Mechanisms of A-type lamin targeting to nuclear ruptures are disrupted in LMNA- and BANF1- associated progerias

Mutations in the genes LMNA and BANF1 can lead to accelerated aging syndromes called progeria. The protein products of these genes, A-type lamins and BAF, respectively, are nuclear envelope (NE) proteins that interact and participate in various cellular processes, including nuclear envelope rupture and repair. BAF localizes to sites of nuclear rupture and recruits NE-repair machinery, including the LEM-domain proteins, ESCRT-III complex, A-type lamins, and membranes. Here, we show that it is a mobile, nucleoplasmic population of A-type lamins that is rapidly recruited to ruptures in a BAF-dependent manner via BAF′s association with the Ig-like β fold domain of A-type lamins. These initially mobile lamins become progressively stabilized at the site of rupture. Farnesylated prelamin A and lamin B1 fail to localize to nuclear ruptures, unless that farnesylation is inhibited. Progeria-associated LMNA mutations inhibit the recruitment affected A-type lamin to nuclear ruptures, due to either permanent farnesylation or inhibition of BAF binding. A progeria-associated BAF mutant targets to nuclear ruptures but is unable to recruit A-type lamins. Together, these data reveal the mechanisms that determine how lamins respond to nuclear ruptures and how progeric mutations of LMNA and BANF1 impair recruitment of A-type lamins to nuclear ruptures.

Deformation of the nucleus by TGFβ1 via the remodeling of nuclear envelope and histone isoforms

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFβ1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFβ1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFβ1-induced compositional changes in the chromatin and nuclear lamina.

Differential expression of lamin B1 in triple negative breast cancer.

Women diagnosed with triple negative breast cancer can benefit neither from endocrine therapy nor from HER2-targeted therapies (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding lamin B1, LMNB1, when comparing the tumor cells of patients with triple negative breast cancer to normal mammary ductal cells (2). LMNB1 was also differentially expressed in bulk tumor in human breast cancer (3). LMNB1 mRNA was present at significantly increased quantities in TNBC tumor cells relative to normal mammary ductal cells. Analysis of human survival data revealed that expression of LMNB1 in primary tumors of the breast was correlated with overall survival in patients with basal-like and luminal A subtype cancer, while within triple negative breast cancer, primary tumor expression of LMNB1 was correlated with overall survival in patients with basal-like 2, immunomodulatory and mesenchymal subtype disease. LMNB1 may be of relevance to initiation, maintenance or progression of triple negative breast cancers.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

Chromosome length and gene density contribute to micronuclear membrane stability

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

Loss of Lamin B1 in Myeloid Neoplasms with 5q Deletion Causes Myeloid-Biased Hematopoiesis and Pelger-Huet Nuclear Anomaly

Hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations and cytogenetic abnormalities leading to myeloid neoplasms (MDS/AML). A subgroup of 10-20% MDS and AML cases undergo complex chromosomal rearrangements associated with TP53 mutations and poor prognosis. One of the most common chromosomal events in high-risk myeloid neoplasms is deletion of chromosome 5q (del(5q)). In contrast to 5q- syndrome, characterized by a distal commonly deleted region (CDR) and good prognosis, the critical CDR in high-risk MDS/AML centers on 5q31. However, the key genes in the high-risk 5q-deleted region that contribute to dysregulation of hematopoiesis, chromosomal instability, and disease progression remain poorly characterized. Progression to high-risk MDS/AML is often accompanied by morphologic dysplasia in the form of Pelger-Huët cells, abnormal neutrophils with bilobed or unilobed nuclei and coarse clumping of the nuclear chromatin. The inherited form of Pelger-Huët anomaly (PHA) is caused by mutations in LBR, which encodes lamin B receptor. Acquired PHA or pseudo-PHA is commonly seen in myeloid malignancies, often in high risk MDS and AML with TP53 mutations. While LBR is not mutated in MDS/AML, its main interaction partner lamin B1/LMNB1 is encoded in the high-risk 5q-deleted region. Lamins organize chromatin into perinuclear compartments and regulate diverse biological processes, including gene expression and DNA repair. The frequent deletion of LMNB1 in 5q-deleted MDS and AML led us to hypothesize that LMNB1 loss drives both nuclear dysmorphology and functional HSC defects in malignant transformation. LMNB1 expression was broadly decreased in MDS and AML, particularly in del(5q) cases, 83% of which had deletions of the LMNB1 locus. We show that in a clinical cohort of MDS patients, the Pelger-Huët nuclear phenotype is strongly associated with del5q. To understand the role of LMNB1, we performed lentiviral shRNA knockdown in normal umbilical cord blood (CB) CD34 + HSPCs followed by transplantation into immunodeficient NSG mice. To study the role of lamin B1 in 5q-deleted MDS, we established an in vitro patient-derived induced pluripotent stem cell (iPSC) model by reprograming an MDS patient with TP53 mutation and complex karyotype, and deriving MDS HSPCs with TP53+/R209fs alone or TP53+/R209fs with an isolated del5q spanning 5q22-5q31.1 encoding LMNB1 (TP53;del5q). Using these model systems we show that LMNB1 loss is both necessary and sufficient to cause Pelger-Huët nuclear morphology in normal and MDS neutrophils. Additionally, we find that LMNB1-deficient human HSCs display increased self-renewal and profound myeloid lineage bias in vitro and in vivo. Single cell RNA expression data from LMNB1-deficient cells in vivo shows mis-expression of lineage-specifying transcription factors in single multipotent progenitors. LMNB1 deficiency also results in poor marking and propagation of unrepaired DNA breaks, leading to genome instability. The role of LMNB1 in maintaining chromatin organization prompted us to use chromosome conformation capture (Hi-C) to visualize the 3D chromatin organization of LMNB1-deficient HSPCs. We show that lamin B1 regulates enhancer-promoter loops suggesting that lamin B1 loss dysregulates HSC function by altering enhancer-promoter interactions at lineage-specifying transcription factor loci. Together, we identify lamin B1 as a novel 5q gene that contributes to malignant transformation by driving enhanced self-renewal, myeloid-bias, and genome instability. Furthermore, lamin B1 deletion is the genetic cause of acquired PHA in myeloid malignancies, providing a potential biomarker for high risk neoplasms. Disclosures Becker: Glycomimetics: Research Funding; CVS Caremark: Consultancy; Abbie: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Cardiff Oncology: Research Funding; SecuraBio: Research Funding.

LEF1 Enhances the Progression of Colonic Adenocarcinoma via Remodeling the Cell Motility Associated Structures

Lymphoid enhancer-binding factor 1 (LEF1) is a key transcription factor mediating the Wnt signaling pathway. LEF1 is a regulator that is closely associated with tumor malignancy and is usually upregulated in cancers, including colonic adenocarcinoma. The underlying molecular mechanisms of LEF1 regulation for colonic adenocarcinoma progression remain unknown. To explore it, the LEF1 expression in caco2 cells was inhibited using an shRNA approach. The results showed that downregulation of LEF1 inhibited the malignancy and motility associated microstructures, such as polymerization of F-actin, β-tubulin, and Lamin B1 in caco2 cells. LEF1 inhibition suppressed the expression of epithelial/endothelial-mesenchymal transition (EMT) relevant genes. Overall, the current results demonstrated that LEF1 plays a pivotal role in maintaining the malignancy of colonic adenocarcinoma by remodeling motility correlated microstructures and suppressing the expression of EMT-relevant genes. Our study provided evidence of the roles LEF1 played in colonic adenocarcinoma progression, and suggest LEF1 as a potential target for colonic adenocarcinoma therapy.

Deformation of the Nucleus by TGFβ1 Via the Remodeling of Nuclear Envelope and Histone Isoforms

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFb1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFb1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed with no observable indications of DNA damage response. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, shuttling between the nucleus and cytoplasm, and clustering at the nuclear periphery. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFb1-induced compositional changes in the chromatin and nuclear lamina.