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IT-901 inhibits growth of human DLBCL cell lines.

c-Rel is constitutively active in a wide range of human lymphoma types.

Analysis of the biochemical efficacy of IT-901 by electrophoretic mobility shift assay (EMSA) and NF-κB DNA binding ELISA.

ROS/RNS induction by IT-­901 treatement in normal tissues.

c-Rel inhibitor treatment is associated with partial recovery of lost body weight during severe acute GVHD.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB–dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease. Cancer Res; 76(2); 377–89. ©2016 AACR.

IT-901 is a more potent c-Rel inhibitor than IT-603.

Effects of IT-901 on lymphocyte subsets in the spleen.

IT-901 has a superior pharmacokinetic profile.

IT-901 is a more potent NF-κB inhibitor than IT-603.

IT-901 mediates anti-lymphoma activity in a xenograft model of EBV-induced lymphoma.

IT-901 has no significant adverse effects on major organ functions and blood cell counts.

Treatment with IT-901 for 14 days does not alter lymphocyte numbers.

Several NF-κB subunits are constitutively active in activated B-like DLBCL cell lines.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB–dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease. Cancer Res; 76(2); 377–89. ©2016 AACR.

The phosphatidylinositol 3 kinase (PI3K)-glycogen synthase kinase β (GSK3β) axis plays a central role in MYC-driven lymphomagenesis, and MYC targeting with bromodomain and extraterminal protein family inhibitors (BETi) is a promising treatment strategy in lymphoma. In a high-throughput combinatorial drug screening experiment, BETi enhance the anti-proliferative effects of PI3K inhibitors in a panel of diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma cell lines. BETi or MYC silencing upregulates several PI3K pathway genes and induces GSK3β S9 inhibitory phosphorylation, resulting in increased β-catenin protein abundance. Further-more, BETi or MYC silencing increases GSK3β S9 phosphorylation levels and β-catenin protein abundance through downregulating the E2 ubiquitin conjugating enzymes UBE2C and UBE2T. In a mouse xenograft DLBCL model, BETi decrease MYC, UBE2C, and UBE2T and increase phospho-GSK3β S9 levels, enhancing the anti-proliferative effect of PI3K inhibitors. Our study reveals prosurvival feedbacks induced by BETi involving GSK3β regulation, providing a mechanistic rationale for combination strategies.

MYC overexpression is a poor prognostic predictor in Diffuse Large B-Cell Lymphoma (DLBCL). MYC-targeting with bromodomain and extraterminal protein family (BET) inhibitors is a promising strategy for the treatment of MYC-driven cancers, including lymphomas. However, preclinical and emerging data from early clinical trials demonstrated a modest antiproliferative activity in vitro and in vivo. We hypothesized that BET inhibition may induce feedback survival mechanisms preventing or attenuating cell death that could be exploited for designing future, more effective, combination strategies. In a high-throughput combinatorial drug screening experiment, we found that phosphatidylinositol 3-kinase (PI3K) pathway inhibitors enhanced the antiproliferative effects of BET inhibitors (JQ1, I-BET 151, CPI-203) with a strong class effect. JQ1 upregulated the mRNA expression of several upstream components of the PI3K pathway, including PIK3CA, PIK3R1, PDK1 in a large panel of DLBCL and Burkitt lymphoma cell lines. These effects translated in increased pathway activation as demonstrated by increased levels of the phosphorylated forms of downstream targets GSK3α/β, TSC2, P70S6K, and by increased concentrations of chemokines known to be regulated by PI3K in cell culture supernatants (CCL3 and CCL4). This effect was reversed by submicromolar doses of the PI3K inhibitor BKM-120. MYC silencing recapitulated the effects of BET inhibitors on PI3K pathway gene expression, activation and chemokine secretion. These data indicate that BET inhibition induces PI3K activation by a MYC-dependent feedback. We also observed transcriptional upregulation of the antiapoptotic gene Myeloid Leukemia 1 (MCL-1) following BET inhibition or MYC depletion, suggesting a second MYC-dependent mechanism. RNAi-mediated MCL-1 silencing or co-treatment with a small molecule MCL-1 inhibitor (UMI-77) enhanced the effects of BET inhibitors in DLBCL cell lines by inducing apoptosis. Using SILAC-based quantitative mass spectrometry, we found that BET inhibitors at submicromolar doses downregulated several E2 ubiquitin conjugating enzymes including UBE2C. RNAi mediated UBE2C knockdown induced MCL-1 upregulation in DLBCL cells. The enhanced in vitro effect of combining BETi and PI3Ki was reproduced in TMD8 mouse xenografts. To our knowledge, this is the first study demonstrating MYC-dependent regulation of the PI3K pathway, MCL-1 and the ubiquitin system upon BET inhibition. Our study revealed previously unknown mechanisms of action of BET inhibitors uncovering novel MYC-dependent survival feedback loops, and providing a framework for future combination strategies. Disclosures Zelenetz: Gilead Sciences: Research Funding.

NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P We next analyzed primary lymphoma cells and found that the c-Rel gene is widely expressed in human B cell malignancies and frequently amplified in DLBCL and EBV-transformed B cells. Importantly, intranuclear analysis of the c-Rel protein demonstrated that this transcription factor can be constitutively active in a wide range of human lymphomas. IT-901 efficiently inhibited growth of EBV-transformed B cells in vitro, and mediated significant anti-lymphoma activity in a xenograft model of EBV-induced lymphoma (P In summary, our findings underscore multiple therapeutic benefits and great potential for clinical translation of a novel c-Rel inhibitor. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.