CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A 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.

Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes.Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis.Although previously proposed to be an adaptor of retinoic acid receptor,here,we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis.We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore,suggesting that phosphorylation may regulate its localization.Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase.Mechanistically,CENP-R phosphorylation disrupts its binding with CENP-U.Thus,we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis.As CENP-R is absent from yeast,we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.