Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using timelapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G 1 , with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype. [Mol Cancer Ther 2008;7(11):3480 -9]

RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry–based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9–driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

Cancer cells can be drug resistant due to genetic variation at multiple steps in the drug response pathway, including drug efflux pumping, target mutation and blunted apoptotic response. These are not discriminated by conventional cell survival assays. Here, we report a rapid and convenient high content cell-imaging assay that measures multiple physiological changes in cells responding to anti-mitotic small-molecule drugs. Our one-step, no-wash assay uses three dyes to stain living cells and is much more accurate for scoring weakly adherent mitotic and apoptotic cells than conventional antibody-based assays. We profiled responses of 33 cell lines to 8 anti-mitotic drugs at multiple concentrations and time points using this assay, and deposited our data and assay protocols into a public database (http://lincs.hms.harvard.edu/). Our data discriminated between alternative mechanisms that compromise drug sensitivity to Paclitaxel, and revealed an unexpected bell-shaped dose-response curve for BI2536, a highly selective inhibitor of Polo-like kinases. Our approach can be generalized, is scalable and should therefore facilitate identification of molecular biomarkers for mechanisms of drug insensitivity in high-throughput screens and other assays.