Three-dimensional telomere profiling predicts risk of progression in smoldering multiple myeloma.

Smoldering multiple myeloma (SMM) is a precursor stage that precedes multiple myeloma (MM). SMM is heterogenous with nearly 40% of patients progressing to MM in the first 5 years. The high rate of progression of SMM patients highlights the need for early intervention, which underscores the importance of identifying SMM patients with the highest risk of progression. Several risk stratification models showed utility in identifying high-risk SMM patients; however, these systems showed limited sensitivity. To date, identifying high-risk SMM patients remains an important clinical need. In this study, we present the 3-dimensional telomere profiling as a structural biomarker capable of stratifying SMM patients as a function of genomic instability. Quantifying telomere dysfunction using the TeloView technology showed utility in risk stratification of cancer patients, particularly hematological malignancies. In this study, we analyzed 168 SMM patients. We report an AUC in ROC analysis of 0.8 using a subset of the patients as a training dataset. We then conducted a blind validation on a different cohort and demonstrated a positive predictive value of 85% and negative predictive value of 73%, with sensitivity and specificity of 83% and 76%, respectively. We examined the correlation between the TeloView prediction and the 20-2-20 scoring system, and cytogenetic abnormalities. We report a correlation of 53% with the 20-2-20 scores and over 60% correlation with cytogenetic abnormalities. The result of this study presents the telomere profiling as an effective biomarker able to stratify SMM patients to their respective risk groups with high sensitivity and specificity.

Three-dimensional nuclear architecture distinguishes thyroid cancer histotypes.

Molecular markers can serve as diagnostic tools to support pathological analysis in thyroid neoplasms. However, because the same markers can be observed in some benign thyroid lesions, additional approaches are necessary to differentiate thyroid tumor subtypes, prevent overtreatment and tailor specific clinical management. This applies particularly to the recently described variant of thyroid cancer referred to as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). This variant has an estimated prevalence of 4.4% to 9.1% of all papillary thyroid carcinomas worldwide. We studied 60 thyroid lesions: 20 classical papillary thyroid carcinoma (CPTC), 20 follicular variant of PTC (FVPTC) and 20 NIFTP. We examined morphological and molecular features to identify parameters that can differentiate NIFTP from the other PTC subtypes. When blindly investigating the nuclear architecture of thyroid neoplasms, we observed that NIFTP has significantly longer telomeres than CPTC and FVPTC. Super-resolved 3D-structured illumination microscopy demonstrated that NIFTP is heterogeneous and that its nuclei contain more densely packed DNA and smaller interchromatin spaces than CPTC and FVPTC, a pattern that resembles normal thyroid tissue. These data are consistent with the observed indolent biological behavior and favorable prognosis associated with NIFTP, which lacks BRAFV600E mutations. Of note, next-generation thyroid oncopanel sequencing was unable to distinguish the thyroid cancer histotypes in our study cohort. In summary, our data suggest that 3D nuclear architecture can be a powerful analytical tool to diagnose and guide clinical management of NIFTP.

Three-dimensional nuclear telomere architecture and differential expression of aurora kinase genes in chronic myeloid leukemia to measure cell transformation.

BACKGROUND: Telomere dysfunction results in aneuploidy, and ongoing chromosomal abnormalities. The three-dimensional (3D) nuclear organization of telomeres allows for a distinction between normal and tumor cells. On the other hand, aurora kinase genes (AURKA and AURKB) play an important role regulating the cell cycle. A correlation between overexpression of aurora kinase genes and clinical aggressiveness has been demonstrated in different types of neoplasias. To better understand cellular and molecular mechanisms of CML evolution, it was examined telomere dysfunction (alterations in the 3D nuclear telomere architecture), and the expression levels of AURKA and AURKB genes in two clinical distinct subgroups of CML samples, from the same patient. METHODS: Eighteen CML patients, in total, 36 bone marrow samples (18 patients, chronic vs. accelerated/blast phase) were eligible for 3D telomeric investigations. Quantitative 3D imaging, cytologic diagnosis and cytogenetic determination of additional chromosomal abnormalities were assessed according to standard protocols. RESULTS: Using TeloView software, two CML subgroups were defined based on their 3D telomeric profiles, reflecting the different stages of the disease (chronic vs. accelerated/blast phase). Statistical analyses showed significant differences between the CML subgroups (p < 0.001). We also found that AURKA and AURKB mRNA were expressed at significantly higher levels in both CML subgroups, when compared with healthy donors. Our findings suggest that the evolution of CML progresses from a low to a high level of telomere dysfunction, that is, from an early stage to a more aggressive stage, followed by disease transformation, as demonstrated by telomere, additional chromosomal abnormalities, and gene expression profile dynamics. CONCLUSIONS: Thus, we demonstrated that 3D telomere organization, in accordance with the genomic instability observed in CML samples were able to distinguish subgroup CML patients. Classifying CML patients based on these characteristics might represent an important strategy to define better therapeutic strategies.

Three-Dimensional Nuclear Telomere Profiling as a Biomarker for Recurrence in Oligodendrogliomas: A Pilot Study.

Mechanisms of recurrence in oligodendrogliomas are poorly understood. Recurrence might be driven by telomere dysfunction-mediated genomic instability. In a pilot study, we investigated ten patients with oligodendrogliomas at the time of diagnosis (first surgery) and after recurrence (second surgery) using three-dimensional nuclear telomere analysis performed with quantitative software TeloView® (Telo Genomics Corp, Toronto, Ontario, Canada). 1p/19q deletion status of each patient was determined by fluorescent in situ hybridization on touch preparation slides. We found that a very specific 3D telomeric profile was associated with two pathways of recurrence in oligodendrogliomas independent of their 1p/19q status: a first group of 8 patients displayed significantly different 3D telomere profiles between both surgeries (p < 0.0001). Their recurrence happened at a mean of 231.375 ± 117.42 days and a median time to progression (TTP) of 239 days, a period defined as short-term recurrence; and a second group of three patients displayed identical 3D telomere profiles between both surgery samples (p > 0.05). Their recurrence happened at a mean of 960.666 ± 86.19 days and a median TTP of 930 days, a period defined as long-term recurrence. Our results suggest a potential link between nuclear telomere architecture and telomere dysfunction with time to recurrence in oligodendrogliomas, independently of the 1p/19q status.

The three-dimensional cancer nucleus.

Research into the three-dimensional (3D) organization of the cancer cell genome started over 100 years ago. We follow an exciting avenue of research in this field, from Hansemann's early observations of aberrant mitoses and nuclei in cancer cells in the late 19th century to Boveri's theory of the cancer cell in the early 20th century, to current views of nuclear organization and its changes in cancer. Molecular and imaging methods go hand in hand with providing us with a better understanding of the spatial nature of the cancer cell genome. This has led to the concept that the structural order of the nucleus can be used as cancer cell biomarker.

Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility.

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost-efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

Characterizing the three-dimensional organization of telomeres in papillary thyroid carcinoma cells.

The relationship between the three-dimensional (3D) nuclear telomere architecture and specific genetic alterations in papillary thyroid carcinoma (PTC), in particular in cancer stem-like cells (CSLCs), has not yet been investigated. We isolated thyrospheres containing CSLCs from B-CPAP, K1, and TPC-1 PTC-derived cell lines, representative of tumors with different genetic backgrounds within the newly identified BRAFV600E -like PTC subgroup, and used immortalized normal human thyrocytes (Nthy-ori 3.1) as control. We performed quantitative fluorescence in situ hybridization, 3D imaging, and 3D telomere analysis using TeloView software to examine telomere dysfunction in both parental and thyrosphere cells. Among the 3D telomere profile, a wide heterogeneity was observed, except for telomere intensity. Our findings indicate that CSLCs of each cell line had longer telomeres than parental cells, according to telomere intensity values, which correlate with telomere length. Indeed, the thyrosphere cells had lower numbers of lower-intensity telomeres (≤5,000 arbitrary fluorescent units, a.u.), compared with parental cancer cells, as well as parental control cells, (p < 0.0001). The B-CPAP thyrospheres showed a decreased number of higher intensity telomeres (>17,000 a.u.) than K1 and TPC-1 cells, as well as control cells (p < 0.0001). By selecting PTC-derived cell lines with different genetic backgrounds characteristic of BRAFV600E -like PTC subgroups, we demonstrate that thyrosphere cells with BRAFV600E and TP53 mutations show shorter telomeres than those harboring RET/PTC or BRAFV600E and wild-type TP53. Hence, our data reveal a trend towards a decrease in telomere shortening in CSLCs, representing the early cancer-promoting subpopulation, as opposed to parental cells representing the tumor bulk cells.

Cementless short-stem total hip arthroplasty in the elderly patient - is it a safe option?: a prospective multicentre observational study.

BACKGROUND: Due to its bone preserving philosophy, short-stem total hip arthroplasty (THA) has primarily been recommended for young and active patients. However, there may be benefits for elderly patients given a less invasive operative technique due to the short curved implant design. The purpose of this study was to compare the clinical and radiological outcomes as well as perioperative complications of a calcar-guided short stem between a young (< 60 years) and a geriatric (> 75 years) population. METHODS: Data were collected in a total of 5 centers, and 400 short-stems were included as part of a prospective multicentre observational study between 2010 and 2014 with a mean follow-up of 49.2 months. Preoperative femur morphology was analysed using the Dorr classification. Clinical and radiological outcomes were assessed in both groups as well as perioperative complications, rates and reasons for stem revision. RESULTS: No differences were found for the mean visual analogue scale (VAS) values of rest pain, load pain, and satisfaction, whereas Harris Hip Score (HHS) was slightly better in the young group. Comparing both groups, none of the radiological parameters that were assessed (stress-shielding, cortical hypertrophy, radiolucency, osteolysis) reached differences of statistical significance. While in young patients aseptic loosening is the main cause of implant failure, in the elderly group particularly postoperative periprosthetic fractures due to accidental fall have to be considered to be of high risk. The incidence of periprosthetic fractures was found to be 0% in Dorr type A femurs, whereas in Dorr types B and C fractures occurred in 2.1 and 22.2% respectively. CONCLUSIONS: Advanced age alone is not necessarily to be considered as contra-indications for calcar-guided short-stem THA, although further follow-up is needed. However, markedly reduced bone quality with femur morphology of Dorr type C seems to be associated with increased risk for postoperative periprosthetic fractures, thus indication should be limited to Dorr types A and B. TRIAL REGISTRATION: German Clinical Trials Register; DRKS00012634 , 07.07.2017 (retrospectively registered).

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations.

Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions.

Near-field infrared nanospectroscopy and super-resolution fluorescence microscopy enable complementary nanoscale analyses of lymphocyte nuclei.

Recent super-resolution fluorescence microscopy (3D-Structured Illumination Microscopy, 3D-SIM) studies have revealed significantly altered nuclear organization between normal lymphocyte nuclei and those of classical Hodgkin's Lymphoma. Similar changes have been found in Multiple Myeloma (MM) nuclei, as well as in a premalignant condition, Monoclonal Gammopathy of Unknown Significance (MGUS). Using 3D-SIM, an increase in DNA-poor and DNA-free voids was evident in reconstructed 3D-SIM images of diseased nuclei at 40 nm pixel resolution (x,y: 40 nm, z: 80 nm). At best, far-field FTIR imaging yields spatially resolved images at ∼500 nm spatial resolution; however, near-field infrared imaging breaks the diffraction limit at a scale comparable to that of 3D-SIM, providing details on the order of 30 nm spatial resolution. We report here the first near-field IR imaging of lymphocyte nuclei, and far-field IR imaging results of whole lymphocytes and nuclei from normal human blood. Cells and nuclei were mounted on infrared-compatible substrates, including CaF2, undoped silicon wafers, and gold-coated silicon wafers. Thermal source far-field FTIR images were obtained with an Agilent-Cary 620 microscope, 15× objective, 0.62 NA and 64 × 64 array Focal Plane Array detector (University of Manitoba), or with a similar microscope equipped with both 15× and 25× (0.81 NA) objectives, 128 × 128 FPA and either thermal source or synchrotron source (single beam) infrared light at the Advanced Light Source (ALS), LBNL, Berkeley CA. Near-field IR spectra were acquired at the ALS, on the in-house SINS equipment, as well as with a Neaspec system, both illuminated with synchrotron light. Finally, some near-field IR spectra and images were acquired at Neaspec GmbH, Germany. Far-field IR spectra of normal cells and nuclei showed the characteristic bands of DNA and proteins. Near-field IR spectra of nuclei showed variations in bands assigned to protein and nucleic acids including single and double-stranded DNA. Near-field IR images of nuclei enabled visualization of protein and DNA distribution in spatially-resolved chromosome territories and nuclear pores.

Impact of adjuvant chemotherapy on patients with ypT0-2 ypN0 rectal cancer after neoadjuvant chemoradiation: a cohort study from a tertiary referral hospital.

BACKGROUND: To investigate the importance of adjuvant chemotherapy in locally advanced rectal cancer (≥ cT3 or N+) staged ypT0-2 ypN0 on final histological work-up after neoadjuvant chemoradiation and radical resection. METHODS: The clinical course of patients with rectal cancer and ypT0-2 ypN0 stages after neoadjuvant chemoradiation and radical resection was analyzed from 1999 to 2012. Patients were divided into two groups depending on whether adjuvant chemotherapy was administered or not. Overall survival, distant metastases, and local recurrence were compared between both groups. RESULTS: Fifty-four patients with adjuvant (ACT) and 50 patients without adjuvant chemotherapy (NACT) after neoadjuvant chemoradiation followed by radical resection for rectal cancer were included in the analysis. Mean follow-up was 68 ± 33.7 months. One patient without adjuvant chemotherapy and none in the ACT group developed a local recurrence. Five patients in the NACT group and three patients in the ACT group had distant recurrences. Median disease-free survival for all patients was 65.5 ± 34.5 months. Multivariate analysis showed adjuvant chemotherapy to be the most relevant factor for disease-free and overall survival. Patients staged ypT2 ypN0 showed a significantly better disease-free survival after application of adjuvant chemotherapy. Disease-free survival in ypT0-1 ypN0 patients showed no correlation to the administration of adjuvant chemotherapy. CONCLUSION: Administration of adjuvant chemotherapy after neoadjuvant chemoradiation and radical resection in rectal cancer improved disease-free and overall survival of patients with ypT0-2 ypN0 tumor stages in our study. In particular, ypT2 ypN0 patients seem to profit from adjuvant treatment.

Super-resolution structure of DNA significantly differs in buccal cells of controls and Alzheimer's patients.

The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p < 0.05) with an overall increase in the measured DNA-free/poor spaces. This represents a significant increase in the interchromatin compartment. We also find that the DNA structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA-containing and DNA-free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD.

Disruption of direct 3D telomere-TRF2 interaction through two molecularly disparate mechanisms is a hallmark of primary Hodgkin and Reed-Sternberg cells.

In classical Hodgkin's lymphoma (cHL), specific changes in the 3D telomere organization cause progression from mononuclear Hodgkin cells (H) to multinucleated Reed-Sternberg cells (RS). In a post-germinal center B-cell in vitro model, permanent latent membrane protein 1 (LMP1) expression, as observed in Epstein-Barr virus (EBV)-associated cHL, results in multinuclearity and complex chromosomal aberrations through downregulation of key element of the shelterin complex, the telomere repeat binding factor 2 (TRF2). Thus, we hypothesized that the three-dimensional (3D) telomere-TRF2 interaction was progressively disturbed during transition from H to RS cells. To this end, we developed and applied for the first time a combined quantitative 3D TRF2-telomere immune fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) technique to monolayers of primary H and RS cells, and adjacent benign internal control lymphocytes of lymph node biopsy suspensions from diagnostic lymph node biopsies of 14 patients with cHL. We show that H and RS cells are characterized by two distinct patterns of disruption of 3D telomere-TRF2 interaction. Disruption pattern A is defined by massive attrition of telomere signals and a considerable increase of TRF2 signals not associated with telomeres. This pattern is restricted to EBV-negative cHL. Disruption pattern B is defined by telomere de-protection due to an impressive loss of TRF2 signals, physically linked to telomeres. This pattern is typical of, but is not restricted to, LMP1+EBV-associated cHL. In the disruption pattern B group, so-called 'ghost' end-stage RS cells, void of both TRF2 and telomere signals, were identified, whether or not associated with EBV. Our findings demonstrate that two molecularly disparate mechanisms converge on the level of 3D telomere-TRF2 interaction in the formation of RS cells.

Distinct and shared three-dimensional chromosome organization patterns in lymphocytes, monoclonal gammopathy of undetermined significance and multiple myeloma.

The consistent appearance of specific chromosomal translocations in multiple myeloma has suggested that the positioning of chromosomes in the interphase nucleus might play a role in the occurrence of particular chromosomal rearrangements associated with malignant transformation. Using fluorescence in situ hybridization, we have determined the positions of selected chromosome pairs (18 and 19, 9 and 22, 4 and 14, 14 and 16, 11 and 14) in interphase nuclei of myeloma cells compared to normal lymphocytes of treatment-naïve patients. All chromosome pairs were arranged in a nonrandom pattern. Chromosomes commonly involved in myeloma-associated translocations (4 and 14, 14 and 16, 11 and 14) were found in close spatial proximity, and this is correlated with the occurrence of overlapping chromosome territories. The spatial distribution of chromosomes may increase the possibility of chromosomal translocations in multiple myeloma.

Plasma microRNA signature is associated with risk stratification in prostate cancer patients.

The aim of this study was to establish a unique expression profile of circulating cell-free microRNAs (miRNAs) capable of differentiating between prostate cancer (PCa) patients with high-risk and intermediate-risk Gleason scores. MiRNA expression profiles were determined in plasma samples from 79 treatment-naïve PCa patients, 1-2 follow-up samples after radical prostatectomy (RP) from 51 out of the 79 PCa patients, and 33 healthy men, using a quantitative real-time PCR-based array containing 48 selected miRNAs. We identified 27 up- and 2 downregulated plasma miRNAs in PCa patients compared with healthy men. Most of the upregulated miRNA levels were also associated with increasing PSA levels and Gleason scores. Particularly, the levels of miR-16 (p = 0.002), miR-148a (p = 0.006) and miR-195 (p = 0.006) significantly correlated with high-risk Gleason scores, whereby miR-148a (p = 0.003) was also significantly associated with increasing PSA values. The high miRNA levels before RP remained increased in the postsurgical plasma samples. Our findings show a network of deregulated plasma miRNAs. In particular, miR-16, miR-148a and miR-195 are involved in the regulation of the PI3K/Akt signaling pathway. These miRNAs may be promising therapeutic targets for high-risk PCa stratification.

LMP1 mediates multinuclearity through downregulation of shelterin proteins and formation of telomeric aggregates.

Hodgkin lymphoma (HL) and Burkitt lymphoma are both germinal center-derived B-cell lymphomas. To assess the consequences of permanent latent membrane protein 1 (LMP1) expression as observed in tumor cells of Epstein-Barr virus (EBV) -associated HL, we analyzed 3-dimensional (3D) telomere dynamics and measured the expression of shelterin proteins at the transcriptional and translational level and their topographic distribution in the EBV-negative Burkitt cell line BJAB stably transfected with an inducible LMP1 system. Stable LMP1 expression led to a highly significant increase of multinucleated cells, nuclear volume, and 3D telomeric aggregates when compared with the LMP1-suppressed BJAB controls. Most importantly, LMP1 induced a significant downregulation of the shelterin components TRF1, TRF2, and POT1 at the transcriptional and translational level, and this downregulation was reversed after resuppression of LMP1. In addition, as revealed by spectral karyotyping, LMP1 induced "outré" giant cells and hypoploid "ghost" cells. This LMP1-induced multinucleation was blocked upon LMP1-independent TRF2 expression. These results show that LMP1-dependent deregulation of telomere stability and nuclear organization via shelterin downregulation, in particular TRF2, favors chromosomal rearrangements. We speculate that telomeric aggregates and ongoing breakage-bridge-fusion cycles lead to disturbed cytokinesis and finally to multinuclearity, as observed in EBV-associated HL.

Small bowel protection in IMRT for rectal cancer : A dosimetric study on supine vs. prone position.

BACKGROUND: This treatment planning study analyzes dose coverage and dose to organs at risk (OAR) in intensity-modulated radiotherapy (IMRT) of rectal cancer and compares prone vs. supine positioning as well as the effect of dose optimization for the small bowel (SB) by additional dose constraints in the inverse planning process. PATIENTS AND METHODS: Based on the CT datasets of ten male patients in both prone and supine position, a total of four different IMRT plans were created for each patient. OAR were defined as the SB, bladder, and femoral heads. In half of the plans, two additional SB cost functions were used in the inverse planning process. RESULTS: There was a statistically significant dose reduction for the SB in prone position of up to 41% in the high and intermediate dose region, compared with the supine position. Furthermore, the femoral heads showed a significant dose reduction in prone position in the low dose region. Regarding the additional active SB constraints, the dose in the high dose region of the SB was significantly reduced by up to 14% with the additional cost functions. There were no significant differences in the dose distribution of the planning target volume (PTV) and the bladder. CONCLUSION: Prone positioning can significantly reduce dose to the SB in IMRT for rectal cancer and therefore should not only be used in 3D conformal radiotherapy but also in IMRT of rectal cancer. Further protection of the SB can be achieved by additional dose constraints in inverse planning without jeopardizing the homogeneity of the PTV.

An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.

BACKGROUND INFORMATION: Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. RESULTS: We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1-102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT(-/-) ES cells and Insi1 retained 26-48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT(-/-) ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT(-/-) ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT(-/-) ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT(-/-) ES cells expressing wild-type mTERT. In addition, in mTERT(-/-) cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. CONCLUSION AND SIGNIFICANCE: An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.

FGFR3 preferentially colocalizes with IGH in the interphase nucleus of multiple myeloma patient B-cells when FGFR3 is located outside of CT4.

Many B-cell malignancies are characterized by chromosomal translocations involving IGH and a proto-oncogene. For translocations to occur, spatial proximity of translocation-prone genes is necessary. Currently, it is not known how such genes are brought into proximity with one another. Although decondensed chromosomes occupy definitive, non-random spaces in the interphase nucleus known as chromosome territories (CTs), chromatin at the edges of CTs can intermingle, and specific genomic regions from some chromosomes have been shown to "loop out" of their respective CTs. This extra-territorial positioning of specific genomic regions may provide a mechanism whereby translocation-prone genes are brought together in the interphase nucleus. FGFR3 and MAF recurrently participate in translocations with IGH at different frequencies. Using 3D, 4-color FISH, and 3D analysis software, we show frequent extra-territorial positioning of FGFR3 and significantly less frequent extra-territorial positioning of MAF. Frequent extra-territorial positioning may be characteristic of FGFR3 in B-cells from healthy adult donors and non-malignant B-cells from patients, but not in hematopoietic stem cells from patients with translocations. The frequency of extra-territorial positioning of FGFR3 and MAF in B-cells correlates with the frequency of translocations in the patient population. Most importantly, in patient B-cells, we demonstrate a significant proportion of extra-territorial FGFR3 participating in close loci pairs and/or colocalizing with IGH. This preliminary work suggests that in patient B-cells, extra-territorial positioning of FGFR3 may provide a mechanism for forming close loci pairs and/or colocalization with IGH; indirectly facilitating translocation events involving these two genes. © 2016 Wiley Periodicals, Inc.

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.