Low Miki Expression in Myelodysplastic Syndromes Results in Polynuclear Cell Formation through Decondensation of Chromosomes in Prolonged Prometaphase

Polynuclear cells (PNCs) are routinely observed in the bone marrow of MDS patients.  They are binuclear, trinuclear or even multinuclear cells with or without micronuclei, the underlying molecular mechanisms for the production of which are largely unknown.  Because loss of the long arm of chromosome 7 (7q-) was reported to be associated with the presence of a higher frequency of PNCs, gene(s) preventing bone marrow cells from carrying such nuclear abnormalities may be located at 7q.

            We previously identified three candidate anti-myeloid tumor suppressor genes, namely Samd9, Samd9L and Miki, from the microdeletion in the 7q21 band frequently detected in JMML patients.  SAMD9L-deficient mice develop MDS resembling human diseases associated with 7q-, most likely through enhancement of cytokine signals (Nagamachi et al., Cancer Cell 2013).  

            Miki (mitotic kinetics regulator) translocates from the Golgi apparatus to mitotic centrosomes coincident with the disappearance of the Golgi body after poly-ADP-ribosylation (PARsylation).  Miki is indispensable for centrosome maturation [the rapid increase of pericentriolar materials (PCM) during prophase and prometaphase], which is required for the production of robust mitotic spindles to move chromosomes promptly (Ozaki et al., Mol. Cell 2012).  Consequently, as observed by time-lapse imaging of HeLa cells expressing histone H2A-GFP, downregulation of Miki by siRNA markedly prolonged the duration of prometaphase to more than several hours (normally around 15 minutes).  Chromosomes were scarcely able to align and cells exited from prometaphase either by cell death or by decondensation of each chromosome.  In the latter, cells with decondensed chromosomes then fused with one another within 30 minutes to form cells with relatively large nuclei, resulting in PNCs containing various sizes of nuclei including micronuclei.  Indeed, reduction of Miki in HeLa cells by siRNA increased the frequency of PNCs from less than 0.5% to 4.5%. 

            To test whether the chaotic chromosome decondensation in prometaphase causes the accumulation of PNCs observed in MDS, we initially used five cell lines derived from MDS associated with 7q-.  PARsylated Miki was barely detectable in these cell lines and we found more cells at prometaphase than at metaphase (the ratio of prometa:meta in the lines ranged from 1.7:1 to 5.7:1).  In contrast, in seven cell lines expressing PARsylated Miki at high levels, mitotic cells in prometaphase were found less frequently or at roughly the same frequency as those in metaphase (prometa:meta ratio 0.6:1 to 1.3:1).  PNCs in five cell lines harboring 7q- were also more frequent (5.9 - 10.2%) than in the seven cell lines expressing high PARsylated Miki (0.8 - 2.4%).  In addition, when we reduced Miki expression levels by shRNA in K562 cells, which express PARsylated Miki at high levels, the prometa:meta ratio increased from 1.1:1 to 3.8:1 and PNCs increased from 0.8% to 8.5%. This suggests that, as in HeLa cells, low expression levels of Miki cause prolongation of prometaphase and increase PNCs in blood cells. 

            Fresh bone marrow preparations from 37 patients with MDS were examined to determine whether Miki mRNA-expression levels influence the prometaphase:metaphase ratio and the frequency of PNCs.  We found a strong negative correlation (R=-0.59, p<0.01) between Miki mRNA expression levels in mononuclear cells of bone marrow samples and the prometa:meta ratio.  We also found a moderate negative correlation (R=-0.4, p<0.05) between PNC frequencies and Miki mRNA expression levels.  In addition, there was a strong positive correlation between prometa:meta ratios and PNC frequencies (R=-0.56, P<0.01). 

            In conclusion, lack of one allele of the Miki gene due to 7q- reduces PARsylated Miki, resulting in the increase of PNCs through decondensation of chromosomes in prolonged prometaphase.  This may contribute to poor outcome of MDS associated with 7q- through increased chromosome instability.