Expression of Aurora-A, -B and -C
First, we assessed expression (,) and differential expression (Supplementary Table S3A,B) of Aurora-A, -B, and -C in primary myeloma cells, normal bone marrow plasma cells, their precursors, as well as normal and myelomatous bone marrow. In our data set, Aurora-A and -B are expressed in 24 % and 3 % of primary myeloma cells and all PPC as well as HMCL. In the Arkansas-data, Aurora-A, -B, and -C are expressed in 48/345 (14 %), 12/345, (3 %) and 0/345 myeloma cell-samples, respectively. The mean expression of Aurora-A and -B is significantly and by several orders of magnitude higher in proliferating plasmablastic cells and cell lines compared to non-proliferating MBC, or BMPC (each P<.001 in TG and VG; Supplementary Table S3A,B, ). Aurora-A and -B are expressed in almost all bone marrow samples of healthy individuals and myeloma patients (). Here, the mean expression of Aurora-B is significantly different (lower) in myelomatous compared to normal bone marrow (4.85 vs. 3.81 arbitrary units, P=.009). A significant stage dependent differential gene expression could be found for Aurora-A between myeloma cells from early- (MGUS and MMI) and advanced stage (MMII and MMIII) patients (P=.01 and .01, in TG and VG, respectively). Aurora-A and -B expression correlates significantly in the VG (rs=0.59, P<.001) and Arkansas-group (rs=0.61, P<.001).
Presence of expression of Aurora-kinase A (AURKA), -B (AURKB), -C (AURKC) as judged by PANP
Presence of expression of Aurora-kinase A (AURKA), -B (AURKB), -C (AURKC) as judged by PANP
Expression of Aurora-A (AURKA), -B (AURKB), and -C (AURKC) as determined by gene expression profiling
Validation of gene expression by qRT-PCR, western blotting and flow cytometry
To validate Aurora-kinase expression detected by gene expression profiling, we performed qRT-PCR, western blotting and flow-cytometric staining. Aurora-A expression in terms of “presence” or “absence” (Ct-value ≥ 35) by qRT-PCR is consistent with results by PANP in 10/11 primary myeloma cell-samples. One sample “absent” by qRT-PCR is judged “marginal” by PANP. Aurora-A expression by GEP strongly correlates with dCt value by qRT-PCR (rs= −0.87, P<.001). Aurora-B expression is consistent with results by PANP in 3/6 samples. All samples are “present” by qRT-PCR but three are judged “absent” by PANP. Aurora-B expression by GEP strongly correlates with dCt value by qRT-PCR (rs= −0.83, P=.06). Aurora-C expression by qRT-PCR is consistent with absence of expression detected by PANP in 5/6 samples. One sample “present” by qRT-PCR (Ct-value 34.8) is judged “absent” by PANP. Aurora-C expression by GEP strongly correlates with the dCt value obtained by qRT-PCR (rs= −0.94, P=.02). Aurora-A and -B expression in HMCL was further validated by western blotting () and intracellular flow cytometry (exemplary data shown in ).
Validation of gene expression by quantitative real-time PCR, western blotting and flow cytometry
Association of Aurora-kinase expression with proliferation and chromosomal aberrations
To investigate the biological impact of Aurora-kinase expression, we assessed the association with proliferation, chromosomal aberrations and presence of subclonal aberrations as detected by iFISH, and a published centrosome-index 49
The expression of Aurora-A correlates with proliferation in terms of the plasma cell labeling index assessed by PI staining (n=66, rs=0.45, P<.001) and the gene expression based proliferation index in TG (rs=0.62, P<.001) and VG (rs=0.87, P<.001). The same holds true for the latter for Aurora-B in the VG (rs=0.64, P<.001).
The absolute number of chromosomal aberrations is not significantly different between myeloma cells expressing or not expressing Aurora-A (mean 5.1 and 5.1, n=105 patients, n=8 FISH-probes tested). Presence/absence of Aurora-A expression does not significantly interrelate to the presence/absence of hyperdiploidy as determined by either CS or CSW, neither does the presence/absence of Aurora-A expression interrelate to the presence of any of the single aberrations t(11;14), t(4;14), or numerical aberrations of 17p13, 9q34, 15q22, 19q13, 4p16, 14q32 or 22q11. Interestingly, in patients with presence of Aurora-A expression, gains of 11q13 (P=.03) and 11q23 (P=.004) are significantly less frequent compared to those with absent Aurora-A expression. The opposite holds true for patients with gain of 1q21 (P=.002) or deletion of 13q14 (P=.03) as well as deletion of 8p21 (P=.03): MMC of patients with present Aurora-A expression show a significantly higher number of these respective aberrations. For a gain 1q21, for which data were also available for the Arkansas-group, the same observation was made (n=244, P<.001). However, subclonal aberrations per se are significantly more frequent in MMC with absent Aurora-A expression (67 with vs. 37 without) compared to present Aurora-A expression (18 vs. 23, P =.03). For single aberrations, subclonal presence (vs. full-clonal or absence of the respective aberration) is significantly more frequent in MMC of patients with absence of Aurora-A expression for gains of 11q13 (P<.03), 11q23 (P<.001), and losses of 13q14 (P=.009). Losses of 17p13 marginally fail significance (P=.06). Losses of 8p21 are significantly (P=.04) more frequent in patients with presence of Aurora-A expression.
The centrosome-index correlates with Aurora A-expression in our series (TG r=.53, P<.001; VG r=.32, P<.001) and the Arkansas-data (r=.43, P<.001). The centrosome-index is significantly predictive for EFS and OAS in the Arkansas-group (EFS P=.005, OAS, P<.001) on which it has been derived, but not our series (EFS P=.09, OAS, P=.99; Supplementary Figure S1).
Prognostic value of Aurora-kinase-expression
Next, we investigated whether the presence of Aurora-kinase expression has a prognostic impact in newly-diagnosed myeloma patients treated with HDT and ASCT.
Presence of Aurora-A expression in MMC is an adverse prognostic factor in terms of EFS and OAS in our data (n=168, EFS P=.003, hazard ratio (HR) 2.02, confidence interval (CI) [1.26,3.25], OAS, P=.03, HR 2.31, CI [1.04,5.15]) and the Arkansas-group (n=345, P=.004, HR 1.74, CI [1.18,2.57], OAS, P=.008, HR 1.85, CI [1.16,2.95]), . The expression-signal of Aurora-A as single continuous variable is significantly predictive for EFS in the VG (n=120, P<.001) and the Arkansas-group (P<.001). The same holds true for OAS in the Arkansas-group (P<.001) and it marginally failed significance for our VG (n=120, P=.06).
Prognostic relevance of Aurora-A expression for two independent groups of patients treated with high-dose therapy and autologous stem cell transplantation
In a Cox-model tested with the international staging system (ISS), presence of Aurora-A expression appears as independent prognostic factor for EFS in our data (Aurora A-expression (P=.003), ISS (P=.009)), and the Arkansas-data (Aurora-A expression (P<.001), ISS (P<.001)). For OAS, Aurora-A expression marginally fails independence (P=.08), ISS (P=.009)) in our data-set, but is significantly independent in the Arkansas-data (Aurora-A expression (P<.001), ISS (P<.001)).
In a Cox-model tested with serum-β2-microglobulin (B2M) as continuous variable, presence of Aurora-A expression appears as independent prognostic factor for EFS in our data (Aurora-A expression (P=.002), B2M (P=.006)), and the Arkansas-data (Aurora-A expression (P<.001), B2M (P<.001)), and OAS (Aurora-A expression (P=.06), B2M (P<.001)), and the Arkansas-data (Aurora-A expression (P<.001), B2M (P<.001)).
Activity of VX680 on myeloma cell lines and primary myeloma cells
Given the expression and prognostic value of Aurora-kinases, we tested the activity of the pan- Aurora-kinase inhibitor VX680 that has previously shown activity on a small series of human myeloma cells, on a large series of 20 myeloma cell lines. VX680 significantly inhibits proliferation of all HMCL investigated (). The maximum inhibition at 10 μM ranges from 64.4 % (HG-1) to 100 % (OPM-2). The concentration to reduce proliferation to half of the control value (IC50) is reached in all myeloma cell lines; it ranges from 0.003–2.715 μM, . No significant correlation could be found between the expression of Aurora-A, -B or hyaluron mediated motility receptor (HMMR) and the IC50 of 12 myeloma cell lines tested (see discussion).
Inhibition of proliferation of myeloma cell lines as well as survival of primary myeloma cells and cells of the bone marrow microenvironment
VX680 significantly inhibits the survival of primary myeloma cells cultivated within their bone marrow microenvironment from 5/5 newly-diagnosed myeloma patients at a concentration of 4 μM (50 - 100 % inhibition, ). At the same dose level, VX680 induces significant but lower toxicity within the bone marrow microenvironment (). Four of four samples, for which sufficient RNA was available, showed an expression of Aurora-A by qRT-PCR.
Next, XG-1 and XG-10 were cultured for 3 days with or without VX680. Cell viability and apoptosis were determined by flow-cytometric analysis of annexin V-binding and PI uptake after 8, 24, 48 and 72 h. Exposure of XG-1 and XG-10 against 1 μM VX680 induces apoptosis after 8 h (XG-1: 23.5 % vs. 24.1 %, XG-10: 16.4 % vs. 13.3 %) to 72 h (XG-1: 17.9 % vs. 85.5 %, XG-10: 9.0 % vs. 49.9 %), , exemplary data shown for XG-10.