Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Distinct uptake and elimination profiles for trastuzumab, human IgG and biocytin-TMR in experimental HER2+ brain metastases of breast cancer.

BACKGROUND: The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the Intramural Periarterial Drainage (IPAD) pathway. METHODS: Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min-48h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD- vascular matrix analyzed in 11,668 metastases. RESULTS: In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. CONCLUSIONS: Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.

Inhibition of MEK Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targe6ng of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

EWSR1's visual modalities are defined by its association with nucleic acids and RNA polymerase II.

We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells.

Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell-derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL. Significance: Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Zinc finger transcription factor CASZ1b is involved in the DNA damage response in live cells.

Zinc finger transcription factor CASZ1b is essential for nervous system development and suppresses neuroblastoma growth. Our previous study showed that CASZ1b interacts with DNA repair proteins, however, whether CASZ1b is involved in the DNA damage response remains unclear. In this study, we investigated the kinetic recruitment of CASZ1b to sites of DNA damage upon induction by laser microirradiation. We find that CASZ1b is transiently recruited to sites of DNA damage in multiple cell lines. Mutagenesis of either the poly-(ADP-ribose) (PAR) binding motif or NuRD complex binding region in CASZ1b significantly reduces the recruitment of CASZ1b to these sites of DNA damage (∼65% and ∼30%, respectively). In addition, treatment of cells with a poly-(ADP-ribose) polymerase (PARP) inhibitor significantly attenuates the recruitment of CASZ1b to these DNA damaged sites. Loss of CASZ1 increases cell sensitivity to DNA damage induced by gamma irradiation as shown by decreased colony formation. Our studies reveal that CASZ1b is transiently recruited to DNA damage sites mainly in a PARP-dependent way and regulates cell sensitivity to DNA damage. Our results suggest that CASZ1b has a role, although perhaps a minor one, in the DNA damage response and ultimately regulating the efficiency of DNA repair during normal development and tumorigenesis.

Dysregulation of Mitochondrial Translation Caused by CBFB Deficiency Cooperates with Mutant PIK3CA and Is a Vulnerability in Breast Cancer.

Understanding functional interactions between cancer mutations is an attractive strategy for discovering unappreciated cancer pathways and developing new combination therapies to improve personalized treatment. However, distinguishing driver gene pairs from passenger pairs remains challenging. Here, we designed an integrated omics approach to identify driver gene pairs by leveraging genetic interaction analyses of top mutated breast cancer genes and the proteomics interactome data of their encoded proteins. This approach identified that PIK3CA oncogenic gain-of-function (GOF) and CBFB loss-of-function (LOF) mutations cooperate to promote breast tumor progression in both mice and humans. The transcription factor CBFB localized to mitochondria and moonlighted in translating the mitochondrial genome. Mechanistically, CBFB enhanced the binding of mitochondrial mRNAs to TUFM, a mitochondrial translation elongation factor. Independent of mutant PI3K, mitochondrial translation defects caused by CBFB LOF led to multiple metabolic reprogramming events, including defective oxidative phosphorylation, the Warburg effect, and autophagy/mitophagy addiction. Furthermore, autophagy and PI3K inhibitors synergistically killed breast cancer cells and impaired the growth of breast tumors, including patient-derived xenografts carrying CBFB LOF and PIK3CA GOF mutations. Thus, our study offers mechanistic insights into the functional interaction between mutant PI3K and mitochondrial translation dysregulation in breast cancer progression and provides a strong preclinical rationale for combining autophagy and PI3K inhibitors in precision medicine for breast cancer. SIGNIFICANCE: CBFB-regulated mitochondrial translation is a regulatory step in breast cancer metabolism and synergizes with mutant PI3K in breast cancer progression.

Morphological and Functional Colonic Defects Caused by a Mutated Thyroid Hormone Receptor α.

Background: Mutations of thyroid hormone receptor α (TRα1) result in resistance to thyroid hormone (RTHα), exhibiting symptoms of retarded growth, delayed bone maturation, anemia, and severe constipation. Using a mouse model of RTHα (Thra1PV/+ mouse), we aimed at understanding the molecular basis underlying the severe constipation observed in patients. Methods: The Thra1PV/+ mouse expresses a strong dominant negative mutant, PV, which has lost T3 binding and transcription activity. Thra1PV/+ mouse faithfully reproduces growth abnormalities and anemia as shown in RTHα patients and therefore is a valid model to examine causes of severe constipation in patients. We used histopathological analysis, confocal fluorescence imaging, transmission electron microscopy (TEM), and gene expression profiles to comprehensively analyze the colonic abnormalities of Thra1PV/+ mouse. Results: We found a significant increase in colonic transit time and decrease stool water content in Thra1PV/+ mouse, mimicking constipation as found in patients. Histopathological analysis showed expanded lamina propria filled with interstitium fluid between crypt columns, enlarged muscularis mucosa, and increased content of collagen in expanded submucosa. The TEM analysis revealed shorter muscle fibers with wider gap junctions between muscle cells, fewer caveolae, and hypoplastic interstitial cells of Cajal (ICC) in the rectal smooth muscles of Thra1PV/+ mice. These abnormal histological manifestations suggested defective intercellular transfer of small molecules, electrolytes, and signals for communication among muscles cells, validated by Lucifer Yellow transferring assays. Expression of key smooth muscle contractility regulators, such as calmodulin, myosin light-chain kinase, and phosphorylated myosin light chain, was markedly lower, and c-KIT signaling in ICC was attenuated, resulting in decreased contractility of the rectal smooth muscles of Thra1PV/+ mice. Collectively, these abnormal histopathological alterations and diminished contractility regulators led to the constipation exhibited in patients. Conclusions: This is the first demonstration that TRα1 mutants could act to cause abnormal rectum smooth muscle organization, defects in intercellular exchange of small molecules, and decreased expression of contractility regulators to weaken the contractility of rectal smooth muscles. These findings provide new insights into the molecular basis underlying constipation found in RTHα patients.

Active DNA demethylation promotes cell fate specification and the DNA damage response.

Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.

Disulfiram Transcends ALDH Inhibitory Activity When Targeting Ovarian Cancer Tumor-Initiating Cells.

Epithelial ovarian cancer (EOC) is a global health burden and remains the fifth leading cause of cancer related death in women worldwide with the poorest five-year survival rate of the gynecological malignancies. EOC recurrence is considered to be driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs). We previously showed that disulfiram, an ALDH inhibitor, effectively targeted TICs compared to adherent EOC cells in terms of viability, spheroid formation, oxidative stress and also prevented relapse in an in vivo model of EOC. In this study we sought to determine whether specific targeting of ALDH isoenzyme ALDH1A1 would provide similar benefit to broader pathway inhibition by disulfiram. NCT-505 and NCT-506 are isoenzyme-specific ALDH1A1 inhibitors whose activity was compared to the effects of disulfiram. Following treatment with both the NCTs and disulfiram, the viability of TICs versus adherent cells, sphere formation, and cell death in our in vitro relapse model were measured and compared in EOC cell lines. We found that disulfiram decreased the viability of TICs significantly more effectively versus adherent cells, while no consistent trend was observed when the cells were treated with the NCTs. Disulfiram also affected the expression of proteins associated with NFκB signaling. Comparison of disulfiram to the direct targeting of ALDH1A1 with the NCTs suggests that the broader cellular effects of disulfiram are more suitable as a therapeutic to eradicate TICs from tumors and prevent EOC relapse. In addition to providing insight into a fitting treatment for TICs, the comparison of disulfiram to NCT-505 and -506 has increased our understanding of the mechanism of action of disulfiram. Further elucidation of the mechanism of disulfiram has the potential to reveal additional targets to treat EOC TICs and prevent disease recurrence.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth.

The tyrosine phosphatase SHP2 is oncogenic in cancers driven by receptor-tyrosine-kinases, and SHP2 inhibition reduces tumor growth. Here, we report that SHP2 is an essential promoter of endothelial cell survival and growth in the remodeling tumor vasculature. Using genetic and chemical approaches to inhibit SHP2 activity in endothelial cells, we show that SHP2 inhibits pro-apoptotic STAT3 and stimulates proliferative ERK1/2 signaling. Systemic SHP2 inhibition in mice bearing tumor types selected for SHP2-independent tumor cell growth promotes degeneration of the tumor vasculature and blood extravasation; reduces tumor vascularity and blood perfusion; and increases tumor necrosis. Reduction of tumor growth ensues, independent of SHP2 targeting in the tumor cells, blocking immune checkpoints, or recruiting macrophages. We also show that inhibiting the Angiopoietin/TIE2/AKT cascade magnifies the vascular and anti-tumor effects of SHP2 inhibition by blocking tumor endothelial AKT signaling, not a target of SHP2. Since the SHP2 and Ang2/TIE2 pathways are active in vascular endothelial cells of human melanoma and colon carcinoma, SHP2 inhibitors alone or with Ang2/TIE2 inhibitors hold promise to effectively target the tumor endothelium.

ZBP1 not RIPK1 mediates tumor necroptosis in breast cancer.

Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. Although several key necroptosis regulators including receptor interacting protein kinase 1 (RIPK1) have been identified, the regulation of tumor necroptosis during tumor development remains elusive. Here, we report that Z-DNA-binding protein 1 (ZBP1), not RIPK1, mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in necrotic tumors. Importantly, ZBP1, not RIPK1, deletion blocks tumor necroptosis during tumor development and inhibits metastasis. We showed that glucose deprivation triggers ZBP1-depedent necroptosis in tumor cells. Glucose deprivation causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.

IDH1 mutations induce organelle defects via dysregulated phospholipids.

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

Thyroid Hormone Receptor α Mutations Cause Heart Defects in Zebrafish.

Background: Mutations of thyroid hormone receptor α1 (TRα1) cause resistance to thyroid hormone (RTHα). Patients exhibit growth retardation, delayed bone development, anemia, and bradycardia. By using mouse models of RTHα, much has been learned about the molecular actions of TRα1 mutants that underlie these abnormalities in adults. Using zebrafish models of RTHα that we have recently created, we aimed to understand how TRα1 mutants affect the heart function during this period. Methods: In contrast to human and mice, the thra gene is duplicated, thraa and thrab, in zebrafish. Using CRISPR/Cas9-mediated targeted mutagenesis, we created C-terminal mutations in each of two duplicated thra genes in zebrafish (thraa 8-bp insertion or thrab 1-bp insertion mutations). We recently showed that these mutant fish faithfully recapitulated growth retardation as found in patients and thra mutant mice. In the present study, we used histological analysis, gene expression profiles, confocal fluorescence, and transmission electron microscopy (TEM) to comprehensively analyze the phenotypic characteristics of mutant fish heart during development. Results: We found both a dilated atrium and an abnormally shaped ventricle in adult mutant fish. The retention of red blood cells in the two abnormal heart chambers, and the decreased circulating blood speed and reduced expression of contractile genes indicated weakened contractility in the heart of mutant fish. These abnormalities were detected in mutant fish as early as 35 days postfertilization (juveniles). Furthermore, the expression of genes associated with the sarcomere assembly was suppressed in the heart of mutant fish, resulting in abnormalities of sarcomere organization as revealed by TEM, suggesting that the abnormal sarcomere organization could underlie the bradycardia exhibited in mutant fish. Conclusions: Using a zebrafish model of RTHα, the present study demonstrated for the first time that TRα1 mutants could act to cause abnormal heart structure, weaken contractility, and disrupt sarcomere organization that affect heart functions. These findings provide new insights into the bradycardia found in RTHα patients.

Phase separation of the Cep63•Cep152 complex underlies the formation of dynamic supramolecular self-assemblies at human centrosomes.

The centrosome is a unique membraneless organelle that plays a pivotal role in the orderly progression of the cell cycle in animal cells. It has been shown that two pericentriolar scaffold proteins, Cep63 and Cep152, generate a heterotetrameric complex to self-assemble into a higher-order cylindrical architecture around a centriole. However, the mechanisms underlying how they reach their threshold concentrations in the vast intracellular space and generate a self-assembled architecture remain mysterious. Here we demonstrate that, like liquid-like assemblies, Cep63 and Cep152 cooperatively generate amorphous aggregates capable of undergoing dynamic turnover and inter-aggregate fusion in vivo and a significant level of internal rearrangemefnt within a condensate in vitro. Consistently, 1,6-hexanediol, a liquid-liquid phase separation disruptor, greatly diminished the ability of endogenous Cep63 and Cep152 to localize to centrosomes. Interestingly, a purified Cep63•Cep152 complex generated either a cylindrical structure or a vesicle-like hollow sphere in a spatially controlled manner. It also formed condensate-like solid spheres in the presence of a macromolecular crowder. At the molecular level, two hydrophobic motifs, one each from Cep63 and Cep152, were required for generating phase-separating condensates and a high molecular-weight assembly. Thus, we propose that the self-assembly of the Cep63•Cep152 complex is triggered by an intrinsic property of the complex undergoing density transition through the hydrophobic-motif-mediated phase separation. Abbreviations: PCM, pericentriolar material; LLPS, liquid-liquid phase separation; MW, molecular-weight; CLEM, correlative light and electron microscopy; WT, wild-type; CMV, cytomegalovirus; FRAP, fluorescence recovery after photobleaching; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; 3D-SIM, three-dimensional structured illumination microscopy; DMEM, Dulbecco's Modified Eagle Medium; PEI Max, Polyethylenimine Max; PBS, phosphate-buffered saline; RT, room temperature; DAPI, 4', 6-diamidino-2-phenylindole; AOTF, acousto-optic tunable filter; LB, Luria broth; OD, optical density; IPTG, isopropyl ß-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Determination of the Expression of PD-L1 in the Morphologic Spectrum of Renal Cell Carcinoma.

Immunotherapy is reportedly an effective form of therapy for some advanced cancers such as lung adenocarcinoma, malignant melanoma and colorectal adenocarcinoma. In renal cell carcinoma (RCC), the role of immunotherapy is under investigation. Programmed Death-Ligand 1 (PD-L1) is a molecule expressed on the surface of certain tumor cells and binds to the Programmed cell death protein 1 (PD-1) on cytotoxic T-cells, an interaction that inhibits the antitumor immune response. The aim of this study is to evaluate PD-L1 expression in the morphologic spectrum of RCC. A total of 172 cases of RCC comprising all types were studied and the PD-L1 was correlated with immune response for CD4 and CD8. Positive membranous staining for PD-L1 was seen in 59 (34%) of the 172 samples. The positive cases were HLRCC (31/53), Type 1 Papillary RCC (10/31), Chromophobe (7/20), Hybrid (3/9), TFE-3 related cancer (3/8), Undifferentiated (3/5), and TFEB tumors (2/2). Clear cell carcinomas, Oncocytomas and SDHB deficient-RCC didn't show any expression of PD-L1; (0/34;0/7;0/3). Our results demonstrated that aggressive forms of RCC such as HLRCC have high expression of PD-L1, in contrast to clear cell renal carcinomas. Our findings support a possible role of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive RCC.