Targeting aurora kinases as therapy in multiple myeloma - PubMed (original) (raw)

Clinical Trial

Targeting aurora kinases as therapy in multiple myeloma

Yijiang Shi et al. Blood. 2007.

Abstract

The aurora kinases facilitate transit from G2 through cytokinesis and, thus, are targets in cancer therapy. Multiple myeloma (MM) is a malignancy characterized by genetic instability, suggesting a disruption of checkpoints that arrest cells at G2M when injury to the mitotic machinery occurs. Since deficient checkpoints would prevent cell cycle arrest and may render cells susceptible to apoptosis in mitosis and since aurora kinases are intermediaries in checkpoint pathways, we tested antimyeloma effects of 2 agents that inhibit aurora kinases. Both inhibited growth of MM lines and primary myeloma samples at nanomolar concentrations while having less of an effect on proliferating lymphocytes and hematopoietic cells. MM cells were not protected by IL-6 or activating mutations of Ras. Antimyeloma effects included induction of tetraploidy followed by apoptosis. Apoptosis correlated with inhibition of aurora activity as shown by reduction of histone 3B phosphorylation. Ectopic expression of aurora A protected MM cells against aurora inhibitors but had no effect on apoptosis induced by bortezomib. As expression of RHAMM in MM contributes to genetic instability, we tested effects of RHAMM. RHAMM overexpression enhanced sensitivity to apoptosis and RHAMM silencing decreased sensitivity. These results suggest potential for aurora kinase inhibitors in MM especially in patients in whom RHAMM is overexpressed.

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Figures

Figure 1

Inhibitory effect of 2 aurora kinase inhibitors against growth of multiple myeloma cells. (A) MM cell lines were exposed to increasing concentrations (shown in key at top of figure) of either VX-680 or ZK inhibitors for 72 hours, after which MTT assays were performed as well as trypan blue exclusion. Bar data are percent inhibition of growth in MTT assays (vs vehicle alone; ie, no VX-680 or ZK), mean of 3 separate experiments where SDs were all less than 5% of the mean. Numbers at the tops of the bars represent percent of cells nonviable above control baseline nonviable percent (5%-15%, mean of 3 experiments). (B) Cells were cultured for 72 hours with increasing concentrations of VX-680 (at 50, 100, and 500 nM with symbols designated as in A), after which trypan blue exclusion was used to assess viable cell yield. Data are percent inhibition of viable cell yield (vs vehicle control), mean of 3 separate experiments. Cell targets are ANBL-6 MM cells, ANBL-6 cells first treated with 1000 U/mL recombinant IL-6 (ANBL-6 + IL-6), ANBL-6 cells stably transfected with an activated N-RAS allele, primary myeloma cells obtained from 2 MM patients (PT #1 and PT #2), peripheral blood lymphocytes (PBLs) from 3 healthy donors (data are means of 3 assays), and isolated B-cell CLL cells from 3 leukemic patients (data are means of 3 separate assays).

Figure 2

Induction of tetraploidy and apoptosis in MM cells. (A) Cell cycle analysis (propidium iodide staining) was performed in OPM-2 and MM1.S MM cell lines after 48-hour exposure to vehicle alone (control), ZK (500 nM), or VX-680 (100 nM). Induction of tetraploidy is shown. (B) Similarly treated OPM-2 and MM1.S cells were assayed at 72 hours for apoptosis by flow cytometric analysis of activated caspase 3 expression. Results are mean percent apoptosis ± SD of 3 separate experiments (control cells treated with vehicle alone showed a mean of 16% positive staining with the caspase 3 antibody).

Figure 3

Effect of inhibitors on histone 3B phosphorylation. (A) OPM-2 or MM1.S cells were exposed to increasing concentrations of the ZK inhibitor, after which immunoblot was performed for expression of total histone 3B or phosphorylated histone 3B (on serine 10). (B-C) Similar immunoblot experiments were performed on OPM-2 and MM1.S cells exposed to increasing concentrations of VX-680. The experiments depicted in panels A-C were repeated 3 times with identical results.

Figure 4

Ectopic aurora A expression inhibits antimyeloma effect of aurora inhibitors. OPM-2 cells were transfected with aurora A (Aur A) or empty vector (EV); cells were selected in vitro and then synchronized by double thymidine block. Four hours after release of the block, lysates were immunoblotted for aurora A expression as well as total histone 3B and phosphorylated histone (on serine 10) as shown in panel A. (B-C) Isogenic transfected cells were treated with increasing concentrations of either ZK (B) or VX-680 (C) and similarly synchronized and immunoblotted for total histone or phosphorylated histone. (D) Aurora A–transfected cells (■) or empty vector control cells (EV, □) were exposed to increasing concentrations of VX-680, ZK, or bortezomib (PS-341) for 48 hours, after which percent inhibition of growth was determined in MTT assays. Results are means of 3 experiments. The percent inhibition induced by VX-680 and ZK was significantly less (P < .05) in aurora A–transfected cells treated with 25, 50, and 200 nM VX-680 and 500 and 1000 nM ZK.

Figure 5

Effect of ectopic RHAMM expression. U266 MM cells were transfected with a RHAMM-EGFP construct (■) or control empty vector (EV, □). (A) Western assay of transfected isogenic cells synchronized by double thymidine block and then immunoblotted for expression of RHAMM, actin, or EGFP. (B) The transfected cells were exposed to increasing concentrations of VX-680 for 72 hours and then assessed for percent inhibition of growth in MTT assays (left panel) and percent apoptosis (right panel, flow cytometric analysis of activated caspase 3 expression). Dark bars are RHAMM transfectants, and open bars are empty vector control cells. Results are means of 3 separate experiments where the SDs were all less than 5% of the mean. Percent of cells demonstrating tetraploidy are shown on top of bars of right panel. The percent inhibition of growth and percent induced apoptosis was significantly greater (P < .05) for RHAMM-transfected cells at all doses of VX-680.

Figure 6

Effect of RHAMM silencing. AF-10 cells were infected with lentivirus expressing shRNA for RHAMM or a scrambled sequence (Control). After selection of transduced clones in blasticidin, the isogenic cell lines were first immunoblotted for expression of endogenous RHAMM (top). Immunoblot was performed 6 hours after synchronizing cells as described in Figure 5. Isogenic cell lines were then exposed to 0, 100, or 200 nM VX-680 for 72 hours, after which percent inhibition was calculated from growth assays and percent apoptosis was assayed by flow cytometric analysis of activated caspase 3 expression (bottom). Results are means of 3 experiments where SDs were less than 5% of the mean for all groups. The degree of percent inhibition and apoptosis in shRNA-expressing cells (■) was significantly less (P < .05) than that of control cells (□).

References

1. Andrews PD, Knatko E, Moore WJ, Swedlow JR. Mitotic mechanics: the auroras come into view. Curr Opin Cell Biol. 2003;15:672–683. - PubMed
1. Katayama H, Brinkley WR, Sen S. The Aurora kinases: Role in cell transformation and tumorigenesis. Cancer Metastasis Rev. 2003;22:451–464. - PubMed
1. Ewart-Toland A, Briassoului P, de Koning JP, et al. Identification of Stk6/STK15 as a candidate low-penetrance tumor susceptibility gene in mouse and human. Nat Genetics. 2003;34:403–412. - PubMed
1. Hirota T, Kunitoku N, Sasayama T, et al. Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell. 2003;114:585–598. - PubMed
1. Dutertre S, Descamps S, Prigent C. On the role of aurora-A in centrosome function. Oncogene. 2002;21:6175–6183. - PubMed

Targeting aurora kinases as therapy in multiple myeloma - PubMed (original) (raw)