P-014: Disruption of chromosome territories in Multiple Myeloma

Abstract

<h3>Background</h3> Multiple Myeloma (MM) is the second most frequent hematologic neoplasm worldwide. MM is currently an incurable disease. MM displays an irregular pattern of gene expression, chromosome alterations and changes in chromosome positioning (relatively to normal plasma cells), which contributes to genomic instability and the heterogeneous phenotype of individual cells observed in MM. Lamin A/C is a nuclear protein that has been shown to play a critical role in the maintenance of genomic stability and nuclear genome architecture. Recently, our group observed that the expression of lamin A/C was upregulated in MM human cell line (MM.1R) and, importantly, in ten primary treatment-naive MM patient samples. Moreover, the lamin A/C protein was found to exhibit an altered 3D spatial organization. Objective: To investigate the role of lamin A/C on nuclear chromosome organization in MM. <h3>Methods</h3> The expression of lamin A/C in two MM cell lines (MM1R and RPMI 8226) was confirmed by western blot (WB) and quantitative immunofluorescence (qIF) analysis. qIF confirmed aberrant 3D lamin A/C protein organization. To evaluate changes in CT, we used whole chromosome painting probes and 3D fluorescence in situ hybridization (FISH) followed by our published quantitative analysis (ChromoView) for chromosomes 4, 9, 11, 14, 16, 18, 19 and 22. We observed differences in CT of chromosomes 9, 16, 18 and 22 towards the nuclear center, CT-19 towards the nuclear periphery in MM.1R and CT-4,9,11,14,18,19, and 22 towards the nuclear periphery in RPMI 8226, all compared to control (B-lymphocytes). To evaluate the role of lamin A/C in affecting CT positions, we downregulated lamin A/C protein levels using two different short interfering RNAs (siRNA), for different regions of lamin A/C mRNA, as well as scrambled siRNA. <h3>Results</h3> Following siRNA but not scrambled siRNA, WB and quantitative image analysis showed for RPMI8226 that lamin A/C protein level was reduced up to ~25% of the endogenous levels at 72-96 hours after transfection, using both siRNAs separately and in combination. After siRNA treatment, we evaluated the effects of lamin A/C downregulation in chromosome positioning. The CT analysis revealed changes in CT4,9,11,14,16,18, and 22 towards the nuclear centre and CT-19 towards the nuclear periphery (p<0.05). Interestingly, we found that during lamin A/C downregulation, CT-11,18 and 22 positions in RPMI 8226 returned to the same nuclear distance as observed in lymphocytes. <h3>Conclusions</h3> Lamin A/C plays a role in genome organization and chromosome positioning. Its disruption alters chromosome positions as described here for the first time. Future investigations will clarify whether these changes affect cell viability, which would suggest that lamin A/C disruption could, in the future, be explored therapeutically for MM.