CCT245718, a dual FLT3/Aurora A inhibitor overcomes D835Y-mediated resistance to FLT3 inhibitors in acute myeloid leukaemia cells

Background: Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are among the most prevalent oncogenic mutations in acute myeloid leukaemia. Inhibitors selectively targeting FLT3 kinase have shown promising clinical activity; their success in the clinic, however, has been limited due to the emergence of acquired resistance.
Methods: CCT245718 was identified and characterised as a dual Aurora A/FLT3 inhibitor through cell-based and biochemical assays. The ability of CCT245718 to overcome TKD-mediated resistance was evaluated in a cell line-based model of drug resistance to FLT3 inhibitors.
Results: CCT245718 exhibits potent antiproliferative activity towards FLT3-ITD + AML cell lines and strongly binds to FLT3-ITD and TKD (D835Y) mutants in vitro.
Activities of both FLT3-ITD and Aurora A are also inhibited in cells. Inhibition of FLT3 results in reduced phosphorylation of STAT5, downregulation of survivin and induction of apoptotic cell death.
Moreover, CCT245718 overcomes TKD-mediated resistance in a MOLM-13-derived cell line containing FLT3 with both ITD and D835Y mutations.
It also inhibits FLT3 signalling in both parental and resistant cell lines compared to FLT3-specific inhibitor MLN518, which is only active in the parental cell line.
Conclusions: Our results demonstrate that CCT245718 is a potent dual FLT3/Aurora A inhibitor that can overcome TKD-mediated acquired resistance

CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for AuroraA inhibitor resistance in breast cancer

Background: Overexpression of Aurora-A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora-A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients.
Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.
Methods: Here, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237.
Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin (MCAK).
Results: Among the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237.
Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore-microtubule (KT-MT) attachment failure, and severe mitotic catastrophe.
We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.
Conclusions: These findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.

Aurora A inhibitor TAS-119 enhances antitumor efficacy of taxanes in vitro and in vivo: preclinical studies as guidance for clinical development and trial design

TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro, TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines.
Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5.
In vivo, TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors.
Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel.
The degree of enhancement calculated as fold of change of the IC50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration.
The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later.
This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination.

Aurora A inhibition disrupts chromosome condensation and spindle assembly during the first embryonic division in pigs.

As common overexpression of Aurora A in various tumors, much attention has focused on its function in inducing cancer, and its value in cancer therapeutics, considerably less is known regarding its role in the first cleavage division of mammalian embryos. Here, we highlight an indispensable role of Aurora-A during the first mitotic division progression of pig embryos just after meiosis.
The expression and spatiotemporal localization of Aurora A were initially assessed in pig embryos during the first mitotic division by Western blot analysis and indirect immunofluorescent staining.
Then, the potential role of Aurora A was further evaluated using a highly selective Aurora A inhibitor, MLN8054, during this mitotic progression in pig embryos.
Aurora-A was found to express and exhibit a specific dynamic intracellular localization pattern during the first mitotic division in pig embryos.
Aurora A was diffused in the cytoplasm at the prophase stage, and then exhibited a dynamic intracellular localization which was tightly associated with the chromosome and spindle dynamics throughout subsequent mitotic phases. Inhibition of Aurora A by MLN8054 treatment led to the failure of the first cleavage, with the majority of embryos being arrested in prophase of the mitotic division.
Further subcellular structure examination showed that Aurora A inhibition not only led to the failure of spindle microtubule assembly, but also resulted in severe defects in chromosome condensation, accompanied by an obvious decrease in p-TACC3(S558) expression during the prophase of the first mitosis.
Together, these results illustrated that Aurora A is crucial for both spindle assembly and chromosome condensation during the first mitotic division in pig embryos, and that the regulation of Aurora A may be associated with its effects on p-TACC3(S558) expression.

Aurora A Inhibition Eliminates Myeloid Cell-Mediated Immunosuppression and Enhances the Efficacy of Anti-PD-L1 Therapy in Breast Cancer

The Aurora A inhibitor alisertib shows encouraging activities in clinical trials against advanced breast cancer. However, it remains unclear whether and how the inflammatory microenvironment is involved in its efficacy.
Here, we demonstrated that inhibition of Aurora-A directly reshaped the immune microenvironment through removal of tumor-promoting myeloid cells and enrichment of anticancer T lymphocytes, which established a tumor-suppressive microenvironment and significantly contributed to the regression of murine mammary tumors. Mechanistically, alisertib treatment triggered apoptosis in myeloid-derived suppressor cells (MDSC) and macrophages, resulting in their elimination from tumors. Furthermore, alisertib treatment disrupted the immunosuppressive functions of MDSC by inhibiting Stat3-mediated ROS production.

Aurora A Inhibitor I

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These alterations led to significant increases of active CD8+ and CD4+ T lymphocytes, which efficiently inhibited the proliferation of tumor cells. Intriguingly, alisertib combined with PD-L1 blockade showed synergistic efficacy in the treatment of mammary tumors.
These results detail the effects of Aurora A inhibition on the immune microenvironment and provide a novel chemo-immunotherapy strategy for advanced breast cancers.
SIGNIFICANCE: These findings show that inhibition of Aurora A facilitates an anticancer immune microenvironment, which can suppress tumor progression and enhance anti-PD-L1 therapy in breast cancer.See related commentary by Rivoltini et al., p. 3169.

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