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

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. Here we have developed an Fc-silenced bispecific IgG1 antibody to CD137 and PD-L1 with monovalent binding specificity to each target. MCLA-145 drives transactivation of CD137 in the vicinity of cells expressing PD-L1, such as in the immunosuppressive tumor microenvironment. The degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells, as demonstrated in transactivation assays whereby reporter T cells were co-cultured with cells expressing different levels of PD-L1. PD-L1 expression as low as 6000 receptors per cell was sufficient to activate CD137 in neighboring T cells. In contrast, MCLA-145 blocked PD-1 signaling without requirement for CD137 binding in a PD-1/PD-L1 reporter assay. CD137 signaling was induced by MCLA-145 in multiple primary human immune cell assays including the mixed lymphocyte reaction, human PBMC, and whole blood SEB stimulation assays. MCLA-145 reversed T cell suppression mediated by M2 macrophages or Tregs, in vitro. In addition, MCLA-145 enhanced Ag-specific expansion and differentiation of human naïve CD8+ T cells in vitro. In vivo, MCLA-145 treatment resulted in significant tumor immune activation and antitumor responses in two separate humanized mouse tumor models. In one model, human T cells expressing NY-ESO specific TCR were adoptively transferred to mice bearing A549 tumors which expressed NY-ESO antigen and human PD-L1. MCLA-145 treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) as compared to T cell only treated mice. In the tumors of MCLA-145 treated mice, the percentage of NY-ESO specific CD8+ T cells were significantly increased compared to controls. In a second model, mice engrafted with human CD34+ cells were implanted with the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and 5 mg/kg induced significant tumor growth inhibition (55 and 57% respectively) as compared to vehicle control or Fc-silenced huIgG1 controls. Additionally, two out of nine animals in the 5 mg/kg MCLA-145-treated group had complete tumor regression. MCLA-145 increased the number of infiltrating CD8+ T cells, as well as the percentage of central memory CD8+ T cells. The cured animals were then re-challenged with MDA-MB-231 tumor cells, and tumors of previously cured mice were rejected as compared to no growth inhibition in treatment-naïve CD34+ NSG mice. In conclusion, these data support the clinical evaluation of MCLA-145 as a novel, PD-L1 dependent CD137 agonist immune therapy. Citation Format: Patrick Mayes, Paul Tacken, Steve Wang, Pieter-Fokko van Loo, Thomas Condamine, Hans van der Maaden, Eric Rovers, Steef Engels, Floris Fransen, Ashwini Kulkarni, Yao-bin Liu, Arpita Mondal, Leslie Hall, Soyeon Kim, Marina Martinez, Shaun O'Brien, Edmund Moon, Steven Albelda, Peggy Scherle, Gregory Hollis, Reid Huber, Mark Throsby, Cecile A. Geuijen. A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137 [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 539.

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

The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumor immune response or to inhibit key proteins in the immunosuppressive tumor milieu. PD-1/PD-L1 axis blockade, CTLA-4 blockade and IDO1 inhibition are examples of the latter approach and have been utilized to reverse the suppressive tumor microenvironment, resulting in clinical benefit for cancer patients. Recent clinical and preclinical data have also demonstrated that combining these approaches results in enhanced therapeutic benefit. Notably, the IDO1-selective inhibitor epacadostat has been shown to increase the efficacy of two checkpoint inhibitors, the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody pembrolizumab, in patients with melanoma. Because both checkpoint receptors and IDO1 serve as negative regulators of the immune response, we also explored the ability of IDO1 inhibition to combine with agents that directly activate T cells through costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily. Rodent active surrogate agonist antibodies to 4-1BB, OX40 and GITR were tested with epacadostat in multiple preclinical models. In the B16-SIY melanoma model that does not express IDO1 in tumor cells, both epacadostat and anti-OX40 had little effect, but the combination resulted in enhanced efficacy. This was associated with increased infiltrates of CD8+ T cells and decreased numbers of FoxP3+ TILs. Increased numbers of SIY-reactive T cells were found in both the tumor and the TDLN post-therapy. In contrast, epacadostat did not provide any enhancement to the activity seen with 4-1BB. Clear combinatorial effects were seen with anti-GITR and epacadostat in the more inflamed, IDO1-expressing PAN02 pancreatic cancer model. These data suggest that IDO1 inhibition can be effective in combination with agents that agonize T cell costimulatory receptors as well as with agents that block coinhibitory receptors. Citation Format: Holly K. Koblish, Brendan Horton, Michael Hansbury, Sybil O'Connor, Kerri Lasky, Christina Stevens, Thomas Condamine, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Horacio Nastri, Gregory Hollis, Reid Huber, Thomas Gajewski, Peggy Scherle. Agonist antibodies targeting OX40 and GITR enhance the activity of the IDO1-selective inhibitor epacadostat in preclinical models [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 2618. doi:10.1158/1538-7445.AM2017-2618

Immune checkpoint blockade has shown considerable therapeutic promise in the clinic. However, single agent activity is compromised by the presence of suppressive myeloid cells, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAM) and polymorphonuclear (PMN) cells, in the tumor microenvironment. Epigenetic alterations can significantly contribute to the development of the immunosuppressive tumor microenvironment and recent data have suggested that combining epigenetic-based therapies with immunotherapeutic agents can lead to improved efficacy in preclinical models. Since Lysine Specific Demethylase 1 (LSD1) has been shown to play a critical role in hematopoiesis, we hypothesized that inhibition of LSD1 could have a direct effect on myeloid cell differentiation and potentially restore normal myelopoiesis in cancer patients. To test this hypothesis, we evaluated INCB059872, a potent, selective and orally available FAD-directed covalent inhibitor of LSD1 in several experimental models. In an in vitro differentiation assay, the majority of CD34+ progenitor cells were driven to a monocytic phenotype in the presence of INCB059872, while control treated cells differentiated toward granulocytic PMN cells. Similar results were observed in vivo. Using the orthotopic 4T1 mammary cancer model, the myeloid compartment was characterized in tumor tissues following treatment with INCB059872. Notably, the population of PMN-MDSC was significantly decreased in tumor tissues following oral administration of INCB059872, whereas the macrophage population was increased. These data suggest that INCB059872 can redirect myeloid differentiation toward monocyte/macrophages and inhibit the differentiation of PMN-MDSC in this syngeneic tumor microenvironment. Consistently, intratumoral T lymphocyte infiltration was increased following INCB059872 treatment. The combination of INCB059872 and α-PD-L1 antibody enhanced anti-tumor efficacy in the 4T1 orthotopic tumor model. Collectively, these data suggest that inhibition of LSD1 with INCB059872 can directly affect myeloid differentiation to reduce the accumulation of myeloid suppressive cells, restoring the tumor microenvironment to be more responsive to PD-1/PD-L1 axis blockade. This study supports the therapeutic potential for the combination of an LSD1 inhibitor with immuno-therapeutic agents to improve overall clinical response in cancer patients. Citation Format: Thomas Condamine, Steve Wang, Melody Diamond, Leslie Hall, Huiqing Liu, Antony Chadderton, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle, Holly Koblish, Sang Hyun Lee. The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2017-4635

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.

The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) was initially identified as preferential integration sites of the Moloney murine leukemia virus in Eμ-myc mice, resulting in perinatal lymphomagenesis. Molecular characterization has revealed that PIM kinases drive cell proliferation and survival in a number of hematological malignancies beyond lymphomas by mediating responses to cytokines, growth factors and cellular stress. Overexpression of various PIM kinase family members in these malignancies has been associated with poor overall survival and with resistance to chemotherapeutic agents. Therefore, development of a pan-PIM inhibitor may be useful in the treatment of hematological malignancies, both as a single agent and in combination with chemotherapy or targeted agents. The in vitro and in vivo activity of INCB053914, a pan-PIM kinase inhibitor, was determined in a panel of acute myelogenous leukemia (AML) cell lines. Greater than half of all AML cell lines tested were sensitive to single agent INCB053914, with anti-proliferative IC50 potencies Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Sybil O'Connor, Qian Wang, Kathy Wang, Lynn Leffet, Maryanne Covington, Krista Burke, Jason Boer, Kevin Bowman, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2015-5416

The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) mediates responses to cytokines and growth factors and drives cell proliferation and survival in a number of hematologic malignancies. Overexpression of PIM kinases in these malignancies, including multiple myeloma (MM), has been associated with poor overall survival. Given the overlapping functions of these kinases, the ability of one family member to compensate for the loss of another as well as the relatively benign phenotype of mice deficient in all three PIM isoforms, discovery of pan-PIM kinase inhibitors is warranted. The in vitro and in vivo activity of the pan-PIM kinase inhibitor, INCB53914, was determined in MM cell lines. The antiproliferative potencies for INCB053914 were To understand the impact of inhibiting the PIM pathway in combination with other pathways dysregulated in hematological malignancies, an unbiased in vitro screen was performed and the potential synergy of INCB053914 in combination with 65 cytotoxic or targeted agents was determined. This screen identified several agents active against the PI3K pathway or which impacted cell cycle progression. In addition, the combinatorial activity of selected targeted agents hypothesized to exhibit significant interactions with the PIM pathway was assessed in vivo. Since PIM family members are STAT regulated genes, enhanced activity may be expected upon combined PIM and JAK inhibition. In fact, synergistic activity was seen with this combination in the INA6 multiple myeloma model, and pharmacodynamic analyses revealed enhanced suppression of both pBAD and c-myc levels in tumors from treated mice. Additionally, c-myc levels are regulated both by PIM and the BET family member, BRD4. The expected synergistic efficacy of PIM and BET inhibitors was also observed in the KMS12 model, again with enhanced reduction in pBAD and c-myc levels in the tumors of treated mice. Taken together, these data support the utility of PIM inhibition in MM patients, both as monotherapy and in combination with other targeted agents. Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Xiaoming Wen, Sybil O'Connor, Valerie Dostalik, Qian Wang, Kathy Wang, Maryanne Covington, Cindy Marando, Kevin Bowman, Jason Boer, Krista Burke, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5414. doi:10.1158/1538-7445.AM2015-5414

Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2), giving rise to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in both B cell development and function, and has now been validated as a therapeutic target in CLL and indolent NHL upon the approval of idelalisib, a selective PI3Kδ inhibitor. However, despite the demonstrated activity of PI3Kδ inhibitors in several subtypes of B cell malignancies, the effectiveness of the approach as a single agent for the treatment of DLBCL, the largest subtype of NHL, has not been demonstrated. We hypothesized that an optimal treatment strategy in this histology would require combined inhibition of PI3Kδ with additional agents that target B cells through distinct signaling pathways. INCB050465 is a novel, small molecule inhibitor of PI3Kδ. In biochemical assays, it potently inhibits PI3Kδ (IC50 = 1 nM at 1 mM ATP) with approximately 20,000-fold selectivity for PI3Kα, PI3Kβ, PI3Kγ and 57 other kinases. In various PI3Kδ functional assays, including B cell proliferation induced by activation of BCR, BAFF-R, IL-4R, CD40 and TLRs as well as T cell differentiation assays, INCB050465 demonstrates potent activity with IC50 values ranging from 0.2 to 2 nM. In addition, INCB050465 blocks the proliferation of several DLBCL and MCL cell lines in vitro (EC50 < 10 nM) and can slow tumor growth in the Pfeiffer model of DLBCL in vivo. In cells, INCB050465 blocks constitutive PI3Kδ signaling, including phosphorylation of Akt and FOXO3a, with potencies similar to those for inhibiting proliferation. More importantly, the ability of INCB050465 to inhibit DLBCL cell proliferation in vitro can be further potentiated by inhibition of PIM (Proviral Integration sites for Moloney virus) protein kinases, which were also found to be important for B cell development and to phosphorylate substrates which overlap with those of the PI3K pathway. The combined inhibition of PI3Kδ by INCB050465 with the potent and selective PIM protein kinase inhibitor, INCB053914, results in profound tumor regression in the Pfeiffer cell model of DLBCL. Taken together, these results provide evidence for cross-talk between the PIM and PI3Kδ pathways, and support a mechanistic rationale for the clinical evaluation of combined PIM and PI3Kd inhibition in B cell malignancies, including DLBCL. Citation Format: Niu Shin, Holly Koblish, Maryanne Covington, Yanlong Li, Kathy Wang, Qian Wang, Patricia Feldman, Leslie Hall, Sybil O'Connor, Xin He, Kamna Katiyar, Yu Li, Eddy W. Yue, Thomas P. Maduskuie, Brent Douty, Song Mei, Yun-Long Li, Chu-Biao Xue, Andrew Combs, Wenqing Yao, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Robert Newton, Kris Vaddi, Reid Huber, Peggy Scherle. INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2015-2671

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