Jeffery A. Ecsedy

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 beta-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 beta-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.

Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

Glioblastoma remains a devastating disease for which novel therapies are urgently needed. Here, we report that the Aurora-A kinase inhibitor alisertib exhibits potent efficacy against glioblastoma neurosphere tumor stem-like cells in vitro and in vivo. Many glioblastoma neurosphere cells treated with alisertib for short periods undergo apoptosis, although some regain proliferative activity upon drug removal. Extended treatment, however, results in complete and irreversible loss of tumor cell proliferation. Moreover, alisertib caused glioblastoma neurosphere cells to partially differentiate and enter senescence. These effects were also observed in glioma cells treated with the Aurora-A inhibitor TC-A2317 or anti-Aurora-A siRNA. Furthermore, alisertib extended median survival of mice bearing intracranial human glioblastoma neurosphere tumor xenografts. Alisertib exerted similar effects on glioblastoma neurosphere cells in vivo and resulted in markedly reduced activated phosphoThr288Aurora-A and increased abnormal mitoses and cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.

// Todd M. Pitts 1, 3, * , Erica L. Bradshaw-Pierce 2, 3, 5, * , Stacey M. Bagby 1 , Stephanie L. Hyatt 1 , Heather M. Selby 1 , Anna Spreafico 1 , John J. Tentler 1, 3 , Kelly McPhillips 1 , Peter J. Klauck 1 , Anna Capasso 1 , Jennifer R. Diamond 1, 3 , S. Lindsey Davis 1, 3 , Aik Choon Tan 1, 3 , John J. Arcaroli 1, 3 , Alicia Purkey 1 , Wells A. Messersmith 1, 3 , Jeffery A. Ecsedy 4 , S. Gail Eckhardt 1, 3 1 Division of Medical Oncology, School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA 2 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA 3 University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 4 Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, Cambridge, MA, USA 5 Takeda California, San Diego, CA, USA * These authors have contributed equally to this work Correspondence to: Todd M. Pitts, email: Todd.Pitts@ucdenver.edu Keywords: colorectal cancer, aurora kinase a Received: January 20, 2016     Accepted: June 17, 2016     Published: July 1, 2016 ABSTRACT Background: The Aurora kinases are a family of serine/threonine kinases comprised of Aurora A, B, and C which execute critical steps in mitotic and meiotic progression. Alisertib (MLN8237) is an investigational Aurora A selective inhibitor that has demonstrated activity against a wide variety of tumor types in vitro and in vivo , including CRC. Results: CRC cell lines demonstrated varying sensitivity to alisertib with IC 50 values ranging from 0.06 to > 5 umol/L. Following exposure to alisertib we observed a decrease in pAurora A, B and C in four CRC cell lines. We also observed an increase in p53 and p21 in a sensitive p53 wildtype cell line in contrast to the p53 mutant cell line or the resistant cell lines. The addition of alisertib to standard CRC treatments demonstrated improvement over single agent arms; however, the benefit was largely less than additive, but not antagonistic. Methods: Forty-seven CRC cell lines were exposed to alisertib and IC 50 s were calculated. Twenty-one PDX models were treated with alisertib and the Tumor Growth Inhibition Index was assessed. Additionally, 5 KRAS wildtype and mutant PDX models were treated with alisertib as single agent or in combination with cetuximab or irinotecan, respectively. Conclusion: Alisertib demonstrated anti-proliferative effects against CRC cell lines and PDX models. Our data suggest that the addition of alisertib to standard therapies in colorectal cancer if pursued clinically, will require further investigation of patient selection strategies and these combinations may facilitate future clinical studies.

Abstract MEK and Aurora A kinase inhibitors (AAK) have profound impacts on cell cycle progression. Inhibition of MEK leads to defects in cell-cycle progression followed by cell cycle arrest and/or apoptosis. These cell-cycle progression defects may in part be due to the role of MEK in the DNA replication or damage checkpoint responses through positive regulation of DNA repair mechanisms. AAK inhibition leads to chromosome congression and segregation defects leading to DNA damage followed by apoptosis or senesence. The overlapping biological effects of inhibition of AAK and MEK kinases in cell cycle progression and chromosomal integrity raise the possibility that inhibiting both targets would provide added benefit over the inhibition of either target alone. The in vivo antitumor activity of TAK-733, an investigational potent, selective, non-ATP-competitive allosteric inhibitor of MEK, in combination with alisertib, an investigational potent, selective, reversible, ATP-competitive inhibitor of Aurora A kinase, was examined in experimental human solid tumor xenograft models including NSCLC (NCI H23 [KRAS and LKB1 mutations]), CRC (SW620 [KRAS, APC, p53 mutations]), and pancreatic cancer (Panc 1 and Capan 1 [KRAS mutations] and BxPC-3 [No MAPK mutations]) models in immunocompromised mice. The previously established maximally efficacious doses for the single agents dosed once daily (QD) orally (PO) (10 mg/kg TAK-733 and 30-mg/kg alisertib) were examined. A lower dose of 20 mg/kg alisertib was also examined in the event the maximally efficacious dose of each agent was not tolerated in combination. The once daily (QD) concurrent oral administration of TAK-733 and alisertib resulted in additive to synergistic antitumor activity and in prolonged inhibition of tumor regrowth after terminating treatment compared to single agent treatment in all xenograft models examined. Concurrent administration of TAK-733 and alisertib daily for 21 days was well tolerated. Treatment with TAK-733 alone and in combination with alisertib clearly inhibited phosphorylated ERK and produced a slight increase in phosphorylated histone H3. The results from the nonclinical models examined demonstrate considerable improvement in nonclinical antitumor activity over either single agent alone and provide a biological rationale for clinical evaluation of a TAK-733/alisertib combination in patients with advanced malignancies. 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 3739. doi:1538-7445.AM2012-3739