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Supplementary Table from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Supplementary Figure from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies.Significance:We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach.See related commentary by Capparelli and Aplin, p. 1413.This article is highlighted in the In This Issue feature, p. 1397

Supplementary Data from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Supplemental Tables 1 & 2 Supplemental Figure Legends Supplemental Figures 1 - 5

Purpose:Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials.Experimental Design:We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations.Results:In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK–STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone.Conclusions:Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer [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 412.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. Significance: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397

Purpose: Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials. Experimental Design: We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations. Results: In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK–STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone. Conclusions: Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations.

BackgroundBlocking the PD-L1 immune checkpoint axis with therapeutic antibodies against either the ligand or PD-1 has proven to be an effective treatment modality for multiple cancer histologies. Small molecules targeting the PD-L1/PD-1 axis represent an alternate modality of blocking this pathway. INCB090244 is a small molecule that blocks the PD-L1/PD-1 interaction and restores T cell function similar to the clinical stage PD-L1 inhibitor INCB086550.MethodsMDA-MB-231 or CHO cells overexpressing PD-L1 were used to investigate effects of INCB090244 on PD-L1 dimerization, and intracellular trafficking. In vivo, CD34+ humanized mice harboring MDA-MB-231 tumors or C57Bl/6 mice bearing GL261 subcutaneous or orthotopic tumors were used to investigate the efficacy, biodistribution, and pharmacodynamic effects of INCB090244. Human specific gene expression changes in tumors from MDA-MB-231 bearing humanized mice were analyzed by RNA sequencing.ResultsIn vitro, INCB090244 potently disrupted the PD-L1:PD-1 interaction, induced PD-L1 dimerization, and inhibited PD-1-mediated negative signaling, resulting in enhanced IFN gamma and IL-2 production in primary human immune cells. Following dimerization, INCB090244 induced internalization of PD-L1 resulting in co-localization with the Golgi apparatus and partial localization in the nucleus. After cell treatment and washing, full restoration of PD-L1 at the cell surface was observed after 5 days of culture in vitro. In vivo, INCB090244 reduced tumor growth in CD34+ humanized mice bearing MDA-MB-231 tumors, to similar levels as atezolizumab. Antitumor activity was completely abrogated in immunodeficient mice, confirming the pharmacologic dependency on a competent immune system. RNA sequencing analysis on tumors from these mice demonstrated similar T cell activation gene signatures as clinical checkpoint blockade antibodies. Biodistribution studies in mice bearing both subcutaneous and orthotopically implanted GL261 glioma tumors demonstrated higher accumulation of INCB090244 in tumor tissue compared to PD-L1 antibodies.ConclusionsINCB090244 effectively disrupted the PD-L1/PD-1 interaction, induced dimerization and internalization of PD-L1, restored immunity in in vitro and in vivo tumor models, and is a suitable surrogate for the clinical candidate INCB086550. RNA sequencing demonstrated T cell activation signatures similar to those observed in patients receiving checkpoint blockade antibodies. Biodistribution studies demonstrated higher subcutaneous and brain tumor penetration by INCB090244 compared to PD-L1 antibodies, suggesting a potential advantage of small molecule PD-L1 inhibitors in accessing intratumoral regions. These data further support the clinical evaluation of small molecule PD-L1 inhibitors as an alternative approach to immune therapy.

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.

A significant population of patients with myelofibrosis (MF) develop anemia and either require red blood cell (RBC) transfusions or have an inadequate response to the currently available therapies and become transfusion-dependent. In patients with MF, elevated levels of serum hepcidin, a key iron regulatory hormone, is associated with increased dependence on RBC transfusions and reduced overall survival. Elevated hepcidin expression has also been observed to cause severe functional iron deficiency anemia and is central to the pathophysiology of anemia of chronic disease. Thus, to ensure proper maintenance of iron homeostasis, hepcidin levels are tightly regulated. Specifically, the production of hepcidin is controlled by the bone morphogenetic protein (BMP) type I receptor ACVR1, a gene that encodes the serine/threonine kinase ALK2. In preclinical models, knockdown or complete loss of ALK2 decreases hepcidin production resulting in elevated serum iron levels. In this study, we report characterization of INCB00928, a novel small molecule inhibitor of ALK2 for the treatment of anemia. INCB00928 was observed to have subnanomolar activity against ALK2 and selectivity over ALK1 and ALK3 in biochemical enzyme assays. In cell-based profiling studies, INCB00928 inhibited ALK2 potently and selectively over ALK1 and ALK3 as determined by the inhibition of ligand-induced SMAD pathway signaling. Importantly, in both an immortalized human liver cell line as well as primary human hepatocytes, INCB00928 inhibited BMP-induced production of hepcidin with nanomolar activity. INCB00928 was also observed to have suitable absorption, distribution, metabolism, and excretion properties to be dosed in in vivo rodent studies. In tumor- and inflammation-induced mouse models of anemia, INCB00928 improved RBC count, hemoglobin, and hematocrit levels while decreasing hepcidin levels in a dose-dependent manner. Additionally, consistent with the improved symptoms of anemia, pSMAD1/5 inhibition was observed in a dose-dependent manner in liver tissues collected from INCB00928-treated mice. In summary, INCB00928 is a potent, selective, and orally available small molecule inhibitor of ALK2, which significantly reduces the production of hepcidin in human liver cells, primary hepatocytes, and in rodent models of anemia. For the majority of patients with MF, the management of anemia remains an unmet need. The preclinical findings from this study suggest ALK2 kinase inhibition with INCB00928 may be a promising novel treatment to reduce the production of hepcidin and improve MF-related anemia in humans, thus warranting further investigation. Disclosures Chen: Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Stubbs:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Pusey:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Wen:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Collins:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Kapilashrami:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Rupar:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Thekkat:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Lin:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Bowman:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Yang:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Diamond:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Yeleswaram:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Kim:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Koblish:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Chen:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Wee:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company.

Antigen recognition and T-cell receptor (TCR) activation are fundamental processes that drive anti-tumor T cell responses. HPK1 has been identified as a negative regulator of TCR activation, as well as BCR activation, and is a potential anticancer target for immuno-oncology. We show that genetic ablation of HPK1 in human T cells resulted in increased cytokine production upon cell stimulation. Jurkat cells with genetic HPK1 knockout were unable to phosphorylate SLP76, a direct downstream target of HPK1, upon cell stimulation which was associated with increased IL-2 production. Genetic deletion of HPK1 in human RAMOS B cells reduced phosphorylation of BLNK upon IgM stimulation and led to increased TNF alpha and TNF beta production. Treatment of HPK1 knockout primary human T cells with pembrolizumab enhanced IFN gamma secretion compared to the knockout cells alone. Based on these genetic data, HPK1 may be an attractive target for immuno-oncology. We describe herein the in vitro and in vivo profile of several small molecule HPK1 inhibitors and report a surprising disparity between in vitro and in vivo findings. In vitro, the HPK1 inhibitors tested phenocopied the genetic data by potently inhibiting pSLP76 and enhancing IL-2 production in Jurkat cells following stimulation. Similarly, HPK1 inhibitors enhanced IL-2 production in human PBMCs, and increased IFN gamma production in combination with atezolizumab in a primary T cell co-culture assay. In vivo, the HPK1 small molecule inhibitors inhibited tumor growth in the MC38 model, which was further enhanced in combination with anti-PD-L1 in 3 different syngeneic models (MC38, CT26 and MBT-2). Despite these results, tumor growth inhibition was not observed in the GL261 glioma syngeneic model whose growth was recently shown to be inhibited when implanted into HPK1 kinase-dead mice. Further, although several in vitro assays demonstrated increased functional cytokine production with HPK1 inhibitor treatment, the compounds did not increase in vivo cytokine production in tumors. Moreover, combining HPK1 inhibitors with a PD-L1 antibody in vivo abolished the anti-PD-L1-induced production of IFN gamma in the CT26 model. In summary, while our genetic data support the role of HPK1 as a negative regulator of T and B cells, the in vitro activity of HPK1 kinase activity inhibitors was not correlated with functional effects in in vivo syngeneic tumor models. These results underline the complexity of interpreting HPK1 biology and also highlight challenges for the development of clinically active compounds targeting this pathway. Citation Format: Yaoyu Chen, Jonathan Rios-Doria, Michelle Pusey, Kerri Lasky, Min Ye, Pramod Thekkat, Karen Gallagher, Kristine Stump, Patricia Conlen, Christine Gardiner, Hui Wang, Alexander Sokolsky, Mark Rupar, Luping Lin, Elham Behshad, Maryanne Covington, Holly Koblish, Oleg Vechorkin, Wenqing Yao, Sunkyu Kim, Yingnan Chen. The role of HPK1 in the regulation of T cell function and anti-tumor immune activity [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 4513.

Phosphatidylinositol 3-kinase delta (PI3K

The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

Blocking the PD-(L)1 immune checkpoint axis with therapeutic antibodies against either the receptor or the ligand has proven to be an effective treatment modality for multiple cancer histologies. We describe the identification and characterization of novel small molecule antagonists of the PD-(L)1 axis that function by inducing dimerization and subsequent internalization of the PD-L1 protein, effectively depleting the ligand from the cell membrane and preventing PD-1 activation on T cells. Compound-dependent PD-L1 dimerization was characterized using several biophysical techniques including fluorescence resonance energy transfer (FRET) measurements, size exclusion chromatography and thermal shift analysis. Experimental evidence demonstrates compound-dependent dimer conformation with slow dissociation kinetics and significantly enhanced thermal stability. Many of the PD-L1-directed small molecules blocked binding of soluble PD-1 to either native PD-L1 expressed on cancer cell lines or PD-L1 expressed in CHO cells with low nanomolar potency. However, only a subset of the small molecules caused loss of cell surface PD-L1 in a time- and concentration-dependent manner. Importantly, there was a strict correlation between the promotion of PD-L1 internalization secondary to dimerization and the induction of an NFAT response element-luciferase reporter gene. Strikingly, only those small molecules that could produce a specific dimeric PD-L1 conformation as measured using FRET were associated with functional activity in cells, suggesting that PD-L1 dimerization was necessary but not sufficient for internalization and cellular activity. A cell-active tool compound (cell binding IC50 Citation Format: Phillip C.C. Liu, Richard Wynn, Liangxing Wu, Alla Volgina, Nina Zolotarjova, Luping Lin, Pramod Thekkat, Alex Margulis, Ronald Klabe, Wenqing Yao, Kaijiong Xiao, Jingwei Li, Xin He, Mark Rupar, Hong Chang, Paul Waeltz, Yanlong Li, Peggy Scherle, Reid Huber, Gregory Hollis. Novel small-molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4483.

The substitution of valine with phenylalanine at amino acid 617 of the Janus kinase 2 (JAK2) gene (JAK2 p.V617F) occurs in a high proportion of patients with myeloproliferative neoplasms (MPNs). The ability to accurately measure JAK2 p.V617F allele burden is of great interest given the diagnostic relevance of the mutation and the ongoing clinical evaluation of JAK inhibitors. A main hurdle in developing quantitative assays for allele burden measurement is the unavailability of accurate standards for both assay validation and use in a standard curve for quantification. We describe our approach to the validation of standards for quantitative assessment of JAK2 p.V617F allele burden in clinical MPN samples. These standards were used in two JAK2 p.V617F assays, which were used to support clinical studies of ruxolitinib (Jakafi(®)) in myelofibrosis, a real-time polymerase chain reaction assay for initial screening of all samples, and a novel single-nucleotide polymorphism typing (SNaPshot)-based assay for samples with less than 5% mutant allele burden. Comparisons of allele burden data from clinical samples generated with these assays show a high degree of concordance with each other and with a pyrosequencing-based assay used for clinical reporting from an independent laboratory, thus providing independent validation to the accuracy of these standards.

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.

High-throughput screening (HTS) of ~50,000 chemical compounds against phosphorylated and unphosphorylated c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF), was carried out in order to compare hit rates, hit potencies and also to explore scaffolds that might serve as potential leads targeting only the unphosphorylated form of the enzyme. The hit rate and potency for the confirmed hit molecules were higher for the unphosphoryalted form of c-Met. While the target of small molecule inhibitor discovery efforts has traditionally been the phosphorylated form, there are now examples of small molecules that target unphosphorylated kinases. Screening for inhibitors of unphosphorylated kinases may represent a complementary approach for prioritizing chemical scaffolds for hit-to-lead follow ups.

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC(50) or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.

Pharmacological inhibition of the oncogenic PI3Kδ pathway has been shown to be efficacious in patients with hematopoietic malignancies. However, its therapeutic application in patients with solid tumors has not yet been tested. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, resulting from the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses. Therefore we explored the immunotherapeutic potential of our PI3Kδ-selective compound, INCB050465, in multiple preclinical tumor models. We demonstrate that INCB050465 can block tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition is not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate the immune-mediated mechanisms, tumors exposed to vehicle or INCB050465 were harvested and analyzed for modulation of gene expression and immune phenotype. INCB050465 was shown to significantly downregulate T cell gene signatures in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. In contrast, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. We next examined INCB050465 in combination with other immune modulators. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T cell-inflamed model by reducing both Treg and M2 macrophages, which then allowed the re-activation of both CD4 and CD8 T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell responses over time. In summary, inactivation of PI3Kδ with a pharmacological inhibitor can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and associated cellular responses may provide a rationale to identify individuals who may benefit most from PI3Kδ inhibitor-based immunotherapy combinations in the clinic.

Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

Inhibitors of the Bromodomain and Extra-Terminal (BET) family of bromodomain containing proteins regulate expression of key cell fate, cell cycle, and survival genes including c-myc. In preclinical models, BET inhibitors have demonstrated significant efficacy in a variety of different oncology indications, including hematological malignancies. Here we describe the preclinical profile of the novel, orally bioavailable BET inhibitor INCB057643 in preclinical models of hematologic malignancies. INCB057643 inhibited binding of BRD2/BRD3/BRD4 to an acetylated histone H4 peptide in the low nM range, and was selective against other bromodomain containing proteins. In vitro analyses showed that INCB057643 inhibited proliferation of human AML, DLBCL, and multiple myeloma cell lines, with a corresponding decrease in MYC protein levels. Cell cycle analyses indicated that G1 arrest and a concentration-dependent increase in apoptosis were seen within 48 hours of treatment with INCB057643. BRD proteins also regulate the expression of many pro-inflammatory genes. Production of several cytokines, including IL-6, IL-10 and MIP-1α, was repressed by INCB057643 in human and mouse whole blood stimulated ex vivo with LPS. Consistent with these effects, analyses of gene expression in cells treated with INCB057643 revealed that pathways involved in cell cycle progression, apoptosis, and IL-6 were among the most significantly altered in vitro. Oral administration of INCB057643 resulted in significant anti-tumor efficacy in xenograft models of AML, myeloma, and DLBCL. Additionally, combining INCB057643 with standard of care agents used for the treatment of DLBCL including rituximab and bendamustine resulted in enhanced anti-tumor efficacy relative to that achieved with single agent therapies at doses that were well tolerated. In addition, many B cell malignancies are reliant on the PI3Kδ pathway for proliferation and survival, suggesting that the combination of INCB057643 with the clinical stage PI3Kδ specific inhibitor INCB050465 may be a rational therapeutic strategy for DLBCL. Compared with single agent BETi or PI3Kδi therapy, the combination significantly potentiated tumor growth inhibition in DLBCL models representative of the ABC subtype (HBL-1), and the double hit GCB subtype (WILL2). These data suggest that clinical exploration of INCB057643 as a monotherapy or in combination in hematologic malignancies is warranted. Citation Format: Matthew C. Stubbs, Thomas Maduskuie, Timothy Burn, Sharon Diamond-Fosbenner, Nikoo Falahatpisheh, Alla Volgina, Nina Zolotarjova, Xiaoming Wen, Patricia Feldman, Mark Rupar, Robert Collins, Cindy Marando, Bruce Ruggeri, Maryanne Covington, Xuesong Mike Liu, Richard Wynn, Swamy Yeleswaram, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Andrew P. Combs, Phillip C. Liu. Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies [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 5071. doi:10.1158/1538-7445.AM2017-5071

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.

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 expression of key survival and cell fate determining genes such as c-myc. Among the various tumor histologies, hematologic malignancies are among the most sensitive cancers to BET inhibition. INCB054329 is a novel, non-benzodiazepine, selective BET inhibitor that is undergoing Phase 1 clinical trials and that has shown encouraging in vitro and in vivo preclinical activity in several models of hematologic malignancy. In the current study, the activity of INCB054329 was evaluated in models of B cell malignancy. INCB54329 effectively inhibited the in vitro growth of a panel of cell lines representing both Hodgkin and non-Hodgkin lymphoma. Treated cells arrested primarily in G1 with sensitive lines also exhibiting dose and time-dependent apoptosis. Within a panel of double-hit lymphoma cell lines, which have activating chromosomal rearrangements in both c-myc and bcl-2, INCB054329 potently inhibited cell growth and was more effective than antagonists of BTK, bcl-2, PIM and PI3Kδ. INCB054329 also showed in vivo efficacy in models of diffuse large B-cell lymphoma (DLBCL). As a single agent, oral administration of INCB054329 inhibited tumor growth in Pfeiffer (GBC) and WILL-2 (GCB, double-hit) subcutaneous xenograft models. The in vivo combination of bendamustine with INCB054329 enhanced anti-tumor efficacy compared with either agent alone in the Pfeiffer model, and the combination was well tolerated. A rational, targeted combination strategy was evaluated involving INCB054329 and a selective, orally active PI3Kδ inhibitor, INCB050465, which is currently in clinical trials in B cell malignancies. Combining INCB054329 with PI3Kδ inhibition markedly enhanced anti-tumor efficacy, increasing the incidence of partial tumor regressions in vivo. In this model, both INCB054329 and INCB050465 treatment led to a reduction in c-Myc protein levels, suggesting a convergence between modulation of BET transcriptional regulation and the PI3Kδ pathway. These data suggest that clinical investigation of INCB054329, both as monotherapy and in combination with standard of care or novel targeted therapies, in several classes of B cell lymphoma, including high risk double hit lymphoma, is warranted. Citation Format: Matthew Stubbs, Robert Collins, Alla Volgina, Mike Liu, Margaret Favata, Mark Rupar, Xiaomng Wen, Richard Sparks, Thomas Maduskuie, Maryanne Covington, Timothy Burn, Bruce Ruggeri, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. Activity of the BET inhibitor INCB054329 in models of lymphoma. [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 3780.

Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [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 C103.

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Bromodomain and Extra Terminal (BET) protein inhibitors have emerged as a potentially effective therapeutic option for multiple tumor types, through their ability to regulate expression of genes necessary for proliferation and survival. For example, multiple myeloma (MM) cells have been shown to be highly sensitive to BET inhibition due in large part to the ability of BET proteins to control transcription of c-myc, an oncogene known to be dysregulated in MM. Likewise, some inflammatory response and cytokine signaling pathways associated with MM (eg. IL-6/JAK/STAT pathway) have also been shown to be reliant on BET proteins. Therefore, inhibition of both BET proteins and the JAK/STAT signaling pathway may be beneficial to MM patients. Here we assess the in vitro and in vivo effects of combining clinical compounds that target BET proteins and JAK in multiple myeloma cell lines. Studies were performed using the potent pan-BET inhibitor INCB054329 and selective JAK1 inhibitors. When tested in cell proliferation assays, the combination of BET and JAK1 inhibitors displayed strong synergistic effects in the IL-6 dependent INA-6 MM cell line in vitro. Western blots also revealed that several pharmacodynamic (PD) markers including c-MYC, PIM-2 and phospho-STAT3 were further repressed with the combination than with single agents alone. Likewise, the c-MYC and p-STAT3 PD markers could also be increasingly repressed in vivo by combined administration of BET and JAK1 inhibitors in the INA-6 mouse xenograft model. In vivo efficacy experiments in the INA-6 model resulted in enhanced, synergistic tumor growth inhibition in the BET/JAK inhibitor cohort as compared with the single drug cohorts. Interestingly, the cytokine independent MM1.S cell line was also sensitive to the BET/JAK inhibitor combination in vivo, while being far less sensitive to JAK1 inhibition as a monotherapy. In the MM1.S model, the c-MYC and p-STAT3 PD markers also behaved as seen in the INA-6 model. Our data indicate that the pharmacological inhibition of BET proteins and JAK1 yields strong combinatorial effects in MM cell lines both in vitro and in vivo. Therefore, dual inhibition of BET proteins and the JAK/STAT signaling pathway may offer a novel therapeutic approach and suggest a potential clinical utility for this drug combination in MM. Citation Format: Matthew C. Stubbs, Xuesong M. Liu, Xiaoming Wen, Jun Li, Valerie Dostalik, Sybil O'Connor, Eian Caulder, Margaret Favata, Mark Rupar, Yu Li, Beth Rumberger, Thomas Maduskuie, Richard Sparks, Nikoo Falahatpisheh, Padmaja Polam, Kris Vaddi, Timothy Burn, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. The BET inhibitor INCB054329 is synergistic with JAK1 inhibition 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 692. doi:10.1158/1538-7445.AM2015-692

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. Genomic analyses of squamous cell lung, gastric and urothelial tumors have revealed recurrent genetic alterations in FGFR1, FGFR2 and FGFR3 genes, respectively. FGFR proteins contribute to the development of malignancies by promoting tumor cell proliferation, survival, and migration and supporting angiogenesis. Therefore targeting FGFR kinases may provide therapeutic benefit to patients with cancers that have genetic alterations in genes encoding components of the FGF-FGFR axis. INCB054828 is a potent inhibitor of FGFR1, FGFR2, and FGFR3 that has selective pharmacological activity against cancer cells with FGFR alterations. In vitro, INCB054828 potently inhibited the kinase activity of recombinant FGFR1, FGFR2 and FGFR3 enzymes and was highly selective against a panel of kinases including VEGFR2. In cellular assays, INCB054828 inhibited the autophosphorylation of FGFR proteins with low nanomolar IC50 values and blocked signal transduction by FGFR to downstream markers of pathway activation. Cancer cell lines that have genetic alterations in FGFR1, FGFR2 and FGFR3 were uniquely sensitive to growth inhibition by INCB054828, with IC50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC50 > 1500 nM). In vivo, once-daily oral administration of INCB054828 inhibited the growth of tumors that are dependent upon FGFR1, FGFR2 and FGFR3 activity at tolerated doses. Suppression of tumor growth was dose-dependent and correlated with pharmacodynamic inhibition of FGFR. Collectively, these preclinical studies demonstrate that INCB054828 potently and selectively inhibits models of FGFR-dependent cancers in vitro and in vivo, supporting the compound's clinical evaluation in patients harboring oncogenic FGFR activation. Citation Format: Phillip CC Liu, Liangxing Wu, Holly Koblish, Kevin Bowman, Yue Zhang, Ronald Klabe, Lynn Leffet, Darlise DiMatteo, Mark Rupar, Karen Gallagher, Michael Hansbury, Colin Zhang, Chunhong He, Paul Collier, Maryanne Covington, Richard Wynn, Swamy Yeleswaram, Kris Vaddi, Timothy Burn, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Preclinical characterization of the selective FGFR inhibitor INCB054828. [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 771. doi:10.1158/1538-7445.AM2015-771

Potent nonpeptidic benzimidazole sulfonamide inhibitors of protein tyrosine phosphatase 1B (PTP1B) were derived from the optimization of a tripeptide containing the novel (S)-isothiazolidinone ((S)-IZD) phosphotyrosine (pTyr) mimetic. An X-ray cocrystal structure of inhibitor 46/PTP1B at 1.8 A resolution demonstrated that the benzimidazole sulfonamides form a bidentate H bond to Asp48 as designed, although the aryl group of the sulfonamide unexpectedly interacts intramolecularly in a pi-stacking manner with the benzimidazole. The ortho substitution to the (S)-IZD on the aryl ring afforded low nanomolar enzyme inhibitors of PTP1B that also displayed low caco-2 permeability and cellular activity in an insulin receptor (IR) phosphorylation assay and an Akt phosphorylation assay. The design, synthesis, and SAR of this novel series of benzimidazole sulfonamide containing (S)-IZD inhibitors of PTP1B are presented herein.

Cancer-associated cachexia (CAC) is a complex syndrome characterized by a detrimental loss of fat and muscle. Approximately 50% of patients suffer from CAC which accounts for a significant proportion of cancer deaths in addition to debilitating constitutional symptoms and poor quality of life. Recently, the Cancer Cachexia Study Group has proposed that systemic inflammation is one of the 3 factors that defines CAC. Consistent with this, we previously demonstrated that inflammatory cytokines are elevated in cachectic mice bearing subcutaneous tumor xenografts. Because many inflammatory cytokines utilize JAK1 and/or JAK2 kinases (JAK1/2) for intracellular signaling, we hypothesized and demonstrated that selective inhibition of JAK1/2 improved tissue preservation and physical performance. Here we extend these studies beyond subcutaneous xenografts and present biochemical data in an effort to better understand the role of JAKs muscle atrophy. Subcutaneous xenograft models are useful tools but may lack host-tumor interactions relevant to CAC. Therefore, we utilized two immune competent models of CAC. Mice inoculated with BaF/3 B-cells transformed with a constitutively active form of JAK2 (JAK2V617F) common in patients with myeloproliferative neoplasms (MPNs) suffer from an aggressive disseminated disease characterized by splenomegaly and weight loss, similar to MPN patients. Compared to normal mice, those inoculated with JAK2V617F expressing cells lost 80% of their fat stores and 15% of skeletal muscle and had severely enlarged spleens (450%). Treatment with an oral selective JAK1/2 inhibitor prevented tissue loss and dramatically reduced spleen size. Similar findings were observed in the ApcMin/+ mouse model of spontaneous colon cancer and CAC. Here, treatment of male mice with a JAK1/2 inhibitor resulted in 120% and 268% increases in fat and muscle stores, respectively. Splenomegaly was also completely eliminated. Importantly, treatment with a JAK1/2 inhibitor markedly improved physical performance as assessed by grip strength and exercise wheel activity (#x003E; 200% of controls). Because JAK activating cytokines are elevated in our models (e.g. IL-6) and have been implicated as causal factors in CAC, we wanted to better understand the impact of aberrant JAK activation on muscle tissue. C2C12 murine myotubes treated with recombinant IL-6 demonstrated increased JAK signaling as measured by elevated levels of pSTAT3 and pSTAT5, canonical substrates of JAK kinases. IL-6 treatment also increased the activity of two intracellular proteases previously linked to muscle catabolism in CAC - cathepsins B and L. Inhibition of JAK1/2 reduced aberrant JAK/STAT signaling in IL-6 treated C2C12 myotubes and in vivo. Moreover, cathepsin activity was decreased explaining, at least in part, the muscle sparing effects of JAK1/2 inhibition observed in vivo. In summary, the data suggest that JAK-activating cytokines are elevated in CAC and contribute to physiological and functional deficits. JAK1/2 inhibition improves signs of inflammation and prevents loss of fat stores and skeletal muscle, the latter, through suppression of cathepsin activity. Clinical exploration of selective JAK inhibition is therefore warranted for the prevention or alleviation of CAC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B266.

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.

Mutants were selected that are incapable of differentiating microcysts, a resting stage formed in response to high osmotic conditions. In the selection procedure amebae that failed to encyst were removed by flotation in 46% Percoll. Genetic crosses among 15 mutant strains were made by means of the macrocyst sexual cycle. Eleven of the strains mapped to three loci. Mutations at two of these loci (cysA and cysB) produced no observable alteration in the aggregation-fruiting pathway, although one set of strains altered at the cysA locus carried defects at a second unlinked site which blocked aggregation. The single strain that defined the third locus (cysC) is aggregateless. These results confirm the conclusion that there are several genes whose function is essential to microcyst development and is exclusive to this pathway. It remains uncertain whether there are other genes whose action is crucial to both encystment and to aggregation/fruiting.