TY - JOUR
T1 - Mass spectrometry– based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells
AU - McDonald, Ian M.
AU - Grant, Gavin D.
AU - East, Michael P.
AU - Gilbert, Thomas S.K.
AU - Wilkerson, Emily M.
AU - Goldfarb, Dennis
AU - Beri, Joshua
AU - Herring, Laura E.
AU - Vaziri, Cyrus
AU - Cook, Jeanette Gowen
AU - Emanuele, Michael J.
AU - Graves, Lee M.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants R01 CA199064 (to L. M. G.), GM083024 and GM102413 (to J. G. C.), and T32 CA009156 (to G. D. G.). This work was also supported in part by a UNC Dissertation Completion Fellowship (to I. M. M.). The authors declare that they have no conflicts of interest with the contents of this article. The con-tent is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by National Institutes of Health Grants R01 CA199064 (to L. M. G.), GM083024 and GM102413 (to J. G. C.), and T32 CA009156 (to G. D. G.). This work was also supported in part by a UNC Dissertation Completion Fellowship (to I. M. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Thomas Bonacci for sharing protocols and reagents, as well as Emily Fennell and Lucas Aponte-Collazo for assistance with experimental troubleshooting. We also thank Dr. Nathanael Gray for the generous gift of the compound HTH-01-091. This research was based in part upon work conducted using the UNC Proteomics Core Facility, which is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.
Publisher Copyright:
© 2020 McDonald et al.
PY - 2020/2/21
Y1 - 2020/2/21
N2 - The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.
AB - The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.
UR - http://www.scopus.com/inward/record.url?scp=85080102596&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.011083
DO - 10.1074/jbc.RA119.011083
M3 - Article
C2 - 31896573
AN - SCOPUS:85080102596
VL - 295
SP - 2359
EP - 2374
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 8
ER -