Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

Background: The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. Methods: In this study, we used cultured cell models to investigate the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Results: Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. Conclusion: These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Modification of the neck-linker of KIF18A alters Microtubule subpopulation preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, posttranslational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to nonkinetochore microtubules at the periphery of the spindle. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker-like state that decreases KIF18A accumulation at the plus-ends of kinetochore microtubules. These findings demonstrate that posttranslational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. In this study, we investigated the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, post-translational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to peripheral microtubules. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker like state that prevents KIF18A from accumulating at the plus-ends of kinetochore microtubules. These findings demonstrate that post-translational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

Chromosomally unstable tumor cells specifically require KIF18A for proliferation.

Chromosomal instability (CIN) is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that compromise the mitotic spindle. Thus, CIN cells may respond differently than diploid cells to treatments that target mitotic spindle regulation. Here, we test this idea by inhibiting a subset of kinesin motor proteins involved in mitotic spindle control. KIF18A is required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but is not required in near-diploid cells. Following KIF18A inhibition, CIN tumor cells exhibit mitotic delays, multipolar spindles, and increased cell death. Sensitivity to KIF18A knockdown is strongly correlated with centrosome fragmentation, which requires dynamic microtubules but does not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.