Jessica Huck

Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase

Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective small-molecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G2/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.

Characterization of Alisertib (MLN8237), an Investigational Small-Molecule Inhibitor of Aurora A Kinase Using Novel In Vivo Pharmacodynamic Assays

Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. Experimental Design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3′-fluoro-3′-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors. Clin Cancer Res; 17(24); 7614–24. ©2011 AACR.

MLN8054, an Inhibitor of Aurora A Kinase, Induces Senescence in Human Tumor Cells Both In vitro and In vivo

Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated β-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft–bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated β-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples. Mol Cancer Res; 8(3); 373–84

MLN8054 and Alisertib (MLN8237): Discovery of Selective Oral Aurora A Inhibitors

The Aurora kinases are essential for cell mitosis, and the dysregulation of Aurora A and B have been linked to the etiology of human cancers. Investigational agents MLN8054 (8) and alisertib (MLN8237, 10) have been identified as high affinity, selective, orally bioavailable inhibitors of Aurora A that have advanced into human clinical trials. Alisertib (10) is currently being evaluated in multiple Phase II and III clinical trials in hematological malignancies and solid tumors.

Translational Exposure–Efficacy Modeling to Optimize the Dose and Schedule of Taxanes Combined with the Investigational Aurora A Kinase Inhibitor MLN8237 (Alisertib)

Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure–efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m2 of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m2 of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents. Mol Cancer Ther; 13(9); 2170–83. ©2014 AACR.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Abstract A164: The small molecule UAE inhibitor TAK-243 (MLN7243) prevents DNA damage repair and reduces cell viability/tumor growth when combined with radiation, carboplatin and docetaxel

Clinical results of VELCADE® (bortezomib) For Injection have prompted evaluation of other enzymes within the ubiquitin proteasome system (UPS) as druggable targets for human cancer. We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), an essential cellular enzyme responsible for activating > 99% of all cellular ubiquitin. Ubiquitin is involved in multiple cellular processes including ubiquitin-dependent protein turnover, cell cycle progression, regulation of apoptosis, protein localization and response to DNA damage. Experiments combining targeted siRNA knockdown with TAK-243 identified DNA damage repair genes necessary for UAE inhibitor-induced cell death. A more focused approach revealed TAK-243 treatment blocked essential monoubiquitination events within the Translesion synthesis (TLS), Fanconi Anemia (FA) and Homologous recombination (HR) pathways. Inhibition of UAE prevented mono-ubiquitin signaling of key mediators within these pathways, including PCNA and FANCD2, by blocking formation of their specific E2-ubiquitin thioesters. In vitro cell-based assays combining TAK-243 with ultraviolet (UV) and radiation, both known to induce DNA damage, yielded inhibition of cell growth and enhanced DNA damage as observed through colony formation assays and Comet assay detection, respectively. Xenograft tumor bearing mice were treated with carboplatin or docetaxel, combined with TAK-243, to evaluate combination benefits in vivo. Synergistic and additive anti-tumor combination benefits were observed in animals treated with TAK-243 + carboplatin and TAK-243 + docetaxel. These important mechanistic in vitro and in vivo studies indicate the dependency of ubiquitination signaling in DNA damage repair and provide a mechanistic rationale for combining radiation, carboplatin or docetaxel with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (ClinicalTrials.gov identifier: NCT02045095). Citation Format: Michael A. Milhollen, Judi Shi, Tary Traore, Jessica Huck, Darshan Sappal, Jennifer Duffy, Eric Lightcap, Yuko Ishii, Jeff Ciavarri, Paul Fleming, Neil Bence, Marc L. Hyer. The small molecule UAE inhibitor TAK-243 (MLN7243) prevents DNA damage repair and reduces cell viability/tumor growth when combined with radiation, carboplatin and docetaxel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A164.

Abstract 3719: TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation

Radiation therapy, as a primary therapy or as a combination partner, is used in half of all worldwide cancer treatments. Research is ongoing to identify agents which potentiate the effects of ionizing radiation (IR) in tumor cells. Because IR causes DNA double strand breaks (DSBs), inhibition of DNA damage repair mechanisms could enhance the effects of radiation. DNA repair at DSBs is mediated by the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways, both of which rely on the post-translational modification of proteins by ubiquitin (Ub). A phosphorylation and ubiqutination cascasde at DSBs results in Ub-dependent recruitment of 53BP1 and BRCA1 complexes. We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), the enzyme responsible for activating > 99% of all cellular Ub. Previously, TAK-243 was shown to inhibit mono-Ub of PCNA and FANCD2, key proteins within the translesion synthesis (TLS) and Fanconi Anemia (FA) DNA repair pathways, and also to inhibit Ub transfer to UBC13, an E2 ubiquitin-conjugating enzyme utilized in DSB repair. We hypothesized that TAK-243 would prevent repair of DSBs and thereby potentiate IR-induced cell death. Here we show that TAK-243 pre-treatment potentiates the effect of IR on HCT-116 cells in a colony formation assay in vitro. To link this combination benefit to the disruption of DNA damage repair, we demonstrate that TAK-243 pre-treatment blocks the IR-induced recruitment of 53BP1 to sites of DNA damage both in vitro and in vivo. In a patient-derived xenograft (PDX) model of non-small cell lung cancer, formation of IR-induced 53BP1 foci is inhibited when TAK-243 is dosed 1 hour before beam-focused radiation exposure. In contrast, levels of IR-induced pH2Ax are not significantly changed by TAK-243 treatment, suggesting that TAK-243 does not prevent formation or detection of DSBs, but rather acts downstream to prevent DNA damage repair. Additive-to-synergistic effects on tumor growth inhibition were observed in several xenograft models treated with the combination of TAK-243 and beam-focused IR, with persistent tumor regressions noted in some NSCLC and breast cancer models. The results of our experiments provide a mechanistic rationale for combining radiation with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (NCT02045095). Citation Format: Michael A. Milhollen, Judy Qiuju Shi, Tary Traore, Jessica Huck, Darshan Sappal, Kenichi Iwai, Akihiro Ohashi, Claudia Rabino, Jennifer A. Duffy, Eric Lightcap, Yuko Ishii, Jeffrey Ciavarri, Neil Bence, Allison J. Berger, Marc L. Hyer. TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation. [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 3719.