Riccardo Colombo

Targeting the Mitotic Checkpoint for Cancer Therapy with Nms-P715, an Inhibitor of Mps1 Kinase.

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.

Synthesis and SAR of new pyrazolo[4,3-h]quinazoline-3-carboxamide derivatives as potent and selective MPS1 kinase inhibitors

The synthesis and SAR of a series of novel pyrazolo-quinazolines as potent and selective MPS1 inhibitors are reported. We describe the optimization of the initial hit, identified by screening the internal library collection, into an orally available, potent and selective MPS1 inhibitor.

Selective Inhibition of Pancreatic Ductal Adenocarcinoma Cell Growth by the Mitotic MPS1 Kinase Inhibitor NMS-P715

Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy. Mol Cancer Ther; 13(2); 307–15. ©2013 AACR.

Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.

Targeting aneuploid cancer cells.

Introduction: Most cancers are characterized by some degree of aneuploidy, although its relevance for tumor initiation or progression and the nature of the initial trigger are still not well understood. It was Theodor Boveri who first suggested a link between aneuploidy and cancer at the beginning of the last century, but it is only recently that the molecular mechanisms involved have started to be uncovered. Areas covered: The molecular mechanisms that are at the origin of aneuploidy and their cellular consequences. Based on these new findings molecular targets have emerged which could lead to a specific treatment of at least some types of aneuploid tumors. Expert opinion: Therapeutic intervention specifically for aneuploid cells is a very promising approach, however, although new promising targets have been spotted they still need to be tested for proof of concept. Targeting the spindle checkpoint could be an interesting approach for cancer therapy, however, as for other mitotic targets, the open question of the therapeutic window and sensitivity of normal hemopoietic cells has to be considered carefully. Future challenges will not only include identifying and validating druggable targets related to the relevant pathways, but also finding predictive biomarkers to define the responding patient population(s).

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3–1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

Destabilizing Aneuploidy by Targeting Cell Cycle and Mitotic Checkpoint Proteins in Cancer Cells

Aneuploidy is one of the major hallmarks of cancer cells and several paths towards aneuploidy have been described. However, the relevance for tumor initiation or progression and how tumors deal with the initial aneuploidy related stress response is still unclear and recent results suggest that aneuploidy can even have tumor suppressive effects under certain conditions. The molecular mechanisms leading to and sustaining growth of aneuploid cells are just at the beginning to be understood and might provide new targets for cancer drug development. We will discuss some of the ideas to specifically kill aneuploid cells by targeting key regulators of mitosis.

Target Identification and Validation in Drug Discovery

The advent of a variety of genomic, proteomic and other system-based scienti fi c approaches has raised the expectations of identifying novel targets for oncology drug discovery. However, the complexity of human genome cancer alterations requires a careful analysis of the function of candidate targets identi fi ed by these efforts. The postulation and testing of a hypothesis that modulation of a protein or pathway will result in a therapeutic effect in a preclinical setting is crucial for target validation activities. In this chapter, we provide an overview on target identi fi cation and validation approaches to interrogate the functional and therapeutic relevance of a candidate cancer drug target as an essential step towards justifying the subsequent investment in drug discovery efforts.

Abstract 1638: Preclinical characterization of the novel TTK kinase inhibitor S81694 for the treatment of triple negative breast cancer

The tyrosine threonine kinase TTK, also known as Monopolar Spindle 1 kinase (MPS1), is a conserved kinase found to be highly expressed in a number of human tumors of different origin. TTK kinase activity plays a critical role in the control of mitosis, regulating the spindle assembly checkpoint (SAC), a mitotic mechanism required for proper chromosome alignment and segregation during cellular division. The activity of this checkpoint has been shown to be proficient and up-regulated in aneuploid tumors, a common feature measured in approximately 90% of solid tumors and 70% of hematological cancers. S81694 (NMS-P153) is a novel potent inhibitor of TTK. It is a tight binder compound with long residence time on the target and is highly selective for TTK against a wide range of tested enzymes (kinases, channels, hormones, GPCRs). In vitro, brief exposure is sufficient to commit cells to death with the compound showing anti-proliferative activity with a demonstrated mechanism of action and marked tumor growth inhibition in preclinical in vivo tumor models associated with a good pharmacokinetic profile in both rodent and non-rodent species. TTK inhibitors are anticipated to be more active in SAC dependent tumors and data in the literature suggests that triple negative breast cancer is among the tumor types potentially highly dependent on TTK activity. Moreover, in accelerating mitosis, TTK inhibitors have a novel mode of action as compared to currently known mitotic inhibitors. To confirm the role of TTK in breast cancer and identify a sensitive patient population to S81694 treatment, translational studies have been conducted on a large panel of human tumor cell lines, including 39 of mammary gland origin. Anti-proliferative activity was measured after 72h continuous treatment or after a brief treatment of 7h followed by 2D colony forming assay to verify the long term effects of a treatment which mimics compound half-life in mice. In addition, active caspase 3 induction was also analyzed to identify cell lines more prone to activate apoptosis upon treatment. Triple negative breast cancer (TNBC) cell lines were confirmed to be particularly sensitive to S81694 and correlation was observed between sensitivity profile in vitro and tumor growth inhibition in vivo. In MDA-MB-231 xenograft and orthotopic models, tumor regression, cured animals and metastasis reduction in several organs including brain and lungs was observed. These results further confirm the role of TTK in triple negative breast cancer and could support the clinical development of S81694 in this indication. Citation Format: Riccardo Colombo, Mike Burbridge, Marianne Rodriguez, Frederique Cantero, Marina Caldarelli, Maria Laura Giorgini, Francesco Sola, Dario Ballinari, Marina Ciomei, Roberta Bosotti, Alessia Montagnoli, Antonella Isacchi, Daniele Donati, Arturo Galvani. Preclinical characterization of the novel TTK kinase inhibitor S81694 for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2015-1638

Abstract 3795: Novel and selective MELK kinase inhibitors active in breast cancer cell lines

Maternal Embryonic Leucine zipper Kinase (MELK) is a serine-threonine kinase implicated in stem cell renewal, override of cell cycle checkpoints, pre-mRNA splicing and resistance to apoptosis, while MELK gene expression levels correlate inversely with poor prognosis in breast cancer, prostate cancer and glioblastoma patients. Moreover, recent findings underlie the oncogenic role of this kinase in triple negative breast cancer (TNBC), a category of high-grade, invasive tumors which lack expression of estrogen receptor (ER) and progesterone receptor (PR) and HER2 amplification and which is resistant to current cytotoxic and targeted therapies. Furthermore, they are highly heterogeneous with respect to genomic alterations, and common therapeutic targets are lacking, although substantial evidence implicates dysregulated kinase signaling. Here, we describe the preclinical characterization of novel, potent and selective ATP-competitive MELK kinase inhibitors identified by means of high-throughput screening of the NMS proprietary compound collection. Leading compounds possess biochemical activity against MELK in the nanomolar range with high selectivity against a panel of 60 further kinases representative of the human kinome. Amongst human tumor cell lines tested in 2-dimensional colony outgrowth assays, marked sensitivity was observed in breast cancer cell lines, with sub-micromolar anti-proliferative activity. This effect was accompanied by dose-dependent induction of apoptosis and by modulation of cellular biomarkers, consistent with a MELK-dependent mechanism of action. Overall, these data provide further evidence that MELK is a promising biological target for the development of novel anticancer therapies. Citation Format: Patrizia Carpinelli, Marisa Montemartini, Nadia Amboldi, Dario Ballinari, Sabrina Cribioli, Marina Ciomei, Riccardo Colombo, Stefania Re Depaolini, Nilla Avanzi, Giulia Canevari, Walter Ceccarelli, Helena Posteri, Maria Gabriella Brasca, Daniele Donati, Eduard Rudolf Felder, Antonella Isacchi, Arturo Galvani, Alessia Montagnoli. Novel and selective MELK kinase inhibitors active in breast cancer cell lines. [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 3795.