The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.
Chromosomal instability (CIN) is a hallmark of cancer that results from errors in chromosome segregation during mitosis. Targeting of CIN-associated vulnerabilities is an emerging therapeutic strategy in drug development. KIF18A, a mitotic kinesin, has been shown to play a role in maintaining bipolar spindle integrity and promotes viability of CIN cancer cells. To explore the potential of KIF18A, a series of inhibitors was identified. Optimization of an initial hit led to the discovery of analogues that could be used as chemical probes to interrogate the role of KIF18A inhibition. Compounds 23 and 24 caused significant mitotic arrest in vivo, which was sustained for 24 h. This would be followed by cell death either in mitosis or in the subsequent interphase. Furthermore, photoaffinity labeling experiments reveal that this series of inhibitors binds at the interface of KIF18A and tubulin. This study represents the first disclosure of KIF18A inhibitors with in vivo activity.
Abstract BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was > 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53mut IGROV-1 cells showed minimal regrowth compared to TP53wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P < .0001 vs. vehicle alone). Notably, co-administration of AMG 900 with docetaxel enhanced efficacy and induced a delay in tumor regrowth compared to docetaxel alone. Single-agent-treated mice showed minimal body weight loss (BWL), whereas combination-treated mice showed moderate BWL (average < 10%) that was largely reversible (2 of 12 animals removed due to toxicity). CONCLUSIONS: AMG 900 alone or in combination with chemotherapy such as paclitaxel may be a promising clinical strategy to treat patients with ovarian cancer. Citation Format: Ondrej Kalous, Dylan Conklin, Kanthinh Manivong, William Wayne, Kelly Hanestad, Jude Canon, Robert Loberg, Gregory Friberg, Erick Gamelin, Florian D. Vogl, Gloria Juan, Angela Coxon, Dennis Slamon, Richard Finn, Marc Payton. Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer. [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 3008.
Abstract Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.
<p>Supplementary Figure S1 shows the cellular effects of AKIs and anti-leukemic agents across a panel of human AML cell lines. Supplementary Figure S2 shows expression of aurora-A and aurora-B protein levels in four AML cell lines. Supplementary Figure S3 shows AMG 900 induces a dose-dependent increase in polyploidy, apoptosis, and p53 protein levels in MOLM-13 cells. Supplementary Figure S4 shows AMG 900 plus Ara-C combination matrix and CI determination in MOLM-13 cells. Supplementary Figure S5 shows AMG 900 induced apoptosis is attenuated by peptide inhibitors of caspases in MOLM-13 cells. Supplementary Figure S6 shows FC gating strategy for annexin-V coupled JC-1 assay. Supplementary Figure S7 shows the anti-proliferative effects of AMG 900 and AZD1152-hQPA on primary human bone marrow mononuclear cells in culture. Supplementary Figure S8 shows a moderate reduction in mouse body weight after AMG 900 treatment.</p>
<p>Supplementary Materials and Methods; Supplementary Figure 1: Effects of AMG 900 on nuclear morphology and centrosome features are consistent with aurora-B inhibition in MDA-MB-231 cell line; Supplementary Figure 2: Elevated expression of ABC transporter gene ABCB1 and ABCB4 in MDA-MB-231 (F11) PTX-r cell line; Supplementary Figure 3: Cell-cycle profiles of TNBC cell lines treated with paclitaxel or ixabepilone.</p>
Abstract Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53 -mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1 -amplified, CDK4–CDK6-inhibitor-resistant and BRCA1 -altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.
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