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.

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.

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.

Aberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration-approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.

Abstract 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.

Abstract Combinatorial therapeutic strategies have achieved improved response rates and durability of responses in several malignancies either by selectively targeting distinct and non-overlapping oncogenic signaling pathways (e.g. PARP and phosphoinositide 3-kinase (PI3K) inhibition in subsets of breast and ovarian cancers), or alternatively, inhibiting distinct nodal points of regulation in common oncogenic signaling pathways (e.g BRaf and MEK inhibition in subsets of melanoma). Recent data suggest that deregulated epigenetic modifications may be just as significant as genetic mutations in driving cancer development and growth by inhibition of tumor suppressor activity and activation of oncogenic pathways. We therefore hypothesized that an epigenetic regulator could potentiate the efficacy of a protein kinase inhibitor to result in robust tumor growth inhibition. We previously reported that the potent and selective LSD1 inhibitor INCB059872 potently inhibited tumor growth in multiple tumor xenograft models of AML and SCLC as a single agent and in a combination with standard of care of agents. In this study, we explored the anti-tumor effect of combining INCB059872 and various signal transduction pathway inhibitors, including the PIM kinase inhibitor INCB053914, the JAK1/2 inhibitor ruxolitinib, or the PI3K delta-selective inhibitor INCB050465 in models of human hematologic malignancies. Each of these therapeutic combinations significantly inhibited tumor growth in the Molm-16 human AML xenograft model. Mechanistic studies suggested that MYC expression levels were downregulated by these combinations both in vitro and in vivo. Treatment with INCB059872 alone or in combination with signal transduction kinase inhibitors significantly downregulated cytokines levels, particularly IL-10, sCD40L, and MCP-1 in Molm-16 tumors. These data suggest that the combination of an LSD1 inhibitor and signal transduction inhibitor can co-regulate key tumor intrinsic and extrinsic pathways involved in paracrine or autocrine signaling in AML. In addition to the improved efficacy observed in AML models, the combination of INCB059872 with the PI3Kdelta inhibitor INCB050465 enhanced tumor growth inhibition in the Will-2 xenograft model (GCB subtype, double hit lymphoma), whereas the activity of these single agents were modest in this particular subtype of lymphoma. Additional mechanistic studies are ongoing to further understand the molecular bases of these observations. Taken together, these data suggest that targeting distinct epigenetic and oncogenic signaling pathways may potentiate anti-tumor efficacy and overcome intrinsic resistance mechanisms in specific hematologic malignancies. Citation Format: Sang Hyun Lee, Matthew Stubbs, Ashish Juvekar, Melody Diamond, Antony Chadderton, Robert Collins, Xiaoming Wen, Holly Koblish, Chunhong He, Liangxing Wu, Richard Wynn, Andrew Combs, Chu-Biao Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. Combination of epigenetic regulation via LSD1 inhibition with signal transduction inhibitors significantly enhances anti-tumor activity 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 2032. doi:10.1158/1538-7445.AM2017-2032

Abstract 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

The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in “double-hit” DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL. Citation Format: Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 143. doi:10.1158/1538-7445.AM2017-143

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.

Abstract 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

JAKs are required for signaling initiated by several cytokines (e. g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFN gamma) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e. g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.