# Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics

> **NIH NIH F31** · WEILL MEDICAL COLL OF CORNELL UNIV · 2021 · $46,036

## Abstract

SUMMARY ABSTRACT
Mitotic kinases and phosphatases are essential for preventing errors in chromosome segregation that can lead
to aneuploidy and cancer. Aurora Kinase A (AurA) is an essential mitotic kinase that is often upregulated in
human cancers. AurA has a well-studied role in assembly of the mitotic spindle apparatus, a complex microtubule
array responsible for separating sister chromatids into two identical daughter cells. It is challenging to study AurA
function outside of spindle assembly because inhibition causes disruption of the mitotic spindle which can mask
other relevant phenotypes. Further, most AurA small molecule inhibitors can be promiscuous. We have
developed a system that circumvents these issues by allowing for specific and conditional inhibition of AurA
activity. Using cultured human cells, we have inactivated endogenous AurA with CRISPR-Cas9 and rescued its
activity with a genetically modified, transgenic AurA allele (AurA-AS). The genetic alteration makes this allele
specifically sensitive to modified ATP competitive inhibitors that cannot inhibit any other cellular kinases. Our
objective is to use this tool to study the connection between AurA and cancer. Specifically, we are interested in
the connection between AurA and centrosome number amplifications, which are common in both solid and
hematological malignancies. Extra centrosomes can create mitotic spindles with more than two poles thus
causing devastating chromosome mis-segregation. AurA inhibition has been associated with formation of
multipolar spindles in the literature. Our preliminary data suggests that AurA activity prevents this multipolar
spindle phenotype by promoting the “clustering” of extra centrosomes into two spindle poles. This led us to our
central hypothesis that AurA phosphorylation is essential for bi-polar spindle formation in the presence of extra
centrosomes. Based on evidence in the literature, we hypothesize that AurA mediates centrosome clustering via
phosphorylation of spindle-pole focusing kinesin HSET (Aim 1) and via promoting localization of mitotic spindle
positioning factor NuMA to the cell cortex (Aim 2). To determine if AurA-phosphorylation of HSET is important
for efficiency of centrosome clustering, we will use in vitro kinase assays to show direct phosphorylation (1.1).
We will use HSET phospho-site mutants to determine if this phosphorylation is relevant to centrosome clustering
(1.2) and to perform in vitro microtubule motility assays to determine how AurA phosphorylation effects HSET’s
activity (1.3). Further, we will use microscopy to determine if loss of AurA impairs mitotic spindle positioning or
NuMA localization to the cell cortex (2.1) and if altered NuMA localization effects efficiency of centrosome
clustering (2.2). All proposed experiments use our established system for studying AurA. This work is of particular
interest as both solid and hematological malignancies often have increased number of centrosomes; by
clusterin...

## Key facts

- **NIH application ID:** 10140625
- **Project number:** 1F31CA257167-01
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Gina M Tomarchio
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 1
- **Project period:** 2020-12-11 → 2023-12-10

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10140625

## Citation

> US National Institutes of Health, RePORTER application 10140625, Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics (1F31CA257167-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10140625. Licensed CC0.

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