TRF2 rescues pathogenic phenotypes in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart; however, these treatments do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that longer telomeres are protective against dilated cardiomyopathy. Here we investigated the role of telomeres as a target for therapy in DMD cardiomyocytes using human induced pluripotent stem cells (iPSCs) to model the disease. Compared to healthy controls, DMD cardiomyocytes exhibited reduced telomere lengths, cell size, nuclear size, and sarcomere density. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends. TRF2 levels are reduced relative to healthy controls in DMD cardiomyocytes. We hypothesized that decreased TRF2 drives telomere attrition and subsequent cardiomyocyte death in the progression of dilated cardiomyopathy. Our data show that TRF2 overexpression prevented telomere attrition and also rescued deficits in cell size, nuclear size, sarcomere density, and calcium handling. These data highlight the benefits of TRF2 upregulation as a potential gene therapy to delay the onset of dilated cardiomyopathy.

Modeling Cell Biological Features of Meiotic Chromosome Pairing

During meiosis, homologous chromosomes become associated side by side in a process known as homologous chromosome pairing. Pairing requires long range chromosome motion through a nucleus that is full of other chromosomes. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we use a coarse-grained molecular dynamics model to investigate how specific features of meiosis, including motor-driven telomere motion, nuclear envelope interactions, and increased nuclear size, affect the rate of pairing and the mitigation/resolution of interlocks. By creating in silico versions of three yeast strains and comparing the results of our model to experimental data, we find that a more distributed placement of pairing sites along the chromosome is necessary to replicate experimental findings. Active motion of the telomeric ends speeds up pairing only if binding sites are spread along the chromosome length. Adding a meiotic bouquet significantly speeds up pairing but does not significantly change the number of interlocks. An increase in nuclear size slows down pairing while greatly reducing the number of interlocks. Interestingly, active forces increase the number of interlocks, which raises the question: How do these interlocks resolve? Our model gives us detailed movies of interlock resolution events which we then analyze to build a step-by-step recipe for interlock resolution. In our model, interlocks must first translocate to the ends, where they are held in a quasi-stable state by a large number of paired sites on one side. To completely resolve an interlock, the telomeres of the involved chromosomes must come in close proximity so that the cooperativity of pairing coupled with random motion causes the telomeres to unwind. Together our results indicate that computational modeling of homolog pairing provides insight into the specific cell biological changes that occur during meiosis.

Control of nuclear size by osmotic forces in Schizosaccharomyces pombe

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount, but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

Aerobic scope does matter in the temperature-size rule but only under optimal conditions

We united theoretical predictions of the factors responsible for the evolutionary significance of the temperature-size rule (TSR). We assumed that (i) the TSR is a response to temperature-dependent oxic conditions, (ii) body size decrease is a consequence of cell shrinkage in response to hypoxia, (iii) this response enables organisms to maintain a wide scope for aerobic performance, and (iv) it prevents a decrease in fitness. We examined three clones of the rotifer Lecane inermis exposed to three experimental regimes: mild hypoxia, severe hypoxia driven by a too high temperature, and severe hypoxia driven by an inadequate oxygen concentration. We compared the following traits in normoxia- and hypoxia-exposed rotifers: nuclear size (a proxy for cell size), body size, specific dynamic action (SDA, a proxy of aerobic metabolism) and two fitness measures, the population growth rate and eggs/female ratio. The results showed that (i) under mildly hypoxic conditions, our causative reasoning was correct, except that one of the clones decreased in body size without a decrease in nuclear size, and (ii) in more stressful environments, rotifers exhibited clone- and condition-specific responses, which were equally successful in terms of fitness levels. Our results indicate the importance of the rule testing conditions. The important conclusions were that (i) a body size decrease at higher temperatures enabled the maintenance of a wide aerobic scope under clone-specific, thermally optimal conditions, and (ii) this response was not the only option to prevent fitness reduction under hypoxia.

Artificial Intelligence-Assisted Mapping of Proliferation Centers in Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma Identifies Patterns That Reliably Distinguish Accelerated Phase and Large Cell Transformation

Background Chronic lymphocytic leukemia (CLL) involving tissues is characterized by sheets of small lymphocytes and vague nodules of larger cells, known as proliferation centers (PCs). CLL can undergo progression to accelerated CLL (aCLL) or further progress to diffuse large B-cell lymphoma, also known as Richter transformation (RT). Distinguishing CLL with many PCs from aCLL or RT can be challenging, particularly in small needle-biopsy specimens. In this study, we sought to design an artificial intelligence (AI)-based tool to automate and enhance the delineation of PCs and provide an objective approach to CLL/SLL acceleration/transformation. Material & Methods We manually annotated 25, 28 and 21 regions of interest (ROIs) encompassing small round PCs and confluent/ expanded PCs of 10 CLL, 12 aCLL, and 8 RT digitized hematoxylin & eosin stained slides, respectively (Figure A1). We analyzed the ROIs with both length and width larger than 2,000 pixels and set the tile length and stride as 1,000 and 100 pixels, respectively (Figure A1), and were able to extract sufficient tiles from each ROI (Figure A2). To recreate PCs, after performing nuclear segmentation via convolutional neural network and quality control, we quantified the nuclear size/intensity of cells occupying each tile (Figure A3). Nuclear size varied from 8 to 108 square micrometers, and nuclear mean intensity varied from 0 to 255. We normalized both the values of nuclear size and mean intensity to 0.0 and 1.0, by subtracting the minimum value and dividing it by the value range length. Nuclear size and mean intensity were represented as S(celli) and Imean(celli), respectively. We estimated the heat value of one tile integrating nuclear and mean intensity using the equationin Figure A2. Results We generated heatmaps based on the heat values per tile inside each ROI from the 3 disease entities (CLL, aCLL and RT), as illustrated in Figures B1-4. Areas with high heat values (in the yellow spectrum) correspond to tiles harboring cells with increased nuclear size and mean intensity (PCs in CLL cases and expanded/ confluent PCs in aCLL and RT cases). In contrast, areas with low heat values (in the blue spectrum) correspond to tiles with decreased nuclear size and mean intensity (small neoplastic lymphocytes surrounding PCs) (Figure B4).We then generated a heat value histogram per tile for each ROI (Figure B5).Based on these results, the two optimal thresholds isolated to obtain the highest separation value among the three disease entities based on the optimal F-score were: 0.228, below which the case was most likely to be CLL, and 0.288, above which the case was most likely to be RT. Cases with heat values ranging between 0.228 and 0.288 were most likely aCLL cases. We then plotted the mean heat value frequencies of the 3 entities: There was a significant difference in the ranges of mean heat value frequencies for CLL, aCLL, and RT, which were 0.168 to 0.233, 0.212 to 0.307, and 0.261 to 0.353, respectively (Figure C). The accuracy and area under the curve diagnostic predictive values using data from nuclear size alone were 0.658 (+/-0.115) and 0.771 (+/-0.096), respectively; and using mean nuclear intensity, 0.679 (+/-0.094) and 0.841 (+/-0.052), respectively; with a noticeable increase using heat value frequencies (integrating the nuclear size and mean nuclear intensity) reaching 0.813 (+/-0.0630) and 0.885 (+/-0.109), respectively. Conclusion We describe a novel AI-based heatmap technique to objectively assess the extent of PCs in CLL, based on the integrative analysis of cell nuclear size and mean nuclear intensity. We suggest that an ROI mean heat value less than 0.228 is predictive of CLL, and more than 0.288 is predictive of RT. aCLL cases demonstrate a mean heat value ranging from 0.228 to 0.288. Using the mean heat value of all cases, we were able to reliably separate the three entities in question with robust diagnostic predictive values. Figure 1 Figure 1. Disclosures Khoury: Stemline Therapeutics: Research Funding; Kiromic: Research Funding; Angle: Research Funding.

The Role of Emerin in Cancer Progression and Metastasis

It is commonly recognized in the field that cancer cells exhibit changes in the size and shape of their nuclei. These features often serve as important biomarkers in the diagnosis and prognosis of cancer patients. Nuclear size can significantly impact cell migration due to its incredibly large size. Nuclear structural changes are predicted to regulate cancer cell migration. Nuclear abnormalities are common across a vast spectrum of cancer types, regardless of tissue source, mutational spectrum, and signaling dependencies. The pervasiveness of nuclear alterations suggests that changes in nuclear structure may be crucially linked to the transformation process. The factors driving these nuclear abnormalities, and the functional consequences, are not completely understood. Nuclear envelope proteins play an important role in regulating nuclear size and structure in cancer. Altered expression of nuclear lamina proteins, including emerin, is found in many cancers and this expression is correlated with better clinical outcomes. A model is emerging whereby emerin, as well as other nuclear lamina proteins, binding to the nucleoskeleton regulates the nuclear structure to impact metastasis. In this model, emerin and lamins play a central role in metastatic transformation, since decreased emerin expression during transformation causes the nuclear structural defects required for increased cell migration, intravasation, and extravasation. Herein, we discuss the cellular functions of nuclear lamina proteins, with a particular focus on emerin, and how these functions impact cancer progression and metastasis.

Nuclear Dynamics and Chromatin Structure: Implications for Pancreatic Cancer

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the ‘gold standard’ for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

Prognostic value of proliferation, PD-L1 and nuclear size in patients with superior sulcus tumours treated with chemoradiotherapy and surgery

AimsThe aim of this study was to determine the relationship between proliferative activity, PD-L1 status and nuclear size changes after preoperative chemoradiotherapy (CRT) and the clinical outcome in patients with superior sulcus tumours.MethodsProliferative activity (MIB-1) and PD-L1 status were estimated by immunohistochemistry in the tumour cells of resection specimen in a series of 33 patients with residual tumour after trimodality therapy for a sulcus superior tumour between 2005 and 2014. A morphometric analysis of both pretreatment and post-treatment tumour materials was also performed. Results were related to disease-free survival and overall survival.ResultsLow proliferative activity (<20% MIB-1) was associated with better overall survival: 2-year overall survival of 73% compared with 43% and 25%, respectively, for moderate (MIB-1 20%–50%) and high (MIB-1 >50%) proliferative activity (p=0.016). A negative PD-L1 status (<1% positive tumour cells) was also associated with better overall survival (p=0.021). The mean nuclear size of normal lung tissue pneumocytes was significantly smaller compared with the mean nuclear size of tumour cells of the resection specimens (median difference −38.1; range −115.2 to 16.0; p<0.001). The mean nuclear size of tumour cells did not differ between pretreatment biopsies and resection specimens (median difference −4.6; range −75.2 to 86.7; p=0.14). Nuclear size was not associated with survival (p=0.82).ConclusionsLow proliferative activity determined by MIB-1 as well as a negative PD-L1 expression are significantly associated with better overall survival in patients with residual tumour after CRT for superior sulcus tumour.

VCP maintains nuclear size by regulating the DNA damage-associated MDC1–p53–autophagy axis in Drosophila

The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.