Daniel Lichtensztejn

The 3D nuclear organization of telomeres marks the transition from Hodgkin to Reed-Sternberg cells

To get an insight into the transition from mononuclear Hodgkin cells (H cells) to diagnostic multinuclear Reed–Sternberg cells (RS cells), we performed an analysis of the three-dimensional (3D) structure of the telomeres in the nuclei of the Hodgkin cell lines HDLM-2, L-428, L-1236 and lymph node biopsies of patients with Hodgkins disease. Cellular localization of key proteins of the telomere-localized shelterin complex, the mitotic spindle and double-stranded DNA breaks was also analyzed. RS cells show significantly shorter and significantly fewer telomeres in relation to the total nuclear volume when compared with H cells; in particular, telomere-poor ‘ghost’ nuclei are often adjacent to one or two nuclei displaying huge telomeric aggregates. Shelterin proteins are mainly cytoplasmic in both H and RS cells, whereas double-stranded DNA breaks accumulate in the nuclei of RS cells. In RS cells, multipolar spindles prevent proper chromosome segregation. In conclusion, a process of nuclear disorganization seems to initiate in H cells and further progresses when the cells turn into RS cells and become end-stage tumor cells, unable to divide further because of telomere loss, shortening and aggregate formation, extensive DNA damage and aberrant mitotic spindles that may no longer sustain chromosome segregation. Our findings allow a mechanistic 3D understanding of the transition of H to RS cells.

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost’ nuclei and very short telomeres

In Epstein–Barr virus (EBV) negative Hodgkins cell lines and classical EBV-negative Hodgkins lymphoma (HL), Reed–Sternberg cells (RS cells) represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. However, the three-dimensional (3D) telomere organization in latent membrane protein 1 (LMP1)-expressing RS cells of EBV-associated HL is not known. We performed a 3D telomere analysis after quantitative fluorescent in situ hybridization on 5 μm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (P<0.0001) and formation of telomere aggregates in RS cells (P<0.0001), when compared with the mononuclear precursor Hodgkin cells (H cells). Telomere-poor or telomere-free ‘ghost’ nuclei were a regular finding in these RS cells. These nuclei and their telomere content strongly contrasted with the corona of surrounding lymphocytes showing numerous midsized telomere hybridization signals. Both H cells and RS cells of two EBV-negative HL cases analyzed in parallel showed 3D telomere patterns identical to those of LMP1-expressing cases. As a major advance, our 3D nuclear imaging approach allows the visualization of hitherto unknown profound changes in the 3D nuclear telomere organization associated with the transition from LMP1-positive H cells to LMP1-positive RS cells. We conclude that RS cells irrespective of LMP1 expression are end-stage tumor cells in which the extent of their inability to divide further is proportional to the increase of very short telomeres, telomere loss, aggregate formation and the generation of ‘ghost’ nuclei.

Different TP53 mutations are associated with specific chromosomal rearrangements, telomere length changes, and remodeling of the nuclear architecture of telomeres

TP53 mutations are the most common mutations in human cancers, and TP53‐R175H and TP53‐R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53‐V143A, TP53‐R175H, and TP53‐R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three‐dimensional (3D) quantitative FISH (Q‐FISH) on interphase and Q‐FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53‐R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53‐R175H and TP53‐V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53‐R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53‐R175H. Taken together, our results indicate that the mutation TP53‐R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major determinants of the TP53‐dependent genomic instability.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation

Upon mitogenic induction of immediate-early genes, phosphorylation of histone H3 at S10 or S28 occurs on different alleles. S28ph depends on CBP/p300-mediated K27ac, whereas H3 acetylated on K9 by PCAF is phosphorylated on S10. The redundant roles of S10ph and S28ph and their random targeting on distinct alleles may enable a fast response.

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.