Richard Wynn

The Janus Kinase 2 Inhibitor Fedratinib Inhibits Thiamine Uptake: A Putative Mechanism for the Onset of Wernicke’s Encephalopathy

The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernickes encephalopathy in myelofibrosis patients. Since Wernickes 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.

High-Throughput Determination of Mode of Inhibition in Lead Identification and Optimization

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 IC50 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 teams 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.

Abstract 3523: Discovery of a novel BET inhibitor INCB054329

Bromodomains (BD) are protein modules that bind acetylated lysine residues and are components of many epigenetic modifiers and transcription factors. The BET (Bromodomain and extra-terminal) family is composed of four members each harboring two tandem BDs. BET proteins are critical regulators of transcription through interactions with complexes including Mediator and p-TEFb at gene promoter and enhancer elements. Studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins is therapeutic in models of cancer and acute inflammation. We describe the preclinical activity of a novel BET inhibitor INCB054329 for the potential treatment of malignant diseases. INCB054329 inhibited binding of BRD2, BRD3 and BRD4 to an acetylated histone H4 peptide with low nanomolar potency. In myeloma cell lines, treatment with INCB054329 inhibited expression of c-MYC and induced HEXIM1. The majority of myeloma, AML, and lymphoma cell lines tested were growth inhibited by INCB054329 with potencies less than 200 nM. Selectivity was seen when compared with nontransformed cells as the potency for growth inhibition of IL-2 stimulated T-cells from normal donors was greater than 1300 nM. Cell cycle analysis revealed treatment-induced G1 arrest. Furthermore in both AML and lymphoma cell lines, INCB054329 induced apoptosis consistent with increased expression of pro-apoptotic regulators. In vivo, oral administration of INCB054329 inhibited tumor growth in several models of hematologic cancers. In the MM1.S multiple myeloma xenograft model, inhibition of tumor growth was correlated with reduction of c-MYC levels. PK-PD analysis showed c-MYC suppression was associated with an IC50 value of less than 100 nM in vivo. In summary these studies demonstrate that INCB054329 is a potent inhibitor of BET transcriptional regulators in models of hematologic malignancies in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Phillip CC Liu, Xuesong Mike Liu, Matthew C. Stubbs, Thomas Maduskuie, Richard Sparks, Nina Zolotarjova, Jun Li, Xiaoming Wen, Margaret Favata, Patricia Feldman, Alla Volgina, Darlise DiMatteo, Robert Collins, Nikoo Falahatpisheh, Padmaja Polam, Yu Li, Maryanne Covington, Sharon Diamond-Fosbenner, Richard Wynn, Timothy Burn, Kris Vaddi, Swamy Yeleswaram, Andrew P. Combs, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Discovery of a novel BET inhibitor INCB054329. [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 3523. doi:10.1158/1538-7445.AM2015-3523

Abstract 5071: Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies

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

Abstract 4712: Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [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 4712.

Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease

Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates. Foremost among the efforts to target bromodomains are those aimed at identifying compounds that interact with the bromodomain and extra‐terminal domain (BET) family of bromodomain‐containing proteins (BRD2, BRD3, BRD4, and BRDT). Inhibitors of these proteins are under clinical development for a large variety of oncologic indications. Described in this unit are several assays to assess the binding potency and selectivity within the BET protein family. Included are AlphaScreen, fluorescence polarization, and thermal shift assays. The strengths and weaknesses of each assay are discussed.

Abstract 4704: The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of human small cell lung cancer

Methylated histone marks on H3K4 and H3K9 are generally coupled with transcriptional activation and repression, respectively. Altered levels of these histone methylation marks lead to abnormal gene expression and are associated with oncogenesis. Lysine specific demethylase 1 (LSD1) catalyzes demethylation of mono and di-methylated lysine 4 and 9 of histone H3 via an FAD-dependent redox-process. Deregulated LSD1 activity perturbs normal gene expression and can lead to cellular transformation. In particular, the function of LSD1 has been reported to maintain stem cell-like gene expression patterns in various cancers, most notably small cell lung cancer (SCLC), a cancer type that is characterized by a highly undifferentiated state and with high unmet medical need. INCB059872 is a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. The effect of INCB059872 in SCLC cell lines was assessed in vitro and in vivo. In these studies, the proliferation of a panel of SCLC cell lines was inhibited by INCB059872, with EC50 values ranging from 47 to 377 nM. Non-tumorigenic cells, such as IL-2 stimulated T cells from normal donors, by contrast, were significantly less sensitive with IC50 values > 10 μM. Oral administration of INCB059872 on once daily (QD) and alternative day (QoD) dosing regimens inhibited tumor growth in the NCI-H526 and NCI-H1417 human SCLC xenograft models. Consistent with previous reports, INCB059872 treatment in these models led to the induction of the FEZ1 and UMODL1 genes relative to vehicle-treated animals, as part of a gene signature in SCLC cell lines that is predictive of LSD1 responsiveness. Moreover, serum levels of the neuroendocrine marker pro-GRP were markedly reduced at all efficacious dosing regimens in the NCI-H1417 human SCLC xenograft model, suggesting that serum pro-GRP levels could be used as a surrogate pharmacodynamic marker of LSD1 inhibition. Currently, the efficacy of INCB059872 in combination with standard of care therapies for SCLC is under evaluation in these preclinical models. These data support the clinical evaluation of INCB059872 in patients with SCLC. Citation Format: Sang Hyun Lee, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Margaret Favata, Chunhong He, Liangxing Wu, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of human small cell lung cancer. [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 4704.

Abstract 4696: The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [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 4696.

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

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

Abstract 771: Preclinical characterization of the selective FGFR inhibitor INCB054828

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 IC 50 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 IC 50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC 50 > 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 compound9s 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