CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer

Abstract

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 Abbreviations: AURKA/Aurora-A, aurora kinase A; AURKB/Aurora-B, aurora kinase B; BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; CHEK1, checkpoint kinase 1; CPC, chromosomal passenger complex; CRISPR, clustered regularly interspaced short palindromic repeats; CSNK1A1, casein kinase 1 alpha 1; GSG2, germ cell-specific gene 2; DAPI, 4′,6-diamidino-2-phenylindole; DDR1, discoidin domain receptor tyrosine kinase 1; EGFP, enhanced green fluorescent protein; EGFP-sgCtrl, cells labeled with EGFP and introduced with a non-targeting sgRNA; ExMD, extends mitotic duration; K-fibers, kinetochore fibers; KMN network, kinetochore null protein 1 (KNL1)-missegregation 12 (MIS12) complex-nuclear division cycle 80 (NDC80) complex; KT, kinetochore; KT-MT, kinetochore-microtubule attachment; MAGeCK, model-based analysis of genome-wide CRISPR/Cas9 knockout; MCAK, mitotic centromere-associated kinesin; mCherry-sgCtrl, cells labeled with mCherry and introduced with a non-targeting sgRNA; mCherry-sgGSG2, cells labeled with mCherry and introduced with a sgRNA targeting GSG2; MINK1, misshapen like kinase 1; MOI, multiplicity of infection; MT, microtubule; NEK1, NIMA related kinase 1; NGS, next-generation sequencing; PCA, principal component analysis; PCM, pericentriolar material; pH3T3, phosphorylated threonine 3 of histone H3; QC, quality control; RB1, retinoblastoma; sgRNAs, single guide RNAs; TCGA, The Cancer Genome Atlas This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

New Insights into the Therapeutic Applications of CRISPR/Cas9 Genome Editing in Breast Cancer

“…However, combining a Haspin inhibitor (CHR-6494) with alisertib reduced cell viability favorably and inhibited its in vivo and in vitro tumor growth by causing extreme microtubule depolymerization, and along with other mechanisms, the mitotic process was disrupted, brought forth by the obliteration of the recruitment of Aurora-B and mitotic centromere associated kinesin to the centromeres. This ultimately demonstrates the use of CHR-6494 as a combinational drug with alisertib and its promising therapeutic benefits [176]. Future implications of breast cancer genes studied using CRISPR/Cas9 are summarized in Table 6.…”

Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition

“…M phase cells were synchronized using nocodazole [ 48 ] unless otherwise indicated. Briefly, cells were treated with 100 ng/ml nocodazole for 14–16 h. For western blot and ATP concentration testing, mitosis cells were collected after shake-off.…”

“…When the tumors reached ~3 mm in diameter, mice were randomized into four groups for treatment with alisertib only (alisertib 15 mg/kg/day), metformin only (metformin 300 mg/kg/day), a combination of metformin and alisertib (metformin 300 mg/kg/day, alisertib 15 mg/kg/day), or a vehicle (1% β-Cyclodextrin with 1% sodium bicarbonate) via oral gavage. Tumor sizes were measured every 2 days and the volumes were calculated by a previously used equation V = (length × width 2 )/2 [ 48 ].…”

“…When the tumors reached ~3 mm in diameter, mice were randomized into groups for treatment with alisertib only (alisertib 15 mg/kg/day), a combination of metformin and alisertib (metformin 300 mg/kg/day, alisertib 15 mg/kg/day), or a vehicle (1% β-Cyclodextrin with 1% sodium bicarbonate) via oral gavage. Tumor sizes were measured every 2 days and the volumes were calculated by a previously used equation V = (length × width 2 )/2 [ 48 ]. Mice were injected intraperitoneally with luciferin (3 mg per mouse, YEASEN, HB181121) and imaged using an in vivo imaging system (Bruker MI) on the 14th day after injection.…”

Design and Synthesis of Newly Synthesized Acrylamide Derivatives as Potential Chemotherapeutic Agents against MCF-7 Breast Cancer Cell Line Lodged on PEGylated Bilosomal Nano-Vesicles for Improving Cytotoxic Activity

“…Breast cancer characteristically displays uncontrolled or abnormal cell proliferation due to excessive microtubule synthesis [11,12]. Knowledge and understanding of this intrinsic property have resulted in the development of chemotherapeutic regimens that act by interfering with the microtubule assembly or disassembly [13].…”