XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc.

Translocation frequencies and chromosomal proximities for selected mouse chromosomes in primary B lymphocytes.

Chromosome positions within the nucleus of mammalian cells are nonrandom and it is assumed that chromosomal neighborhoods affect the probability of translocations. Four chromosomes can be involved in c-myc-activating chromosomal translocations in mouse plasmacytoma (PCT): the c-myc gene on mouse chromosome 15 can be juxtaposed to either one of the immunoglobulin (Ig) loci on chromosomes 12 (IgH), 16 (Igλ), or 6 (Igκ). In the BALB/c mouse, the translocation between chromosomes 12 and 15, T(12;15), is most common (90%) while the other two possible translocations, T(6;15) and T(16;15), are much less common (<10%). In contrast, in the BALB/cRb6.15 mouse, T(6;15) is found with the same frequency as T(12;15). We, therefore, examined the distance between chromosomes 15 and 12, 6, and 16 in primary mouse B lymphocytes in order to examine the effect of the chromosome proximity on the translocation frequency. We performed three-dimensional fluorescent in situ hybridization (3D-FISH) with chromosome paints. We acquired three-dimensional image stacks with 90 slices per stack and used constrained iterative deconvolution. The nucleus and chromosomes were segmented from this image stack and the interchromosomal distances were measured. Chromosomes 6 and 15 were found in close proximity in BALB/cRb6.15 mice (82%), whereas they did not share this neighborhood relationship in BALB/c mice. No other chromosome combinations showed such a high percentage of close proximities in either mouse strain. Chromosome positions contribute to translocation frequencies in mouse PCTs. The BALB/cRb6.15 mouse data argue for a proximity relationship of chromosomes that engage in illegitimate recombination. These positions are not, however, the only contributing factor as the T(12;15) translocation preference in BALB/c mice could not be supported by significantly elevated proximity of chromosomes 12 and 15 versus 12 and 16 or 12 and 6. Moreover, while there is a significant increase in T(6;15) in BALB/cRb6.15 mice, T(12;15) still occurs in this mouse strain.

c-Myc-induced extrachromosomal elements carry active chromatin.

Murine Pre-B lymphocytes with experimentally activated MycER show both chromosomal and extrachromosomal gene amplification. In this report, we have elucidated the size, structure, and functional components of c-Myc-induced extrachromosomal elements (EEs). Scanning electron microscopy revealed that EEs isolated from MycER-activated Pre-B+ cells are an average of 10 times larger than EEs isolated from non-MycER-activated control Pre-B- cells. We demonstrate that these large c-Myc-induced EEs are associated with histone proteins, whereas EEs of non-MycER-activated Pre B- cells are not. Immunohistochemistry and Western blot analyses using pan-histone-specific, histone H3 phosphorylation-specific, and histone H4 acetylation-specific antibodies indicate that a significant proportion of EEs analyzed from MycER-activated cells harbors transcriptionally competent and/or active chromatin. Moreover, these large, c-Myc-induced EEs carry genes. Whereas the total genetic make-up of these c-Myc-induced EEs is unknown, we found that 30.2% of them contain the dihydrofolate reductase (DHFR) gene, whereas cyclin C (CCNC) was absent. In addition, 50% of these c-Myc-activated Pre-B+ EEs incorporated bromodeoxyuridine (BrdU), identifying them as genetic structures that self-propagate. In contrast, EEs isolated from non-Myc-activated cells neither carry the DHFR gene nor incorporate BrdU, suggesting that c-Myc deregulation generates a new class of EEs.