molecule PI3Kδ selective inhibitor currently being evaluated in clinical trials for the inflammatory indications Sjogren’s syndrome and autoimmune hemolytic anaemia (NCT03627065 & NCT03538041). Objectives: To quantify the efficacious potential of INCB050465, a selective PI3Kδ inhibitor, in a preclinical model of systemic lupus erythematosus with kidney pathology. Methods: The MRL/lpr mouse model spontaneously develops multiple inflammatory phenotypes that mimic human SLE pathologies, including kidney damage resulting in proteinuria, cutaneous skin lesions and anti-double stranded DNA (anti-dsDNA) antibodies. Kidney sections were stained with hematoxylin and eosin (H&E) and assigned a composite score based on glomerulus, crescents, protein casts, interstitial inflammation and vasculitis by a rater unaware of the treatment groups2. Flow cytometry was performed on splenocytes to characterize B cell and T cell subsets. Anti-dsDNA antibodies were determined by commercial ELISA. Results: In vitro enzymatic selectivity screening revealed INCB050465 potently inhibited the PI3Kδ kinase enzyme (IC50 = 1.1 ± 0.5 nM), with ∼20,000-fold selectivity for the other PI3K family member enzymes. Proteinuria identified spontaneous kidney damage in 85%+ of vehicle treated mice by 21 weeks of age, and kidney histology revealed gross tissue enlargement associated with structural abnormalities. In contrast, therapeutic INCB050465 treatment (0.3 – 3.0 mg/kg, b.i.d.) dose-dependently reduced the incidence of proteinuria (p Conclusion: INCB050465, a selective PI3Kδ inhibitor, was highly efficacious against immune-mediated mechanisms associated with lupus nephritis, but not cutaneous lupus disease manifestations in this preclinical model. Treatment-induced preservation of kidney tissue integrity corresponded to specific B cell subset modulation and autoantibody reduction. Selective PI3Kδ inhibitor treatment did not induce lymphocyte depletion. Reference: [1] Grande, et al. Experimental models of lupus nephritis. Contrib Nephrol. 2011; 169:183-97. [2] Chan, et al. The roles of B cells in MRL/lpr murine lupus. Ann N Y Acad Sci. 1997Apr5;815:75-87. Disclosure of Interests: Brittany Fay Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Monika Scuron Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Niu Shin Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Yan-ou Yang Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Eddy Yue Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Andrew Combs Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Eduardo Huarte Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Melissa Parker Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation., Paul Smith Shareholder of: The author is an employee and/or shareholder of Incyte Corporation., Employee of: The author is an employee and/or shareholder of Incyte Corporation.

Pharmacological modulation of the Janus kinase (JAK) family has achieved clinically meaningful therapeutic outcomes for the treatment of inflammatory and hematopoietic diseases. Several JAK1 selective compounds are being investigated clinically to determine their anti-inflammatory potential. We used recombinant enzymes and primary human lymphocytes to assess the JAK1 specificity of itacitinib (INCB039110) and study inhibition of signal transducers and activators of transcription (STAT) signaling. Rodent models of arthritis and inflammatory bowel disease were subsequently explored to elucidate the efficacy of orally administered itacitinib on inflammatory pathogenesis. Itacitinib is a potent and selective JAK1 inhibitor when profiled against the other JAK family members. Upon oral administration in rodents, itacitinib achieved dose-dependent pharmacokinetic exposures that highly correlated with STAT3 pharmacodynamic pathway inhibition. Itacitinib ameliorated symptoms and pathology of established experimentally-induced arthritis in a dose-dependent manner. Furthermore, itacitinib effectively delayed disease onset, reduced symptom severity, and accelerated recovery in three distinct mouse models of inflammatory bowel disease. Low dose itacitinib administered via cannula directly into the colon was highly efficacious in TNBS-induced colitis but with minimal systemic drug exposure, suggesting localized JAK1 inhibition is sufficient for disease amelioration. Itacitinib treatment in an acute graft-versus-host disease (GvHD) model rapidly reduced inflammatory markers within lymphocytes and target tissue, resulting in a marked improvement in disease symptoms. This is the first manuscript describing itacitinib as a potent and selective JAK1 inhibitor with anti-inflammatory activity across multiple preclinical disease models. These data support the scientific rationale for ongoing clinical trials studying itacitinib in select GvHD patient populations.

Cancer patients have higher levels of arginase and lower levels of arginine in plasma than healthy subjects. The importance of arginine in maintaining the viability and function of T cells and NK cells is well established. Thus, inhibition of arginase to increase arginine is being investigated clinically as a potential immunotherapeutic approach for the treatment of cancer. Exogenous arginine is also known to be critical for arginine auxotrophic tumor cells which lack the expression of ASS1 and ASL, two key enzymes in the urea cycle that are responsible for the production of arginine. Accordingly, PEGylated arginase has been clinically tested to starve tumors that are arginine auxotrophic. However, the majority of tumor cells are arginine prototrophic based on the expression of ASS1 and ASL, and the effect of arginine on these cells has not been fully explored. In this study, we investigate the effect of exogenous arginine at the pathophysiologically relevant concentrations on the arginine prototrophic tumor cells. Compared to arginine at 100 μM (normal plasma concentration), arginine restriction at 20 μM (plasma concentration detected in some cancer patients) reduced the cell cycle S-phase entry and proliferation signaling in the cells of different tumor types. Importantly, the potencies of multiple chemotherapies, including oxaliplatin, 5-fluorouracil, gemcitabine, and cisplatin, were also reduced by the lower level of arginine (up to ~270 fold) in these tumor cells. In line with the well-established negative correlation between ER stress and autophagy and sensitivity to chemotherapies, we found that L-arginine restriction stimulated the eIF2α/ATF4/CHOP signaling axis, suggesting activation of integrated stress response, ER stress, and autophagic pathways. Blockade of autophagy using either 3-methyladenine, an inhibitor of early stage autophagy, or hydroxychloroquine, an inhibitor of late stage autophagy, partially reduced the arginine restriction-induced chemo-resistance in the cancer cells. Taken together, these data suggest that reduction in arginine through enhanced arginase activity in the tumor microenvironment may represent a metabolic response by the host to slow tumor growth, with the inhibitory effect on the anti-tumor function of immune cells being an unfortunate collateral consequence. Elevating arginine by arginase inhibition may not only take away the metabolic brake on immune cells, but also help restore the sensitivity of tumor cells to chemotherapies. Citation Format: Mingming Gao, Kathy He Wang, Qian Wang, Hao Liu, Valerie Roman, Timothy Burn, Maryanne Covington, Holly Koblish, Niu Shin. L-arginine regulates chemotherapy sensitivity via ER stress and autophagic pathways in arginine prototrophic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2648.

Phosphatidylinositol 3-kinase delta (PI3K

The histamine H₄ receptor mediates several histamine-induced cellular functions of leukocytes, including cell migration and cytokine production. Recent studies suggest that histamine signaling through the histamine H₄ receptor can also have anti-pruritic and anti-nociceptive functions. 1-(7-(2-amino-6-(4-methylpiperazin-1-yl) pyrimidin-4-yl)-3, 4-dihdroisoquinolin-2(1H)-yl)-2-cyclopentylethanone (INCB38579) is a novel small molecule antagonist of the human and rodent histamine H₄ receptors with at least 80-fold selectivity over the human histamine H₁, H₂ and H₃ receptors, and has good pharmacokinetic properties in rats and mice. The compound is potent in inhibiting histamine binding to and signaling through the recombinant human, mouse and rat histamine H₄ receptors and blocks the histamine-induced migration of human and mouse dendritic cells, as well as the cell shape change and migration of human eosinophils. INCB38579 and histamine may have separate but overlapping binding sites on the human histamine H₄ receptor. This novel inhibitor is efficacious when evaluated in two previously established in vivo models for histamine H₄ receptor activity (histamine-induced itch in mice and carrageenan-induced acute inflammatory pain in rats). When examined in formalin-induced pain models, INCB38579 significantly reduces the sustained inflammatory pain experienced by rats and mice. A good correlation between the protein binding adjusted potency from in vitro studies and its analgesic effect in vivo was observed. These results suggest that INCB38579 can serve as a useful tool for pharmacologic characterization of the histamine H₄ receptor and further support the hypothesis that targeting the histamine H₄ receptor may provide new therapeutic agents for various chronic inflammatory diseases, including inflammatory pain.

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.

Indoleamine 2,3-dioxygenase-1 (IDO1; IDO) mediates oxidative cleavage of tryptophan, an amino acid essential for cell proliferation and survival. IDO1 inhibition is proposed to have therapeutic potential in immunodeficiency-associated abnormalities, including cancer. Here, we describe INCB024360, a novel IDO1 inhibitor, and investigate its roles in regulating various immune cells and therapeutic potential as an anticancer agent. In cellular assays, INCB024360 selectively inhibits human IDO1 with IC50 values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)–cell growth, increases IFN-γ production, and reduces conversion to regulatory T (Treg)–like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86high DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells. Consistent with its effects in vitro, administration of INCB024360 to tumor-bearing mice significantly inhibits tumor growth in a lymphocyte-dependent manner. Analysis of plasma kynurenine/tryptophan levels in patients with cancer affirms that the IDO pathway is activated in multiple tumor types. Collectively, the data suggest that selective inhibition of IDO1 may represent an attractive cancer therapeutic strategy via up-regulation of cellular immunity.

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (Kd) of approximately 5 nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.

The histamine H 4 receptor shows generally restricted expression in immune cells and has been shown to mediate histamine induced-chemotaxis, suggesting that it may play a role in inflammation. This has led to the discovery and characterization of histamine H 4 receptor antagonists from multiple chemical scaffolds. These antagonists have shown efficacy in numerous preclinical models of inflammation, further supporting the critical role of the histamine H 4 receptor in inflammatory diseases, including rheumatoid arthritis, asthma, pruritis and inflammatory pain. The first histamine H 4 receptor antagonists have entered into clinical trials and data are expected soon to establish the importance of targeting this receptor as a novel therapeutic in inflammation. This review focuses on the histamine H 4 receptor antagonists that have been identified to date.

The histamine H₄ receptor mediates several histamine-induced cellular functions of leukocytes, including cell migration and cytokine production. Recent studies suggest that histamine signaling through the histamine H₄ receptor can also have anti-pruritic and anti-nociceptive functions. 1-(7-(2-amino-6-(4-methylpiperazin-1-yl) pyrimidin-4-yl)-3, 4-dihdroisoquinolin-2(1H)-yl)-2-cyclopentylethanone (INCB38579) is a novel small molecule antagonist of the human and rodent histamine H₄ receptors with at least 80-fold selectivity over the human histamine H₁, H₂ and H₃ receptors, and has good pharmacokinetic properties in rats and mice. The compound is potent in inhibiting histamine binding to and signaling through the recombinant human, mouse and rat histamine H₄ receptors and blocks the histamine-induced migration of human and mouse dendritic cells, as well as the cell shape change and migration of human eosinophils. INCB38579 and histamine may have separate but overlapping binding sites on the human histamine H₄ receptor. This novel inhibitor is efficacious when evaluated in two previously established in vivo models for histamine H₄ receptor activity (histamine-induced itch in mice and carrageenan-induced acute inflammatory pain in rats). When examined in formalin-induced pain models, INCB38579 significantly reduces the sustained inflammatory pain experienced by rats and mice. A good correlation between the protein binding adjusted potency from in vitro studies and its analgesic effect in vivo was observed. These results suggest that INCB38579 can serve as a useful tool for pharmacologic characterization of the histamine H₄ receptor and further support the hypothesis that targeting the histamine H₄ receptor may provide new therapeutic agents for various chronic inflammatory diseases, including inflammatory pain.

C-C chemokine receptor 5 (CCR5) is a clinically proven target for inhibition of HIV-1 infection and a potential target for various inflammatory diseases. In this article, we describe 5-[(4-{(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl}-4-methylpiperidin-1-yl)carbonyl]-4,6-dimethylpyrimidine dihydrochloride (INCB9471), a potent and specific inhibitor of human CCR5 that has been proven to be safe and efficacious in viral load reduction in phase I and II human clinical trails. INCB9471 was identified using a primary human monocyte-based radioligand competition binding assay. It potently inhibited macrophage inflammatory protein-1β-induced monocyte migration and infection of peripheral blood mononuclear cells by a panel of R5-HIV-1 strains. The results from binding and signaling studies using incremental amounts of INCB9471 demonstrated INCB9471 as a noncompetitive CCR5 inhibitor. The CCR5 residues that are essential for interaction with INCB9471 were identified by site-specific mutagenesis studies. INCB9471 rapidly associates with but slowly dissociates from CCR5. When INCB9471 was compared with three CCR5 antagonists that had been tested in clinical trials, the potency of INCB9471 in blocking CCR5 ligand binding was similar to those of 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R0-2-(methyloxy)-1-[4-(trifluoromethyl) phenyl]ethyl}-1-piperazingyl)-1-piperidinyl]carbonyl}pyrimidine (SCH-D; vicriviroc), 4-{[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxyl)methyl]-2, 5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140; aplaviroc), and 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)cyclohexanecarboxamide (UK427857; maraviroc). Its inhibitory activity against CCR5-mediated Ca(2+) mobilization was also similar to those of SCH-D and 873140. Further analysis suggested that INCB9471 and UK427857 may have different binding sites on CCR5. The significance of two CCR5 antagonists with different binding sites is discussed in the context of potentially overcoming drug-resistant HIV-1 strains.

We report the discovery of a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist (3S,4S)-N-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine (19). After evaluation in 28-day toxicology studies, compound 19 (INCB10820/PF-4178903) was selected as a clinical candidate.

To identify a CCR5 antagonist as an HIV-1 entry inhibitor, we designed a novel series of indane derivatives based on conformational considerations. Modification on the indane ring led to the discovery of compound 22a (INCB9471) that exhibited high affinity for CCR5, potent anti-HIV-1 activity, high receptor selectivity, excellent oral bioavailability, and a tolerated safety profile. INCB9471 has entered human clinical trials.

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 O'Connor, 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

The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) mediates responses to cytokines and growth factors and drives cell proliferation and survival in a number of hematologic malignancies. Overexpression of PIM kinases in these malignancies, including multiple myeloma (MM), has been associated with poor overall survival. Given the overlapping functions of these kinases, the ability of one family member to compensate for the loss of another as well as the relatively benign phenotype of mice deficient in all three PIM isoforms, discovery of pan-PIM kinase inhibitors is warranted. The in vitro and in vivo activity of the pan-PIM kinase inhibitor, INCB53914, was determined in MM cell lines. The antiproliferative potencies for INCB053914 were To understand the impact of inhibiting the PIM pathway in combination with other pathways dysregulated in hematological malignancies, an unbiased in vitro screen was performed and the potential synergy of INCB053914 in combination with 65 cytotoxic or targeted agents was determined. This screen identified several agents active against the PI3K pathway or which impacted cell cycle progression. In addition, the combinatorial activity of selected targeted agents hypothesized to exhibit significant interactions with the PIM pathway was assessed in vivo. Since PIM family members are STAT regulated genes, enhanced activity may be expected upon combined PIM and JAK inhibition. In fact, synergistic activity was seen with this combination in the INA6 multiple myeloma model, and pharmacodynamic analyses revealed enhanced suppression of both pBAD and c-myc levels in tumors from treated mice. Additionally, c-myc levels are regulated both by PIM and the BET family member, BRD4. The expected synergistic efficacy of PIM and BET inhibitors was also observed in the KMS12 model, again with enhanced reduction in pBAD and c-myc levels in the tumors of treated mice. Taken together, these data support the utility of PIM inhibition in MM patients, both as monotherapy and in combination with other targeted agents. Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Xiaoming Wen, Sybil O'Connor, Valerie Dostalik, Qian Wang, Kathy Wang, Maryanne Covington, Cindy Marando, Kevin Bowman, Jason Boer, Krista Burke, 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 multiple myeloma. [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 5414. doi:10.1158/1538-7445.AM2015-5414

Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2), giving rise to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in both B cell development and function, and has now been validated as a therapeutic target in CLL and indolent NHL upon the approval of idelalisib, a selective PI3Kδ inhibitor. However, despite the demonstrated activity of PI3Kδ inhibitors in several subtypes of B cell malignancies, the effectiveness of the approach as a single agent for the treatment of DLBCL, the largest subtype of NHL, has not been demonstrated. We hypothesized that an optimal treatment strategy in this histology would require combined inhibition of PI3Kδ with additional agents that target B cells through distinct signaling pathways. INCB050465 is a novel, small molecule inhibitor of PI3Kδ. In biochemical assays, it potently inhibits PI3Kδ (IC50 = 1 nM at 1 mM ATP) with approximately 20,000-fold selectivity for PI3Kα, PI3Kβ, PI3Kγ and 57 other kinases. In various PI3Kδ functional assays, including B cell proliferation induced by activation of BCR, BAFF-R, IL-4R, CD40 and TLRs as well as T cell differentiation assays, INCB050465 demonstrates potent activity with IC50 values ranging from 0.2 to 2 nM. In addition, INCB050465 blocks the proliferation of several DLBCL and MCL cell lines in vitro (EC50 < 10 nM) and can slow tumor growth in the Pfeiffer model of DLBCL in vivo. In cells, INCB050465 blocks constitutive PI3Kδ signaling, including phosphorylation of Akt and FOXO3a, with potencies similar to those for inhibiting proliferation. More importantly, the ability of INCB050465 to inhibit DLBCL cell proliferation in vitro can be further potentiated by inhibition of PIM (Proviral Integration sites for Moloney virus) protein kinases, which were also found to be important for B cell development and to phosphorylate substrates which overlap with those of the PI3K pathway. The combined inhibition of PI3Kδ by INCB050465 with the potent and selective PIM protein kinase inhibitor, INCB053914, results in profound tumor regression in the Pfeiffer cell model of DLBCL. Taken together, these results provide evidence for cross-talk between the PIM and PI3Kδ pathways, and support a mechanistic rationale for the clinical evaluation of combined PIM and PI3Kd inhibition in B cell malignancies, including DLBCL. Citation Format: Niu Shin, Holly Koblish, Maryanne Covington, Yanlong Li, Kathy Wang, Qian Wang, Patricia Feldman, Leslie Hall, Sybil O'Connor, Xin He, Kamna Katiyar, Yu Li, Eddy W. Yue, Thomas P. Maduskuie, Brent Douty, Song Mei, Yun-Long Li, Chu-Biao Xue, Andrew Combs, Wenqing Yao, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Robert Newton, Kris Vaddi, Reid Huber, Peggy Scherle. INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL. [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 2671. doi:10.1158/1538-7445.AM2015-2671

The PIM (Proviral Integration sites for Moloney virus) family of constitutively active serine/threonine kinases consists of three members (PIM1, PIM2 and PIM3) that phosphorylate substrates involved in both apoptosis and cellular metabolism. As downstream targets of the JAK/STAT pathway and by having overlapping substrates with the PI3K/AKT pathway, PIM kinases integrate signals from multiple pathways important for the survival and proliferation of malignant cells. Overexpression of PIM kinases has been reported in human hematological cancers with each isoform showing a distinct expression pattern among the various malignancy subtypes, including MM, AML, ALL and NHL. Although the three family members possess overlapping functions that are mutually compensatory, mice deficient for all three PIM kinases exhibit minor phenotypic abnormalities, with smaller overall size at maturity and decreased cellular responses to cytokines. These data suggest that pan-inhibition of the PIM kinase family will have a favorable toxicity profile and may be required for therapeutic benefit in hematological malignancies. INCB053914 is a novel, ATP-competitive, small molecule, pan-inhibitor of PIM kinases. In biochemical assays, INCB053914 is potent against PIM1, PIM2 and PIM3 and highly selective. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50 values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. Following treatment with INCB053914, induction of PIM kinase protein levels was noted in human cancer cell lines. Importantly, this compensatory response does not reduce the activity of the molecule, as effects on downstream signaling and efficacy remain intact when PIM kinase levels are elevated. Induction of PIM kinases after INCB53914 treatment was also evident in whole blood from healthy volunteers and in primary blast cells isolated from patients diagnosed with AML, further supporting the need for pan-PIM kinase inhibition. Taken together, these results indicate that INCB053914 has potent and selective activity against PIM kinases and may be useful as monotherapy or in combination with other agents for treating hematologic malignancies that are dependent on PIM kinase activity for growth and survival. Citation Format: Niu Shin, Maryanne Covington, Richard Wynn, Yanlong Li, Alexander Margulis, Qian Wang, Kathy Wang, Cindy Marando, Patricia Feldman, Lynn Leffet, Karen Gallagher, Xin He, Hong Chang, He Zhang, Hao Feng, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Timothy Burn, Kris Vaddi, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Gregory Hollis, Robert Newton, Reid Huber, Peggy Scherle, Holly Koblish. Characterization of INCB053914, a novel pan-PIM kinase inhibitor. [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 5397. doi:10.1158/1538-7445.AM2015-5397

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.

The histamine H4 receptor is a novel G-protein-coupled receptor with a unique pharmacological profile. The distribution of H4 mRNA suggests that it may play a role in the regulation of immune function, particularly with respect to allergy and asthma. To define the histamine-binding site of this receptor, molecular modeling and site-directed mutagenesis were used to predict and alter amino acids residing in the histamine-binding pocket. The effects of these alterations on histamine binding and receptor activation were then assessed. Our results indicate that Asp94 (3.32) in transmembrane region (TM) 3 and Glu182 (5.46) in TM5 are critically involved in histamine binding. Asp94 probably serves as a counter-anion to the cationic amino group of histamine, whereas Glu182 (5.46) interacts with the N(tau) nitrogen atom of the histamine imidazole ring via an ion pair. In contrast, Thr178 (5.42) and Ser179 (5.43) in TM5 are not significantly involved in either histamine binding or receptor activation. These results resemble those for the analogous residues in the H1 histamine receptor but contrast with findings regarding the H2 histamine receptor. Our results also demonstrate that Asn147 (4.57) in TM4 and Ser320 (6.52) in TM6 play a role in receptor activation but are not involved in histamine binding. Taken together, these data indicate that although histamine seems to bind to the H4 receptor in a fashion similar to that predicted for the other histamine receptor subtypes, there are also important differences that can probably be exploited for the discovery of novel H4-selective compounds.

Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma, and can be classified into activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL according to gene expression profiling. In both subtypes, dysregulation of the B cell receptor (BCR) signaling pathway plays a critical role in tumorigenesis and survival. BCR activation induces phosphorylation of CD79A/B by Src family tyrosine kinases, which leads to the activation of the BTK and PI3Kδ/Akt pathways. Oncogenic mutations have been identified in DLBCL impacting CD79A/B (BCR complex), PTEN (negative regulator of PI3K), CARD11 and MYD88 (NF-kB activating factors) function. The influence of these mutations on the DLBCL phenotype appears to be further modified by aberrant cytokine signaling and JAK/Stat pathway activation. High levels of JAK/Stat-activating cytokines such as IL6 and IL10 are present in DLBCL, and patients with high serum IL10 have been shown to have shorter event-free survival and a higher international Prognostic Index score. Therefore, concurrent inhibition of both the BCR and JAK/Stat pathways may provide an attractive therapeutic approach in DLBCL. We have developed and characterized a potent and selective PI3Kδ inhibitor INCB040093 and have established that DLBCL cells are sensitive to INCB040093 treatment. We demonstrate that IL10 activates the JAK/Stat signaling pathway in DLBCL cells, which renders the cells resistant to INCB040093 in cell proliferation assays. This resistance can be reversed by co-treatment with a JAK1/JAK2 inhibitor. Although INCB040093 induces apoptosis in DLBCL cells, concurrent activation of the JAK/Stat pathway by IL10 prevents INCB040093-induced apoptosis, an effect that can be abrogated by a JAK1/JAK2 inhibitor. Similar resistance to IL-10 is observed with the BTK inhibitor, ibrutinib, which can also be reversed by a JAK1/JAK2 inhibitor. Interestingly, a JAK1 selective inhibitor is as effective as a JAK1/JAK2 inhibitor in reversing the IL10-mediated resistance to PI3Kδ and BTK inhibition. Combination treatment blocks both PI3K/Akt and JAK/Stat signaling pathways and results in the down-regulation of pro-survival proteins, including Pim2 and c-myc. These data illustrate the functional cross-talk that exists in DLBCL between the BCR and JAK/Stat signaling pathways, and support the clinical evaluation of combined BCR and JAK inhibition as a novel approach to targeted therapy. Citation Format: Xuesong (Mike) Liu, Margaret Favata, Jun Li, Niu Shin, Kathy He Wang, Qian Wang, Yun-Long Li, Andrew Combs, Chu-Biao Xue, Robert Newton, Kris Vaddi, Peggy Scherle. JAK inhibition reverses IL10-mediated resistance to B cell receptor (BCR) pathway inhibition in DLBCL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4207. doi:10.1158/1538-7445.AM2014-4207

Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate, giving rise to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and functions by mediating the signaling of key receptors on B cells including BCR, BAFF-R, IL-4R, CD40 and TLRs. In addition, increased malignant B cell proliferation and survival has been associated with aberrant activation of PI3Kδ, and data from recent clinical studies using idelalisib, a PI3Kδ specific inhibitor, indicate that the enzyme is a therapeutic target for the treatment of B cell lymphomas. INCB40093 is a novel, potent small molecule inhibitor of PI3Kδ with 74 to > 900-fold selectivity over the other PI3K family members. It also does not inhibit (< 30% inhibition) a broad panel of 196 other kinases when tested at a concentration of 1 μM. INCB40093 blocks the proliferation of primary B cells induced by the activation of PI3Kδ-mediated signaling through several key receptors (e.g. BCR, CD40). More importantly, INCB40093 is potent (IC50 values of ∼10-100 nM) in cell-based assays relevant to the pathogenesis of B cell malignancies, such as PI3Kδ-mediated signaling and growth of human B cell lines. This effect is not due to general cytotoxicity because 10 μM INCB40093 had no significant effect on the growth of non-lymphoid cell lines. Compared to the inhibition of primary B cell proliferation, INCB40093 is at least 50 times less potent in assays that measure human T cell or NK cell proliferation, and neutrophil and macrophage function, suggesting that the impact of INCB40093 on the human immune system will likely be restricted to B cells. In vivo, the effects of INCB40093 were evaluated in two human hematological cancer xenograft models. In the Pfeiffer model of non-Hodgkin's lymphoma, oral administration of INCB40093 inhibited tumor growth as a single agent. INCB40093 also potentiated the anti-tumor effects of the clinically relevant chemotherapeutic agent, bendamustine. The tumor growth inhibitory effects of INCB40093 were also demonstrated in a myeloma tumor xenograft model, INA-6.Tu1. Taken together, pharmacological data obtained in both in vitro and in vivo model systems support the potential utility of orally administered INCB40093 in the treatment of B cell malignancies. Citation Format: Niu Shin, Kathy Wang, Leslie Hall, Qian Wang, Gengjie Yang, Yanlong Li, Yun-Long Li, Maryanne Covington, Jordan Fridman, Robert Newton, Peggy Scherle. INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2014-4531

The histamine H4 receptor (H4) has been considered as an attractive therapeutic target for a number of diseases, such as allergy, asthma, atopic dermatitis and inflammatory pain. INCB38579 is a novel human and rodent active H4 receptor antagonist, with at least 50-fold selectivity over the human H1, H2 and H3 receptors and good pharmacokinetic properties in rats and mice. The compound potently inhibits histamine binding to and signaling through recombinant human and mouse H4 and blocks histamine-induced migration of human and mouse dendritic cells, as well as cell shape change and migration of human eosinophils. This novel H4 inhibitor is efficacious in blocking histamine-induced pruritus in mice and carrageenan-induced acute inflammatory pain in rats. Interestingly, when examined in a formalin pain model, INCB38579 could significantly reduce the relatively long lasting inflammation associated pain. A good correlation between the predicted protein binding adjusted potency from in vitro studies and the analgesic effect in vivo was observed. These results suggest that INCB38579 can serve as a useful tool for pharmacologic characterization of H4 and further supports the hypothesis that the H4 receptor is a target for the development of new therapeutics for various inflammatory diseases.

Indoleamine 2,3-dioxygenase (IDO or IDO1) mediates the oxidation of tryptophan, an amino acid essential for cell proliferation and survival. Inhibition of IDO activity or expression has shown therapeutic potential in preclinical models of immunodeficiency-associated abnormalities including cancer. Here we report the identification of INCB024360, a novel, potent and selective small molecule inhibitor of IDO1, and the investigation of its effects on the proliferation and activation of various immune cells in vitro as well as its anti-tumor activity in preclinical tumor models. In multiple cell-based assays, INCB024360 potently inhibits human IDO1 with an IC50 of approximately 10 nM - the most potent IDO1 inhibitor published thus far. INCB024360 is IDO1 selective and has little activity against other related enzymes including IDO2, TDO or tryptophan transporters. In co-culture systems of human allogeneic lymphocytes with either dendritic cells or tumor cells, inhibition of IDO1 by INCB024360 increases the proliferation of T and NK cells, as well as IFN- production. INCB024360 addition also reduces the generation of regulatory T cells. Interestingly, IDO1 expression promotes dendritic cell apoptosis while addition of INCB024360 reverses this and increases the number of CD86high cells within the IDO+ DC population, potentially representing a novel mechanism by which IDO1 inhibition can promote T cell activation. In vivo, INCB024360 inhibits tryptophan catabolism in tumors and tumor draining lymph nodes and controls tumor growth either alone or in combination with chemotherapeutic agents in multiple tumor models. The ability to reduce tumor growth is dependent on a functional immune system, consistent with the proposed mechanism of action. Finally, an analysis of kynurenine/tryptophan levels in patient blood samples affirms that the IDO activity is elevated in multiple tumor types. Collectively, these data suggest that INCB024360, a potent IDO1 selective inhibitor, has the potential to be a novel and effective immunotherapeutic agent for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C106.