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Diffuse large B-cell lymphoma (DLBCL) is the most common of the non–Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic. Mol Cancer Ther; 11(9); 2045–53. ©2012 AACR.

e13579 Background: During phase-1 oncology trials, early pharmacodynamic (PD) signals are often interpreted as predicting later outcomes of antitumor activity (E). A major complication in making th...

We explored the location and function of the human cIAP1 protein, a member of the inhibitor of apoptosis protein (IAP) family. Unlike family member X-linked IAP (XIAP), which was predominantly cytoplasmic, the cIAP1 protein localized almost exclusively to nuclei in cells, as determined by immunofluorescence microscopy and subcellular fractionation methods. Interestingly, apoptotic stimuli induced nuclear export of cIAP1, which was blocked by a chemical caspase inhibitor. In dividing cells, cIAP1 was released into the cytosol early in mitosis, then reaccumulated in nuclei in late anaphase and in telophase, with the exception of a pool of cIAP1 that associated with the midbody. Survivin, another IAP family member, and cIAP1 were both localized on midbody microtubules at telophase, and also interacted with each other during mitosis. Cells stably overexpressing cIAP1 accumulated in G2-M phase and grew slower than control-transfected cells. These cIAP1-overexpressing cells also exhibited cytokinesis defects over 10 times more often than control cells and displayed a mitotic checkpoint abnormality with production of polyploid cells when exposed to microtubule-targeting drugs nocodazole and paclitaxel (Taxol). Our findings demonstrate a role for overexpressed cIAP1 in genetic instability, possibly by interfering with mitotic functions of Survivin. These findings may have important implications for cancers in which cIAP1 overexpression occurs.

Presage technology enables simultaneous analysis of multiple cancer drug candidates, drug concentrations, and drug combinations within a single living tumor. The platform employs arrayed tumor microinjection technology that delivers multiple spatially defined “threads” of drug directly into discreet portions of a tumor. This enables rapid, reliable, and internally controlled cross comparisons of multiple cancer therapeutics using screening quantities of drug in an in vivo setting in which the local tumor microenvironment is maintained. Here, as an example, we apply the platform to investigate tumor responsiveness to an inhibitor of the ubiquitin activating enzyme UBA1 from Millennium Pharmaceuticals. The ubiquitin activating enzyme UBA1 regulates ubiquitin activation and subsequent polyubiquitination of proteins necessary for their degradation by the proteasome, and functionally impacts cell signaling, DNA damage repair and cell cycle progression. In vivo tumor responses were evaluated in two human xenograft models, WSU-DLCL2 and MCF-7, grown as flank tumors in immune-compromised mice. Microdosing of multiple concentrations of the UBA1 inhibitor (UBAi) into both models led to localized, easily detectable, and drug concentration-dependent biomarker changes indicative of ubiquitin pathway perturbation in the area proximal to injected drug. This included loss of poly-ubiquitin, accompanied by the expected accumulation of cMyc in tumor regions exposed to the UBAi. Localized time-dependent tumor cell death responses were observed following pathway perturbation as quantified by staining for cleaved caspase-3 and gamma-H2AX staining. Furthermore, pathological evidence of UBAi-induced cell death was clearly visible in both tumor models upon histological examination of H&E stained slides. These results highlight the capacity of the Presage platform to perform multiplexed drug studies in live tumor models. This capability could readily be expanded for use in validating additional biomarker hypotheses, indication finding studies, or for efficient identification of novel drug combinations. Citation Format: Beryl A. Hatton, Marc Grenley, Nathan Hedin, Nathan Caffo, Marc L. Hyer, Mark Manfredi, Stephen Blakemore, Richard A. Klinghoffer, Neil Bence. Arrayed microinjection of a ubiquitin activating enzyme inhibitor induces PD biomarker effects predictive of in vivo tumor responses to systemic drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4153. doi:10.1158/1538-7445.AM2013-4153

Abstract 2733 Aurora A kinase (AAK) is a serine/threonine protein kinase that is essential for normal mitotic progression. MLN8237, an investigational, selective inhibitor of AAK, is being evaluated in multiple Phase 1 and 2 clinical studies in patients with solid cancers and hematological malignancies. In early clinical trials in heme-lymphatic malignancy, MLN8237 dosed as a single agent has demonstrated antitumor activity (Padmanabhan ASH 2010; Goldberg ASH 2010). In order to identify potential combination partners for MLN8237 for clinical evaluation, previous preclinical efforts demonstrated that MLN8237 combined with rituximab leads to significant antitumor activity in a panel of in vivo diffuse large B-cell lymphoma (DLBCL) models (Huck et. al. ASH 2008; Zhang et, al. AACR 2009). In the present preclinical studies, a novel combination treatment of MLN8237 and vincristine was tested in two xenograft models of DLBCL, WSU and OCI-LY19. Mice were dosed for three weeks with MLN8237 orally daily (QD) and with vincristine intravenously weekly (QW). In both tumor models, single agent dose dependent antitumor activity was observed with MLN8237 and vincristine; however, tumors gradually re-grew after the treatment. Combined MLN8237 and vincristine treatment led to additive or synergistic antitumor activity and resulted in significant tumor growth delay even after discontinuing treatment. In the OCI-Ly19 model, MLN8237 (20 mg/kg) with vincristine (0.5mg/kg or 1 mg/kg) resulted in complete cures with no tumor re-growth out to >120 day. In the WSU model, MLN8237 (20mg/kg) combined with vincristine (1mg/kg) led to tumor regression during the treatment period and significant tumor growth delay (TGD=38). Body weight loss (BWL) was observed in the combination arms in a dose dependent manner in an acceptable range (maximum ∼10%); however body weight recovered quickly after treatment termination. The plasma and tumor pharmacokinetic profiles of MLN8237 (20 and 3 mg/kg) and vincristine (1 mg/kg) in the WSU model demonstrated decreased MLN8237 exposure when co-administered with vincristine. Vincristine exposures were not affected by co-administration with MLN8237. Additional preclinical studies are planned to evaluate the antitumor activity of MLN8237 combined with vincristine and rituximab. Preclinical data showing the antitumor activity of the combination of MLN8237 and vincristine provides the basis for its clinical evaluation as a treatment option for aggressive DLBCL. Disclosures: No relevant conflicts of interest to declare.

Millennium Pharmaceuticals, Inc. is dedicated to the discovery and development of novel oncology therapeutics in the area of protein homeostasis. Here we report the identification and characterization of compounds that target the ubiquitin activating enzymes, UBA1 and UBA6. These compounds are mechanism based inhibitors that inactivate the ubiquitin E1 enzymes by forming a ubiquitin compound adduct that remains tightly associated with the E1 adenylate binding site. Treatment of cells with these inhibitors results in cellular effects consistent with known Uba1 biology including rapid loss of E2 ubiquitin thioesters, loss of total ubiquitin conjugates, and accumulation of many ubiquitin proteasome system substrates. Following prolonged treatment, cells primarily arrest in the G2 phase of the cell cycle and ultimately undergo apoptosis. Reflecting the extensive cellular roles of ubiquitin, the compounds also impact global protein turnover, ER stress and DNA damage repair. UBA1 inhibition impairs ubiquitination of PCNA and the Fanconia Anemia protein FANCD2 leading to defective repair of UV induced DNA damage. UBA1 inhibition impacts numerous biological pathways relevant to cancer, results in apoptosis in vitro and is capable of inhibiting tumor growth in mouse xenografts in vivo. These data implicate UBA1 as a target for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C82.

PLK1 is a serine/threonine mitotic kinase that plays a key role in mitotic cell cycle progression, and its over-expression has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which reduces cell viability and inhibits cell proliferation in a broad range of human tumor cells. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in human xenograft models grown in rodents. MLN0905 rapidly and extensively distributed to xenograft tumor with a high tumor-to-plasma exposure ratio. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. MLN0905 demonstrated robust anti-tumor activity (partial and complete responses) in a variety of solid and hematological human xenograft models, including cancers derived from colon (HCT-116, HT29), NSCLC (Calu-6), Ovarian (SKOV3), and Lymphoma (OCI-LY19, OCI-LY10, and the primary lymphoma PHTX-22L). Significant anti-tumor activity was observed when dosing on either a continuous (daily) or intermittent schedule, underscoring the dosing flexibility with this compound. In the SKOV3 and Calu-6 xenograft models, MLN0905 yielded a synergistic anti-tumor response when combined with the standard of care therapy Taxane. This is the first report of a PLK1 inhibitor synergizing with taxane therapy in vivo. In summary, our findings indicate MLN0905 has good drug-like pharmacokinetic properties, modulates the biomarker phospho-Histone H3, and yields significant anti-tumor activity in multiple xenograft models. These preclinical data support further evaluating MLN0905 as a novel anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2011-3573

Abstract 3922 Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30 percent of newly diagnosed cases. Current treatment options for this disease are effective, but not always curative; therefore experimental therapies are being investigated. Diffuse large B-cell lymphoma cells typically over-express the serine/threonine mitotic kinase PLK1, which plays a key role in mitotic cell cycle progression, and has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL lines. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in mouse models harboring human DLBCL disease. MLN0905 has drug-like pharmacokinetic properties and an acceptable toxicity profile making it a good clinical candidate. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. The anti-tumor activity of MLN0905 was evaluated in three human subcutaneous xenograft models OCI-LY10, OCI-LY19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant anti-tumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The anti-tumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI-LY19) setting to better mimic DLBCL in humans. In this disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with Rituximab in the disseminated OCI-LY19 model. Combining Rituximab and MLN0905 provided both a synergistic anti-tumor effect and a synergistic survival advantage. Our findings indicate for the first time that PLK1 inhibition leads to pharmacodynamic pH3 modulation and significant anti-tumor activity in multiple models of DLBCL. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anti-cancer agents in the clinic. Disclosures: Shi: Millennium: The Takeda Oncology Company: Employment. Lasky:Millennium: The Takeda Oncology Company: Employment. Shinde:Millennium: The Takeda Oncology Company: Employment. Duffey:Millennium: The Takeda Oncology Company: Employment. Stringer:Millennium: The Takeda Oncology Company: Employment. Qian:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Liu:Millennium: The Takeda Oncology Company: Employment. Rao:Millennium: The Takeda Oncology Company: Employment. Vos:Millennium: The Takeda Oncology Company: Employment. D'Amore:Millennium: The Takeda Oncology Company: Employment. Hyer:Millennium: The Takeda Oncology Company: Employment.

8553 Background: Aurora A kinase is a serine/threonine protein kinase that is essential for the successful transit of cells through mitosis. MLN8237 is a selective small molecule inhibitor of Aurora A kinase that has demonstrated anti-tumor activity in animal models of solid human tumors. In this study we explored the anti-tumor effect of MLN8237 in vivo in pre-clinical models of human Diffuse Large B-cell Lymphoma (DLBCL) both as a single agent and in combination with the anti-CD20 monoclonal antibody Rituximab. Methods: Three human DLBCL models were examined in SCID mice. In two of the models (Ly19 & WSU) the tumor cells expressed a constitutively active luciferase, enabling tumor burden analysis in either a subcutaneous or disseminated setting. The third model was a primary DLBCL recently obtained from a patient. Tumor bearing animals were treated for 21 days with MLN8237 (QD PO dosing at 3–20mg/kg), Rituximab (Q7D IV dosing at 10 mg/kg), or the two agents combined. Tumor burden was measured in the disseminated models as a function of luciferase-induced photon flux, and in the subcutaneous models using vernier calipers. Results: MLN8237 induced anti-tumor activity that was dose-dependent in all three models. In LY19 disseminated model, 3 mg/kg of MLN8237 combined with Rituximab induced synergistic anti-tumor activity (n=2); while 10 mg/kg MLN8237 (dosed QD) combined with Rituximab was additive. Importantly, combining MLN8237 with Rituximab led to complete cures in 100% of the animals. In the WSU model, combining MLN8237(3–10mg/kg) with Rituximab resulted in additive tumor growth inhibition. The mean survival endpoint was significantly longer (p=0.003 and [Table: see text]

Aurora A kinase is a serine/threonine protein kinase that is essential for normal transit of cells through mitosis. In many tumor types the Aurora A gene is amplified and/or the protein is over-expressed. The Aurora A small-molecule inhibitor MLN8237 demonstrated robust tumor growth inhibition in xenograft models of solid tumors grown subcutaneously (S.C.) in immunocompromised mice. Here we explored the antitumor activity of MLN8237 in models of diffuse large B-cell lymphoma (DLBCL) both in vitro and in vivo. In vivo three established DLBCL xenograft models (OCI-Ly7, OCI-Ly19, and WSU-DLCL2; all cells expressing luciferase) and a primary DLBCL tumor model PHTX-22-06 were tested using MLN8237 at different doses. Rituximab, an anti-CD20 monoclonal antibody that is active against CD20+ malignant B cells and is a standard of care agent was used for comparison. Using these model systems, tumor cells were injected either I.V. (to evaluate disseminated disease), or S.C. in severe combined immunodeficient mice (SCID). Animals were dosed orally for 21 days with MLN8237 (QD or BID) at various doses, or Rituximab dosed at 10mg/kg IV (once/week) and tumor growth inhibition was monitored using either bioluminescent imaging for the disseminated models or vernier calipers for the S.C. models. Tumor growth inhibition by MLN8237 was dose dependent with 20 mg/kg bid being the most efficacious dose (TGI>100% in both disseminated OCI-Ly19 and WSU models). All animals in the OCI-Ly19 disseminated model 20 mg/kg BID treatment group demonstrated regressions and remained disease free until the end of the study, day 65. In this study the Rituximab treated animals were euthanized on day 31 due to a high level of tumor burden. In the primary tumor model, PHTX-22-06, MLN8237 dosed at 20 mg/kg BID was also the most efficacious with a TGI of 95%. Moreover, tumor growth inhibition was durable as determined by prolonged tumor growth delay (>50 days). Significant efficacy was achieved in all models tested, whether grown as disseminated or subcutaneous models. A noted increase in durability of response was observed with MLN8237 treatment when compared with previous data from solid tumor models. In vitro, MLN8237 treatment increased levels of apoptosis in the OCI-Ly19 cells in comparison to the solid tumor cell line HCT-116 (colon). Greater Annexin V positive cells and greater cleaved PARP and Caspase-3 signals were detected in the MLN8237 treated OCI-Ly19 cells when compared to HCT-116 cells. The demonstration of robust and durable anti-tumor activity in preclinical models treated with MLN8237 provides the basis for its clinical evaluation as a treatment option for DLBCL. MLN8237 is currently in multiple Phase I clinical trials.