Abstract PR001: KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability
Authors: Lynes Maureen S. , Ghisolfi Laura , Khan Sanjoy , Hotz Taylor , Sparling Brian A. , Zablocki Mary-Margaret , Gotur Deepali , Boriack-Sjodin P. Ann , Duncan Kenneth W. , Blakemore Stephen J. , Silver Serena J. , Copeland Robert A. 
Published: June 2024

Abstract
KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling synthetic lethality target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX020, inhibits KIF18A ATPase activity with an IC50 of 0.014 µM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 &gt; 10 µM) and EG5 (IC50 5.87 µM). ATX020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 0.053 µM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In these sensitive cell lines, ATX020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX020 exhibit fragmented nuclei and malformed mitotic spindles. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX020 treatment, cell division is unaffected, and no induction of Annexin V, phospho-Histone H3, and gamma-H2AX is observed. This selective dependency is also observed in vivo, where administration of ATX020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer.
Citation Format: Maureen S. Lynes, Laura Ghisolfi, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR001.