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.

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 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.

Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser558 by Aurora A: a novel pharmacodynamic method for measuring Aurora A activity.

Aurora A is a serine/threonine protein kinase essential for normal mitotic progression. Aberrant increased expression of Aurora A, which occurs frequently in human cancers, results in abnormal mitoses leading to chromosome instability and possibly tumorigenesis. Consequently, Aurora A has received considerable attention as a potential target for anticancer therapeutic intervention. Aurora A coordinates several essential mitotic activities through phosphorylation of a variety of proteins, including TACC3, which modulates microtubule stabilization of the mitotic spindle. Recent studies identified a conserved serine in Xenopus (Ser(626)) and Drosophila (Ser(863)) TACC3 orthologues that is phosphorylated by Aurora A. We show that this conserved serine on human TACC3 (Ser(558)) is also phosphorylated by Aurora A. Moreover, phosphorylation of TACC3 by Aurora A in human cells is essential for its proper localization to centrosomes and proximal mitotic spindles. Inhibition of Aurora A with the selective small molecule inhibitor MLN8054 in cultured human tumor cells resulted in mislocalization of TACC3 away from mitotic spindles in a concentration-dependent manner. Furthermore, oral administration of MLN8054 to nude mice bearing HCT-116 human tumor xenografts caused a dose-dependent mislocalization of TACC3 away from spindle poles that correlated with tumor growth inhibition. As TACC3 localization to mitotic spindles depends on Aurora A-mediated phosphorylation, quantifying TACC3 mislocalization represents a novel pharmacodynamic approach for measuring Aurora A activity in cancer patients treated with inhibitors of Aurora A kinase.

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 G(2)/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.

Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes.

PURPOSE: High-level expression of the telomerase reverse transcriptase (hTERT) in >85% of human cancers, in contrast with its restricted expression in normal adult tissues, points to hTERT as a broadly applicable molecular target for anticancer immunotherapy. CTLs recognize peptides derived from hTERT and kill hTERT+ tumor cells of multiple histologies in vitro. Moreover, because survival of hTERT+ tumor cells requires functionally active telomerase, hTERT mutation or loss as a means of escape may be incompatible with sustained tumor growth. EXPERIMENTAL DESIGN: A Phase I clinical trial was performed to evaluate the clinical and immunological impact of vaccinating advanced cancer patients with the HLA-A2-restricted hTERT I540 peptide presented with keyhole limpet hemocyanin by ex vivo generated autologous dendritic cells. RESULTS: As measured by peptide/MHC tetramer, enzyme-linked immunospot, and cytotoxicity assays, hTERT-specific T lymphocytes were induced in 4 of 7 patients with advanced breast or prostate carcinoma after vaccination with dendritic cells pulsed with hTERT peptide. Tetramer-guided high-speed sorting and polyclonal expansion achieved highly enriched populations of hTERT-specific cells that killed tumor cells in an MHC- restricted fashion. Despite concerns of telomerase activity in rare normal cells, no significant toxicity was observed. Partial tumor regression in 1 patient was associated with the induction of CD8+ tumor infiltrating lymphocytes. CONCLUSIONS: These results demonstrate the immunological feasibility of vaccinating patients against telomerase and provide rationale for targeting self-antigens with critical roles in oncogenesis.

Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction.

The identification of antigens associated with tumor destruction is a major goal of cancer immunology. Vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor generates potent, specific, and long-lasting antitumor immunity through improved tumor antigen presentation by dendritic cells and macrophages. A phase I clinical trial of this immunization strategy in patients with disseminated melanoma revealed the consistent induction in distant metastases of dense T and B cell infiltrates that effectuated substantial tumor necrosis and fibrosis. To delineate the target antigens of this vaccine-stimulated tumor destruction, we screened a melanoma cDNA expression library with postimmunization sera from a long-term responding patient (K030). High-titer IgG antibodies recognized melanoma inhibitor of apoptosis protein (ML-IAP), a caspase antagonist containing a single baculoviral IAP repeat and a COOH-terminal RING domain. Although K030 harbored antibodies to ML-IAP at the time of study entry, multiple courses of vaccination over 4 years increased antibody titers and elicited isotype switching. Moreover, lymphocyte infiltrates in necrotic metastases included CD4+ and CD8+ T cells specific for ML-IAP, as revealed by proliferation, tetramer, enzyme-linked immunospot, and cytotoxicity analysis. Whereas melanoma cells in densely infiltrated lesions showed strong ML-IAP expression by immunohistochemistry, lethal disease progression was associated with the loss of ML-IAP staining and the absence of lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a target for immune-mediated tumor destruction, but that antigen-loss variants can accomplish immune escape.