kinesin-5 inhibitor sensitivity
5747267
An optimized lentivirus mediated RNAi screen reveals kinase modulators of kinesin 5 inhibitor sensitivity.
Abstract: Induction of RNA interference (RNAi) in human cells has enabled comprehensive functional annotation of the human genome via reverse genetic screens. Here we describe an optimized semiautomated method to produce, titrate, and screen large collections of short hairpin RNA (shRNA) containing lentiviral vectors. We also present results from a pilot lentiviral RNAi screen for kinases whose silencing modulates sensitivity to a mitotic spindle protein kinesin 5 inhibitor (kinesin 5i) Our screen identified three distinct serine/threonine kinase 6 shRNA vectors within our library as enhancers of kinesin 5i mediated HT29 cell growth inhibition. In contrast, three distinct shRNAs targeting cell division cycle 2/cyclin dependent kinase 1 resulted in kinesin 5i resistance. These results demonstrate the feasibility of screening with large collections of lentiviral vectors to identify drug enhancers and suppressors.
Assay and drug development technologies
Richard A Klinghoffer Brian S Roberts James E Annis Jason P Frazier Patrick Lewis Peter S Linsley Michele A Cleary
17586514
Mitotic Slippage and Expression of Survivin Are Linked to Differential Sensitivity of Human Cancer Cell Lines to the Kinesin 5 Inhibitor Monastrol
The mitotic Kinesin 5 motor proteins crosslink and slide apart antiparallel spindle microtubules, thus performing essential functions in mitotic spindle dynamics. Specific inhibition of their function by monastrol like small molecules has been examined in clinical trials as anticancer treatment, with only partial success. Thus, strategies that improve the efficiency of monastrol like anticancer drugs are required. In the current study, we examined the link between sensitivity to monastrol and occurrence of mitotic slippage in several human cell lines. We found that the rank of sensitivity to monastrol, from most sensitive to least sensitive, is: AGS>HepG2>Lovo>Du145>=HT29. We show correlation between the sensitivity of a particular cell line to monastrol and the tendency of the same cell line to undergo mitotic slippage. We also found that in the monastrol resistant HT29 cells, prolonged monastrol treatments increase mRNA and protein levels of the chromosomal passenger protein survivin. In contrast, survivin levels are not increased by this treatment in the monastrol sensitive AGS cells. We further show that over expression of survivin in the monastrol sensitive AGS cells reduces mitotic slippage and increases resistance to monastrol. Finally, we show that during short exposure to monastrol, Si RNA silencing of survivin expression reduces cell viability in both AGS and HT29 cells. Our data suggest that the efficiency of anti cancer treatment with specific kinesin 5 inhibitors may be improved by modulation of expression levels of survivin.
PloS one
Hila Asraf Rachel Avunie-Masala Michal Hershfinkel Larisa Gheber
8921302
An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin 5 Inhibitor
Kinesin 5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin 5, developed as potential anti cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome wide siRNA screen for enhancers and suppressors of a Kinesin 5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers) and vice versa (suppressors) and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology.
PloS one
Melody Tsui Tiao Xie James D Orth Anne E Carpenter Stewart Rudnicki Suejong Kim Caroline E Shamu Timothy J Mitchison
8123590
Cell type variation in responses to antimitotic drugs that target microtubules and kinesin 5.
To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin 5 (also known as KSP, Eg5, Kif11) Using automated microscopy with markers for mitosis and apoptosis (high content screening) we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation. We found no correlation between levels of mitotic arrest and apoptosis. Apoptosis depended on entry into mitosis and occurred both from within mitosis and after exit. Response to the three drugs strongly correlated, although paclitaxel caused more apoptosis in some cell lines at similar levels of mitotic arrest. Molecular investigations showed that sensitivity to apoptosis correlated with loss of an antiapoptotic protein, XIAP, during the drug response, but not its preresponse levels, and to some extent also correlated with activation of the p38 and c Jun NH(2) kinase pathways. We conclude that variation in sensitivity to antimitotic drugs in drug naive cell lines is governed more by differences in apoptotic signaling than by differences in mitotic spindle or spindle assembly checkpoint proteins and that antimitotics with different mechanisms trigger very similar, but not identical, responses.
Cancer research
Jue Shi James D Orth Timothy J Mitchison
56177685
Involvement of C terminal truncation mutation of kinesin 5 in resistance to kinesin 5 inhibitor
Cultured cells easily develop resistance to kinesin 5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin 12/KIF15, or by acquiring mutations in the N terminal motor domain of kinesin 5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S trityl L cysteine (STLC) one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U 2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10 fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11 independent property of the cells derived from HOS, those derived from U 2OS still required KIF11 for their growth and, intriguingly, expressed a C terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs) All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U 2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C terminal truncated KIF11 contributes to the STLC resistance of the U 2OS derived cells.
PloS one
Eri Saeki Shinji Yasuhira Masahiko Shibazaki Hiroshi Tada Minoru Doita Tomoyuki Masuda Chihaya Maesawa
3978285
Kinesin 5 inhibitor resistance is driven by kinesin 12
The kinesin 5 Eg5 is essential for mitotic progression, and the lethal effects of Eg5 inhibitors make these inhibitors attractive candidates for chemotherapy drugs. Sturgill et al. show that kinesin 12 and a nonmotile Eg5 mutant form an alternative spindle assembly pathway that provides resistance to Eg5 inhibitors.
The Journal of cell biology
Emma G Sturgill Stephen R Norris Yan Guo Ryoma Ohi
23970554
Niche Based Screening in Multiple Myeloma Identifies a Kinesin 5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.
Novel therapeutic approaches are urgently required for multiple myeloma (MM) We used a phenotypic screening approach using co cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non competitive kinesin 5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5 microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.
Cell reports
Shrikanta Chattopadhyay Alison L Stewart Siddhartha Mukherjee Cherrie Huang Kimberly A Hartwell Peter G Miller Radhika Subramanian Leigh C Carmody Rushdia Z Yusuf David B Sykes Joshiawa Paulk Amedeo Vetere Sonia Vallet Loredana Santo Diana Cirstea Teru Hideshima Vlado Dancik Max M Majireck Mahmud M Hussain Shambhavi Singh Ryan Quiroz Jonathan Iaconelli Rakesh Karmacharya Nicola J Tolliday Paul A Clemons Malcolm A S Moore Andrew M Stern Alykhan F Shamji Benjamin L Ebert Todd R Golub Noopur S Raje David T Scadden Stuart L Schreiber
22573430
Rapid induction of apoptosis during Kinesin 5 inhibitor induced mitotic arrest in HL60 cells.
Small molecule inhibitors of Kinesin 5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti cancer drug candidates. Clinical trials of K5Is revealed dose limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl 2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (DPsm) before mitochondrial outer membrane permeabilization (MOMP) Interestingly, this loss of DPsm was not blocked by overexpressing Bcl 2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of DPsm as an upstream event of MOMP.
Cancer letters
Yangzhong Tang James D Orth Tiao Xie Timothy J Mitchison
9960383
Chromosome 20q Amplification Regulates in Vitro Response to Kinesin 5 Inhibitor
We identified gene expression signatures predicting responsiveness to a Kinesin 5 (KIF11) inhibitor (Kinesin 5i) in cultured colon tumor cell lines. Genes predicting resistance to Kinesin 5i were enriched for those from chromosome 20q, a region of frequent amplification in a number of tumor types. siRNAs targeting genes in this chromosomal region identified AURKA, TPX2 and MYBL2 as genes whose disruption enhances response to Kinesin 5i. Taken together, our results show functional interaction between these genes, and suggest that their overexpression is involved in resistance to Kinesin 5i. Furthermore, our results suggest that patients whose tumors overexpress AURKA due to amplification of 20q will more likely resist treatment with Kinesin 5 inhibitor, and that inactivation of AURKA may sensitize these patients to treatment.
Cancer informatics
Aimee L Jackson Mao Mao Sumire V Kobayashi Teresa Ward Matthew C Biery Hongyue Dai Steven R Bartz Peter S Linsley
211245592
Structure of Microtubule Trapped Human Kinesin 5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy
Summary Kinesin 5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin 5 inhibitor GSK 1, which allosterically promotes tight microtubule binding. GSK 1 inhibits monomeric human kinesin 5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule bound motor GSK 1 at 3.8 A overall resolution. The structure reveals that GSK 1 stabilizes the microtubule binding surface of the motor in an ATP like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK 1 is located between helix a4 and helix a6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein ligand docking, we characterized the kinesin 5 GSK 1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.
Structure
Alejandro Pena Aaron Sweeney Alexander D Cook Julia Locke Maya Topf Carolyn Ann Moores