Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism.

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

Integrating leiomyoma genetics, epigenomics, and single-cell transcriptomics reveals causal genetic variants, genes, and cell types.

Uterine fibroids (UF), that can disrupt normal uterine function and cause significant physical and psychological health problems, are observed in nearly 70% of women of reproductive age. Although heritable genetics is a significant risk factor, specific genetic variations and gene targets causally associated with UF are poorly understood. Here, we performed a meta-analysis on existing fibroid genome-wide association studies (GWAS) and integrated the identified risk loci and potentially causal single nucleotide polymorphisms (SNPs) with epigenomics, transcriptomics, 3D chromatin organization from diverse cell types as well as primary UF patient's samples. This integrative analysis identifies 24 UF-associated risk loci that potentially target 394 genes, of which 168 are differentially expressed in UF tumors. Critically, integrating this data with single-cell gene expression data from UF patients reveales the causal cell types with aberrant expression of these target genes. Lastly, CRISPR-based epigenetic repression (dCas9-KRAB) or activation (dCas9-p300) in a UF disease-relevant cell type further refines and narrows down the potential gene targets. Our findings and the methodological approach indicate the effectiveness of integrating multi-omics data with locus-specific epigenetic editing approaches for identifying gene- and celt type-targets of disease-relevant risk loci.

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism.

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTS: Paired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONS: Our findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

Engineered MED12 mutations drive leiomyoma-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.

Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the MED12 gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.

Tumor Cell Infiltration into the Brain in Glioblastoma: From Mechanisms to Clinical Perspectives.

Glioblastoma is the most common and malignant primary brain tumor, defined by its highly aggressive nature. Despite the advances in diagnostic and surgical techniques, and the development of novel therapies in the last decade, the prognosis for glioblastoma is still extremely poor. One major factor for the failure of existing therapeutic approaches is the highly invasive nature of glioblastomas. The extreme infiltrating capacity of tumor cells into the brain parenchyma makes complete surgical removal difficult; glioblastomas almost inevitably recur in a more therapy-resistant state, sometimes at distant sites in the brain. Therefore, there are major efforts to understand the molecular mechanisms underpinning glioblastoma invasion; however, there is no approved therapy directed against the invasive phenotype as of now. Here, we review the major molecular mechanisms of glioblastoma cell invasion, including the routes followed by glioblastoma cells, the interaction of tumor cells within the brain environment and the extracellular matrix components, and the roles of tumor cell adhesion and extracellular matrix remodeling. We also include a perspective of high-throughput approaches utilized to discover novel players for invasion and clinical targeting of invasive glioblastoma cells.

Generation of TRAIL-resistant cell line models reveals distinct adaptive mechanisms for acquired resistance and re-sensitization.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell-specific apoptosis, making it a prime therapeutic candidate. However, many tumor cells are either innately TRAIL-resistant, or they acquire resistance with adaptive mechanisms that remain poorly understood. In this study, we generated acquired TRAIL resistance models using multiple glioblastoma (GBM) cell lines to assess the molecular alterations in the TRAIL-resistant state. We selected TRAIL-resistant cells through chronic and long-term TRAIL exposure and noted that they showed persistent resistance both in vitro and in vivo. Among known TRAIL-sensitizers, proteosome inhibitor Bortezomib, but not HDAC inhibitor MS-275, was effective in overcoming resistance in all cell models. This was partly achieved through upregulating death receptors and pro-apoptotic proteins, and downregulating major anti-apoptotic members, Bcl-2 and Bcl-xL. We showed that CRISPR/Cas9 mediated silencing of DR5 could block Bortezomib-mediated re-sensitization, demonstrating its critical role. While overexpression of Bcl-2 or Bcl-xL was sufficient to confer resistance to TRAIL-sensitive cells, it failed to override Bortezomib-mediated re-sensitization. With RNA sequencing in multiple paired TRAIL-sensitive and TRAIL-resistant cells, we identified major alterations in inflammatory signaling, particularly in the NF-κB pathway. Inhibiting NF-κB substantially sensitized the most resistant cells to TRAIL, however, the sensitization effect was not as great as what was observed with Bortezomib. Together, our findings provide new models of acquired TRAIL resistance, which will provide essential tools to gain further insight into the heterogeneous therapy responses within GBM tumors. Additionally, these findings emphasize the critical importance of combining proteasome inhibitors and pro-apoptotic ligands to overcome acquired resistance.

TRAIL-conjugated silver nanoparticles sensitize glioblastoma cells to TRAIL by regulating CHK1 in the DNA repair pathway.

OBJECTIVES: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers apoptosis in cancer cells, but not in normal cells. Resistance of glioblastoma cells to TRAIL is a major obstacle for successful clinical treatment of TRAIL. Thus, there is an essential requirement for novel approaches to sensitize TRAIL resistance. Silver nanoparticles (AgNPs) are one of the most promising nanomaterials that show immense antitumor potential via targeting various cellular and molecular processes; however, the effects of AgNPs on TRAIL sensitivity in cancer cells remain unclear. Therefore, we hypothesized that TRAIL-conjugated AgNPs (TRAIL-AgNPs) can overcome TRAIL resistance through inducing death receptor activation in glioblastoma cells, but not normal cells. METHODS: In this study, the therapeutic effect of TRAIL-AgNPs is investigated by analyzing the cell viability, caspase activity, and CHK1 gene expression in T98 G TRAIL-Sensitive (TS) and T98 G TRAIL-Resistant (TR) glioblastoma cells. RESULTS: It is found that TRAIL-AgNPs are more toxic compared to TRAIL and AgNPs treatments alone on TR cells. While TRAIL and AgNPs alone do not enhance the caspase activity, conjugation of TRAIL to AgNPs increases the caspase activity in TR cells. Moreover, the TRAIL-AgNPs-treated TR cells show less CHK1 expression compared to the TRAIL treatment. CONCLUSION: These results suggest that TRAIL sensitivity of TR cells can be enhanced by conjugation of TRAIL with AgNPs, which would be a novel therapeutic approach to sensitize TRAIL resistance.

Drug Repositioning Screen on a New Primary Cell Line Identifies Potent Therapeutics for Glioblastoma.

Glioblastoma is a malignant brain cancer with limited treatment options and high mortality rate. While established glioblastoma cell line models provide valuable information, they ultimately lose most primary characteristics of tumors under long-term serum culture conditions. Therefore, established cell lines do not necessarily recapitulate genetic and morphological characteristics of real tumors. In this study, in line with the growing interest in using primary cell line models derived from patient tissue, we generated a primary glioblastoma cell line, KUGBM8 and characterized its genetic alterations, long term growth ability, tumor formation capacity and its response to Temozolomide, the front-line chemotherapy utilized clinically. In addition, we performed a drug repurposing screen on the KUGBM8 cell line to identify FDA-approved agents that can be incorporated into glioblastoma treatment regimen and identified Topotecan as a lead drug among 1,200 drugs. We showed Topotecan can induce cell death in KUGBM8 and other primary cell lines and cooperate with Temozolomide in low dosage combinations. Together, our study provides a new primary cell line model that can be suitable for both in vitro and in vivo studies and suggests that Topotecan can offer promise as a therapeutic approach for glioblastoma.