Mengkun Zhang

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

Phase I assessment of new mechanism-based pharmacodynamic biomarkers for MLN8054, a small-molecule inhibitor of Aurora A kinase

The mitotic kinase Aurora A is an important therapeutic target for cancer therapy. This study evaluated new mechanism-based pharmacodynamic biomarkers in cancer patients in two phase I studies of MLN8054, a small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received MLN8054 orally for 7 consecutive days in escalating dose cohorts, with skin and tumor biopsies obtained before and after dosing. Skin biopsies were evaluated for increased mitotic cells within the basal epithelium. Tumor biopsies were assessed for accumulation of mitotic cells within proliferative tumor regions. Several patients in the highest dose cohorts showed marked increases in the skin mitotic index after dosing. Although some tumors exhibited increases in mitotic cells after dosing, others displayed decreases, a variable outcome consistent with dual mechanisms of mitotic arrest and mitotic slippage induced by antimitotics in tumors. To provide a clearer picture, mitotic cell chromosome alignment and spindle bipolarity, new biomarkers of Aurora A inhibition that act independently of mitotic arrest or slippage, were assessed in the tumor biopsies. Several patients, primarily in the highest dose cohorts, had marked decreases in the percentage of mitotic cells with aligned chromosomes and bipolar spindles after dosing. Evidence existed for an exposure-effect relationship for mitotic cells with defects in chromosome alignment and spindle bipolarity that indicated a biologically active dose range. Outcomes of pharmacodynamic assays from skin and tumor biopsies were concordant in several patients. Together, these new pharmacodynamic assays provide evidence for Aurora A inhibition by MLN8054 in patient skin and tumor tissues.

Targeting Aurora A kinase activity with the investigational agent alisertib increases the efficacy of cytarabine through a FOXO‐dependent mechanism

Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A link between Aurora A expression and sensitivity to ara‐C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara‐C. Accordingly, alisertib significantly potentiated the antileukemic activity of ara‐C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro‐apoptotic effects of the alisertib/ara‐C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara‐C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara‐C that warrants further investigation.

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.

Abstract 3073: Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931

Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. Kinase activity of CDC7 is controlled by its binding protein DBF4 in a cell-cycle dependent manner. Here we developed a potent CDC7 inhibitor TAK-931 (IC50 120-fold selectivity of TAK-931 for CDC7 kinase inhibition compared to other kinase inhibitions. Treatment with TAK-931 suppressed the cellular MCM2 phosphorylation at Ser40, resulting in a delayed S phase progression, checkpoint activation, apoptosis, and potent growth suppression in various cancer cell lines. Furthermore, oral administration of TAK-931 as a single agent caused a significant antitumor activity in multiple xenograft models which include both cell line-based xenografts and patient-derived xenograft (PDX) models. These results demonstrate that TAK-931 is a highly potent and selective inhibitor of CDC7 kinase, and causes a potent antiproliferation both in vitro and in vivo studies using various cancer cells. Next, to identify potential predictive biomarkers to guide patient-selection strategies, in vitro cell panel screening of TAK-931 using was tested for its ability to antiproliferation in 246 cell lines, which includes both solid and hematological cancer cells. TAK-931 inhibited proliferation of multiple cancer cell lines, with mean concentration producing a half-maximal response (EC50) values ranging from 29.1 nM to > 30 μM (median = 554.5 nM). While the wide range of TAK-931 antiproliferative spectrum was observed, neither doubling speed nor CDC7 expression profile did predict tTAK-931 sensitivity in cancer cell lines. A correlative study of the tumor genetic mutations in relation to antiproliferative activity that KRAS mutant cancer cells were more sensitive to TAK-931 compared to KRAS non-mutant cell lines (p 60% TGI. Our findings suggest that the KRAS-mutant pancreatic tumors could be the potential candidate for the TAK-931 target indication. Citation Format: Kenichi Iwai, Tadahiro Nambu, Osamu Kurasawa, Noriko Uchiyama, Ryo Dairiki, Yukiko Yamamoto, Satoru Nishizawa, Mengkun Zhang, Yuko Ishii, Huifeng Niu, Akihiro Ohashi. Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931 [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 3073. doi:10.1158/1538-7445.AM2017-3073