Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

How Much Are Borrowers Willing to Pay to Remove Uncertainty Surrounding Mortgage Defaults?

Using a large, non-student sample, we assess and differentiate between borrowers' Risk Aversion and Ambiguity Aversion levels and their willingness to pay to resolve a mortgage default settlement negotiation. Ambiguity Aversion is found to be negatively associated with willingness to pay for borrowers with high financial literacy in both the gain and loss domains, whereas personality traits matter more for borrowers with low financial literacy. This finding is important to policymakers in that they should adopt differential resolution strategies for defaulting borrowers based on these intervening variables.

Reducing Strategic Forbearance under the CARES Act: an Experimental Approach Utilizing Recourse Attestation.

The Coronavirus Aid, Relief, and Economic Security (CARES) Act was passed in response to both the global pandemic's immediate negative and expected long-lasting impacts on the economy. Under the Act, mortgage borrowers are allowed to cease making payments if their income was negatively impacted by Covid-19. Importantly, borrowers were not required to demonstrate proof of impaction, either currently or retrospectively. Exploring the economic implications of this policy, this study uses an experimental design to first identify strategic forbearance incidence, and then to quantify where the forborne mortgage payment dollars were spent. Our results suggest strategic mortgage forbearance can be significantly reduced, saving taxpayers billions of dollars in potential losses, simply by requiring a 1-page attestation with lender recourse for borrowers wishing to engage in COVID-19 related mortgage payment cessation programs. Additionally, we demonstrate the use of these forborne mortgage payments range from enhancing the financial safety net for distressed borrowers by increasing precautionary savings, to buying necessities, to equity investing and debt consolidation.

Three-dimensional evaluation of subsurface damage in optical glasses with ground and polished surfaces using FF-OCT.

Subsurface damage (SSD) induced during conventional manufacturing of optics contributes mainly to a reduction in the performance and quality of optics. In this paper, we propose the application of full-field optical coherence tomography (FF-OCT) as a high-resolution and nondestructive method for evaluation of SSD in optical substrates. Both ground and polished surfaces can be successfully imaged, providing a path to control SSD throughout the entire optics manufacturing process chain. Full tomograms are acquired for qualitative and quantitative analyses of both surface and SSD. The main requirements for the detection of SSD are addressed. Data processing allows the removal of low-intensity image errors and the automatic evaluation of SSD depths. OCT scans are carried out on destructively referenced glass samples and compared to existing predictive models, validating the obtained results. Finally, intensity projection methods and depth maps are applied to characterize crack morphologies. The experiments highlight differences in crack characteristics between optical glasses SF6 and HPFS7980 and illustrate that wet etching can enhance three-dimensional imaging of SSD with FF-OCT.

BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs.

The differentiation of several T- and B-cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodelling. Here we report that promyelocytic leukaemia zinc finger (PLZF), the BTB-zinc finger (BTB-ZF) transcription factor directing the innate-like effector program of natural killer T-cell thymocytes, is prominently associated with cullin 3 (CUL3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF transports CUL3 to the nucleus, where the two proteins are associated within a chromatin-modifying complex. Furthermore, PLZF expression results in selective ubiquitination changes of several components of this complex. CUL3 was also found associated with the BTB-ZF transcription factor BCL6, which directs the germinal-centre B cell and follicular T-helper cell programs. Conditional CUL3 deletion in mice demonstrated an essential role for CUL3 in the development of PLZF- and BCL6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit CUL3 to alter the ubiquitination pattern of their associated chromatin-modifying complex. We propose that this new function is essential to direct the differentiation of several T- and B-cell effector programs, and may also be involved in the oncogenic role of PLZF and BCL6 in leukaemias and lymphomas.

Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.

BACKGROUND: Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. METHODS: Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. RESULTS: The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. CONCLUSIONS: ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.

Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS and AML.

Splicing modulation is a promising treatment strategy pursued to date only in splicing factor-mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we showed that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing factor 3B subunit 1 (SF3B1) H3B-8800 and E-7107. We identified drug-induced alterations in splicing, and corresponding reduced gene expression, of a number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We found that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators due to their long length and large number of exons per transcript. Furthermore, we demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators not only resulted in impaired DNA damage response and accumulation of DNA damage, but it sensitized cells to subsequent killing by poly(ADP-ribose) polymerase (PARP) inhibitors and chemotherapy and led to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML.

Extended outlook: description, utilization, and daily applications of cloud technology in radiology.

OBJECTIVE: The purpose of this article is to discuss the concept of cloud technology, its role in medical applications and radiology, the role of the radiologist in using and accessing these vast resources of information, and privacy concerns and HIPAA compliance strategies. CONCLUSION: Cloud computing is the delivery of shared resources, software, and information to computers and other devices as a metered service. This technology has a promising role in the sharing of patient medical information and appears to be particularly suited for application in radiology, given the field's inherent need for storage and access to large amounts of data. The radiology cloud has significant strengths, such as providing centralized storage and access, reducing unnecessary repeat radiologic studies, and potentially allowing radiologic second opinions more easily. There are significant cost advantages to cloud computing because of a decreased need for infrastructure and equipment by the institution. Private clouds may be used to ensure secure storage of data and compliance with HIPAA. In choosing a cloud service, there are important aspects, such as disaster recovery plans, uptime, and security audits, that must be considered. Given that the field of radiology has become almost exclusively digital in recent years, the future of secure storage and easy access to imaging studies lies within cloud computing technology.

Modification of Polymeric Surfaces with Ultrashort Laser Pulses for the Selective Deposition of Homogeneous Metallic Conductive Layers.

In recent years, the demand for highly integrated and lightweight components has been rising sharply, especially in plastics processing. One strategy for weight-saving solutions is the development of conductive tracks and layouts directly on the polymer housing parts in order to be able to dispense with the system integration of additional printed circuit boards (PCB). This can be conducted very advantageously and flexibly with laser-based processes for functionalizing polymer surfaces. In this work, a three-step laser-based process for subsequent selective metallization is presented. Conventional injection molded components without special additives serve as the initial substrate. The Laser-Based Selective Activation (LSA) uses picosecond laser pulses to activate the plastic surface to subsequently deposit palladium. The focus is on determining the amount of deposited palladium in correlation to the laser and scan parameters. For the first time, the dependence of the metallization result on the accumulated laser fluence (Facc) is described. The treated polymer parts are characterized using optical and scanning electron microscopy as well as a contact-type profilometer.

Reprogramming RNA processing: an emerging therapeutic landscape.

The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape.

Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275.

Towards improved cancer diagnosis and prognosis using analysis of gene expression data and computer aided imaging.

With the increasing cost effectiveness of whole slide digital scanners, gene expression microarray and SNP technologies, tissue specimens can now be analyzed using sophisticated computer aided image and data analysis techniques for accurate diagnoses and identification of prognostic markers and potential targets for therapeutic intervention. Microarray analysis is routinely able to identify biomarkers correlated with survival and reveal pathways underlying pathogenesis and invasion. In this paper we describe how microarray profiling of tumor samples combined with simple but powerful methods of analysis can identify biologically distinct disease subclasses of breast cancer with distinct molecular signatures, differential recurrence rates and potentially, very different response to therapy. Image analysis methods are also rapidly finding application in the clinic, complementing the pathologist in quantitative, reproducible, detection, staging, and grading of disease. We will describe novel computerized image analysis techniques and machine learning tools for automated cancer detection from digitized histopathology and how they can be employed for disease diagnosis and prognosis for prostate and breast cancer.

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone.

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a "cold" tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer.

Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators.

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased Inner Retinal Layer Reflectivity in Eyes With Acute CRVO Correlates With Worse Visual Outcomes at 12 Months.

Purpose: To determine if inner retinal layer reflectivity in eyes with acute central retinal vein occlusion (CRVO) correlates with visual acuity at 12 months. Methods: Macular optical coherence tomography (OCT) scans were obtained from 22 eyes of 22 patients with acute CRVO. Optical intensity ratios (OIRs), defined as the mean OCT reflectivity of the inner retinal layers normalized to the mean reflectivity of the RPE, were measured from the presenting and 1-month OCT image by both manual measurements of grayscale B-scans and custom algorithmic measurement of raw OCT volume data. OIRs were assessed for association with final visual outcome. Cohort subgroup division for analysis was determined statistically. Results: Eyes with poorer final visual acuity (≥20/70) at 1 year were more likely to have a higher ganglion cell layer OIR than eyes with better final visual acuity (<20/70) at 1 month (manually: 0.591 to 0.735, P = 0.006, algorithmically: 0.663 to 0.799, P = 0.014). At 1 month, eyes with a poorer final visual acuity demonstrated a higher variance of OIR measurements (algorithmically: 0.087 vs. 0.160, P = 0.002) per scan than eyes with better final visual acuity. Conclusions: In acute CRVO, ganglion cell layer changes at 1 month, including increased reflectivity and increased heterogeneity of reflectivity signal as expressed as OIR and OIR variance, were associated with a poorer visual prognosis at 1 year. Technique calibration with larger sample sizes and automated integration into OCT platforms will be necessary to determine if OIR can be a clinically useful prognostic tool.

Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types.

Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis.

Immunosuppressive Therapy Improves Both Short- and Long-Term Prognosis in Patients With Virus-Negative Nonfulminant Inflammatory Cardiomyopathy.

BACKGROUND: Inflammatory cardiomyopathy (infl-CMP) is characterized by increased cardiac inflammation in the absence of viruses, ischemia, valvular disease, or other apparent causes. Studies addressing the efficacy of immunosuppressive therapy in patients with infl-CMP are sparse. This study retrospectively investigates whether immunosuppressive agents on top of heart failure therapy according to current guidelines improves cardiac function and long-term outcome in patients with infl-CMP. METHODS AND RESULTS: Within the Innsbruck and Maastricht Cardiomyopathy Registry, a total of 209 patients fulfilled the criteria for infl-CMP using endomyocardial biopsy (≥14 infiltrating inflammatory cells/mm2). A total of 110 (53%) patients received immunosuppressive therapy and 99 (47%) did not. To correct for potential selection bias, 1:1 propensity score matching was used on all significant baseline parameters, resulting in a total of 90 patients per group. Baseline characteristics did not significantly differ between both patient groups, reflecting optimal propensity score matching. After a median follow-up of 31 (15-47) months, immunosuppressive therapy resulted in an improved long-term outcome (eg, heart transplantation-free survival) as compared with standard heart failure therapy alone (Log-rank P=0.043; hazard ratio, 0.34 [95% CI, 0.17-0.92]) and in a significant larger increase of left ventricular ejection fraction after a mean of 12 months follow-up, as compared with patients receiving standard heart failure treatment only (12.2% versus 7.3%, respectively; P=0.036). CONCLUSIONS: To conclude, this study suggests that immunosuppressive therapy in infl-CMP patients results in an improved heart transplantation-free survival as compared with standard heart failure therapy alone, underscoring the urgent need for a large prospective multicenter trial.

Genome-wide CRISPR-Cas9 Screen Identifies Leukemia-Specific Dependence on a Pre-mRNA Metabolic Pathway Regulated by DCPS.

To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy.

Splicing modulation sensitizes chronic lymphocytic leukemia cells to venetoclax by remodeling mitochondrial apoptotic dependencies.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eµ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.