Ray Liu

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

Novel DNA Damage Checkpoints Mediating Cell Death Induced by the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

MLN0905, a Small-Molecule PLK1 Inhibitor, Induces Antitumor Responses in Human Models of Diffuse Large B-cell Lymphoma

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.

Abstract B92: Nedd8-activating enzyme inhibitor MLN4924 provides synergy in nonclinical models with mitomycin C through interactions with ATR, BRCA1/BRCA2 and chromatin dynamics pathways.

MLN4924 is an investigational small molecule inhibitor of the Nedd8-activating enzyme (NAE) currently in Phase 1 clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standards of care, including other DNA damaging agents. We studied the interaction of MLN4924 with other DNA damaging agents in a panel of 4 cell lines and found that mitomycin C, cisplatin, carboplatin, cytarabine, ultraviolet radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in at least 1 cell line. Further testing in xenograft-bearing mice demonstrated synergy of MLN4924 with mitomycin C and with carboplatin, and additivity with gemcitabine. Based in part on this data, MLN4924 is currently being evaluated in a Phase 1b trial ([NCT01862328][1]) with 3 combination arms: MLN4924 + carboplatin and paclitaxel, MLN4924 + gemcitabine, and MLN4924 + docetaxel. To evaluate the mechanism of synergy between MLN4924 and mitomycin C, in vitro experiments with RNAi were performed. Depletion of genes within the ATR and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924, results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may be applied to additional combinations currently under clinical evaluation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B92. Citation Format: Eric S. Lightcap, Khristofer Garcia, Jonathan L. Blank, David C. Bouck, Xiaozhen J. Liu, Greg Hather, Allison Berger, Katherine Cosmopoulos, Michael P. Thomas, Mike Kuranda, Michael D. Pickard, Ray Liu, Syamala Bandi, Peter G. Smith. Nedd8-activating enzyme inhibitor MLN4924 provides synergy in nonclinical models with mitomycin C through interactions with ATR, BRCA1/BRCA2 and chromatin dynamics pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B92. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01862328&atom=%2Fmolcanther%2F12%2F11_Supplement%2FB92.atom

Abstract 2791: Just how noisy is xenograft data? Using growth rate modeling and bootstrapping to optimize xenograft study design.

The evaluation of tumor growth inhibition in xenograft models, generated by subcutaneous implantation of human cancer cell lines, is an integral component of the drug discovery and development process. While xenograft data is ubiquitous, and opinions on its utility are just as common, systematic examinations of signal-to-noise ratio, overall precision of xenograft data and the best metric to report tumor growth inhibition are, remarkably, absent from the literature. To this end, we present a thorough retrospective analysis of xenograft studies from a large in-house database of 225 human xenograft efficacy studies performed from 2006 to present, using a wide range of anti-neoplastic drugs. Individual xenograft tumor growth curves were fit to an exponential model, and a novel measure for data analysis was developed (the model-fitted T/C ratio on Day 21). This novel measure possesses several advantages over a traditional (raw) T/C ratio, notably the use of all available data (not just Day 21), the absence of bias due to informative right-censoring (nonrandom removal of mice in the control group whose tumor volume reached a pre-defined humane endpoint), and the absence of bias due to the inherent slowing of growth kinetics in the control group. The model-fitted T/C ratio on Day 21 was then compared to the raw T/C ratio. For each pair of comparisons between treatment group and control, bootstrapping was used to determine the mean (μ) and standard deviation (σ) of the T/C ratio (either model-fitted or raw). A Z-score measure, (1-μ)/σ, was computed as a measure of the signal-to-noise ratio of the xenograft studies, for 1167 comparisons. For xenograft studies conducted with 10 mice per group over 21 days, the model-fitted T/C ratio outperformed the raw T/C ratio in terms of median Z-score (7.1 and 5.4 respectively). When the number of mice in each group was reduced to 6, and the study length was decreased from 21 to 14 days, a high Z-score (5.1) was still achieved for the model-fitted T/C ratio. The power of xenograft study comparisons for T/C in the range of 0.35 to 0.45 was examined and found to be 0.95 for studies with 6 animals per group using the model-fitted T/C measure. Finally, the misclassification frequency (fraction of comparisons where the treatment was misclassified using a binary cutoff of efficacious vs. non-efficacious) was calculated, and found to be 0.04 using the model-fitted T/C. Our calculations demonstrate that switching to a model-fitted T/C makes more efficient use of the data, yielding considerable cost savings while maintaining a high power and low misclassification rate. The exact degree of benefit from this novel measure and alternative design may vary for other animal facilities, since the noise levels could vary. However, the methods developed here to evaluate potential designs should still be widely applicable, and represent a general approach for the optimization of xenograft studies. Citation Format: Jill Donelan, Greg Hather, Ray Liu, Syamala Bandi, Wen Chyi Shyu, Mark Manfredi, Arijit Chakravarty. Just how noisy is xenograft data? Using growth rate modeling and bootstrapping to optimize xenograft study design. [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 2791. doi:10.1158/1538-7445.AM2013-2791

Abstract 3104: Genome-wide screen for modulators of cell death induced by the NEDD8-activating enzyme inhibitor MLN4924

The NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of ubiquitin E3 ligases termed the cullin-RING ligases (CRLs) through conjugation of the cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrate proteins targets them for degradation by the proteasome. In this way, NAE regulates the stability of proteins required for cancer cell growth and survival. MLN4924 is an investigational small molecule that is a potent and selective inhibitor of NAE in Phase I clinical trials. In order to investigate the primary genetic determinants that confer sensitivity of cells to NAE inhibition, we have performed a genome-wide synthetic lethal RNAi screen using MLN4924 in the A375 melanoma cell line. We have also investigated the biological consequences of NAE inhibition by studying the regulation of protein and transcript levels in MLN4924-treated A375 cells using large-scale quantitative proteomics and gene expression profiling, respectively. The RNAi screen has identified 123 genes whose down-regulation modulates MLN4924-induced cell death, and approximately one-third of these interfere with components of the NEDD8 pathway itself, the cell cycle and apoptotic machinery, and DNA damage-response pathways. Of these genes, 99 were subsequently assessed using high throughput FACS analysis for their contribution to the major phenotype induced by MLN4924. The results emphasize replication, p53, BRCA1/BRCA2, and transcription-coupled repair as being particularly important for MLN4924-induced cell death. In addition, the interactions of 80 genes with roles in cell cycle and DNA damage repair were explored with proteasome inhibitors and 12 DNA damaging agents, demonstrating that MLN4924 induces DNA damage by a distinct mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3104. doi:1538-7445.AM2012-3104

Abstract 3573: A small molecule PLK1 inhibitor, MLN0905, yields broad anti-tumor activity in human xenograft tumor models, and synergizes with taxane therapy

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 397: The antitumor activity of the investigational drug MLN9708 in genetically engineered mouse models of plasma cell malignancy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: The proteasome inhibitor VELCADE® (bortezomib) is an important component of the chemotherapeutic strategy in treating multiple myeloma, a plasma cell malignancy (PCM). While genetically engineered mouse models (GEMMs) of cancer often accurately recapitulate their human disease counterparts, their usage in drug discovery settings has been very limited. Here we describe the antitumor activity of a second generation proteasome inhibitor, the investigational drug MLN9708, in the iMycCα/Bcl-XL GEMMs of PCM, in which neoplastic plasma cell development is driven by enforced expression of the Myc and Bcl-XL transgenes. Materials: MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma; MLN2238 was used for all studies below. We previously demonstrated that double transgenic iMycCα/Bcl-XL (C57BL6/FVB) mice develop de novo PCM with short onset (135 days) and full penetrance (100%). We derived a plasma cell tumor (PCT) cell line, DP54, from the bone marrow of a syngeneic mouse previously inoculated with an iMycCα/Bcl-XL tumor. DP54 PCT cells were stably transfected with the firefly luciferase gene, clonally isolated, and designated as DP54-Luc cells. Methods: Nine-week-old iMycCα/Bcl-XL (C57BL6/FVB) mice were untreated or treated with bortezomib (1.2 mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (18 mg/kg IV BIW) for 6 consecutive weeks and monitored for tumor-free survival for an additional 25 weeks. To establish disseminated and intraosseous mouse models of iMycCα/Bcl-XL PCM, freshly dissociated DP54-Luc cells were aseptically injected into the lateral tail veins and the bone marrow space of the upper right tibia, respectively, of immunocompromised mice. Once tumor growth was established, mice were randomized and treated with vehicle (5%HPbCD), bortezomib (0.8 mg/kg IV BIW) or MLN2238 (13 mg/kg IV BIW) for 3-4 consecutive weeks. The doses used represent the maximum tolerated dose for each drug in each mouse strain. Results: In the iMycCα/Bcl-XL GEMM of de novo PCM, treatment with bortezomib or MLN2238 significantly prolonged tumor-free survival (+27 and +36 days, respectively; p<0.0001) and decreased plasma immunoglobulin levels compared to untreated controls. In the models of iMycCα/Bcl-XL PCM, treatment with bortezomib and MLN2238 significantly reduced disease burden as measured by IVIS® bioluminescent imaging. Conclusion: GEMMs of cancer have often been viewed as promising alternatives to traditional subcutaneous xenograft models, yet data to support their wider use in drug discovery settings are sparse. Here we demonstrated that carefully implemented GEMM studies can be integrated as an important part of the drug-discovery paradigm. MLN9708 is currently in clinical development for both hematologic and solid tumor indications. Safety and efficacy have not been established. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 397.