MATRIN3 deficiency triggers autoinflammation via cGAS-STING activation.

Interferon-stimulated genes (ISGs) comprise a program of immune effectors important for host immune defense. When uncontrolled, ISGs play a central role in interferonopathies and other inflammatory diseases. The mechanisms responsible for turning on ISGs are not completely known. By investigating MATRIN3 (MATR3), a nuclear RNA-binding protein mutated in familial ALS, we found that perturbing MATR3 results in elevated expression of ISGs. Using an integrative approach, we elucidate a pathway that leads to activation of cGAS-STING. This outlines a plausible mechanism for pathogenesis in a subset of ALS, and suggests new diagnostic and therapeutic approaches for this fatal disease.

TGF-ß and BMP signaling are associated with the transformation of glioblastoma to gliosarcoma and then osteosarcoma.

Background: Gliosarcoma, an isocitrate dehydrogenase wildtype (IDH-WT) variant of glioblastoma, is defined by clonal biphasic differentiation into gliomatous and sarcomatous components. While the transformation from a glioblastoma to gliosarcoma is uncommon, the subsequent transformation to osteosarcoma is rare but may provide additional insights into the biology of these typically distinct cancers. We observed a patient initially diagnosed with glioblastoma, that differentiated into gliosarcoma at recurrence, and further evolved to osteosarcoma at the second relapse. Our objective was to characterize the molecular mechanisms of tumor progression associated with this phenotypic transformation. Methods: Tumor samples were collected at all 3 stages of disease and RNA sequencing was performed to capture their transcriptomic profiles. Sequential clonal evolution was confirmed by the maintenance of an identical PTEN mutation throughout the tumor differentiation using the TSO500 gene panel. Publicly available datasets and the Nanostring nCounter technology were used to validate the results. Results: The glioblastoma tumor from this patient possessed mixed features of all 3 TCGA-defined transcriptomic subtypes of an IDH-WT glioblastoma and a proportion of osteosarcoma signatures were upregulated in the original tumor. Analysis showed that enhanced transforming growth factor-ß (TGF-ß) and bone morphogenic protein signaling was associated with tumor transformation. Regulatory network analysis revealed that TGF-ß family signaling committed the lineage tumor to osteogenesis by stimulating the expression of runt-related transcription factor 2 (RUNX2), a master regulator of bone formation. Conclusions: This unusual clinical case provided an opportunity to explore the modulators of longitudinal sarcomatous transformation, potentially uncovering markers indicating predisposition to this change and identification of novel therapeutic targets.

Cell-free chromatin immunoprecipitation to detect molecular pathways in heart transplantation.

Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.

Cell-free Chromatin Immunoprecipitation to detect molecular pathways in Physiological and Disease States.

Patient monitoring is a cornerstone in clinical practice to define disease phenotypes and guide clinical management. Unfortunately, this is often reliant on invasive and/or less sensitive methods that do not provide deep phenotype assessments of disease state to guide treatment. This paper examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) to define molecular gene sets in physiological and heart transplant patients taking immunosuppression medications. We show cfChIP-seq reliably detect gene signals that correlate with gene expression. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of the relevant immune and non-immune molecular pathways that were predominantly downregulated in immunosuppressed heart transplant patients compared to healthy controls. cfChIP-seq also identified tissue sources of cfDNA, detecting greater cell-free DNA from cardiac, hematopoietic, and other non-hematopoietic tissues such as the pulmonary, digestive, and neurological tissues in transplant patients than healthy controls. cfChIP-seq gene signals were reproducible between patient populations and blood collection methods. cfChIP-seq may therefore be a reliable approach to provide dynamic assessments of molecular pathways and tissue injury associated to disease.

Creatine riboside is a cancer cell-derived metabolite associated with arginine auxotrophy.

The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.

Host gene expression modulated by Zika virus infection of human-293 cells.

The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58, CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.

Cross-resistance of cisplatin selected cells to anti-microtubule agents: Role of general survival mechanisms.

Although the first line of therapy for epithelial ovarian cancer typically consists of taxane-platinum combination therapy, many patients develop a platinum-resistant tumor within a year. Several previous studies have looked at this cross-resistance between cisplatin and anti-microtubule drugs, but their findings have been somewhat conflicting. Here, we developed cisplatin-resistant cell lines that are resistant to low and high levels of cisplatin and explored the effects of three anti-microtubule drugs (paclitaxel, vincristine, and colchicine) on the parental and cisplatin-resistant cells. We found that cells resistant to lower levels of cisplatin were no more resistant to anti-microtubule drugs than parental cells, while cells that were resistant to higher levels of cisplatin had a subpopulation of cells that were cross-resistant to anti-microtubule drugs, clarifying discrepancies within the field. We then isolated this subpopulation by applying selective pressure with anti-microtubule drugs and performed RNA sequencing and gene set enrichment analysis to identify resistance mechanisms. This subpopulation was found to express increased levels of pro-survival TNF/NFκB signaling, among other enriched pathways, suggesting that cross-resistance was due to more general survival mechanisms found in the cisplatin-selected cells.

Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients.

Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1ß and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.

Whole-Exome Profiling of NSCLC Among African Americans.

INTRODUCTION: Lung cancer incidence is higher among African Americans (AAs) compared with European Americans (EAs) in the United States, especially among men. Although significant progress has been made profiling the genomic makeup of lung cancer in EAs, AAs continue to be underrepresented. Our objective was to chart the genome-wide landscape of somatic mutations in lung cancer tumors from AAs. METHODS: In this study, we used the whole-exome sequencing of 82 tumor and noninvolved tissue pairs from AAs. Patients were selected from an ongoing case-control study conducted by the National Cancer Institute and the University of Maryland. RESULTS: Among all samples, we identified 178 significantly mutated genes (p < 0.05), five of which passed the threshold for false discovery rate (p < 0.1). In lung adenocarcinoma (LUAD) tumors, mutation rates in STK11 (p = 0.05) and RB1 (p = 0.008) were significantly higher in AA LUAD tumors (25% and 13%, respectively) compared with The Cancer Genome Atlas EA samples (14% and 4%, respectively). In squamous cell carcinomas, mutation rates in STK11 (p = 0.002) were significantly higher among AA (8%) than EA tumors from The Cancer Genome Atlas (1%). Integrated somatic mutation data with CIBERSORT (Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts) data analysis revealed LUAD tumors from AAs carrying STK11 mutations have decreased interferon signaling. CONCLUSIONS: Although a considerable degree of the somatic mutation landscape is shared between EAs and AAs, discrete differences in mutation frequency in potentially important oncogenes and tumor suppressors exist. A better understanding of the molecular basis of lung cancer in AA patients and leveraging this information to guide clinical interventions may help reduce disparities.

Protein kinase A inhibitor proteins (PKIs) divert GPCR-Gαs-cAMP signaling toward EPAC and ERK activation and are involved in tumor growth.

The PKA-inhibitor (PKI) family members PKIα, PKIß, and PKIγ bind with high affinity to PKA and block its kinase activity, modulating the extent, and duration of PKA-mediated signaling events. While PKA is a well-known regulator of physiological and oncogenic events, the role of PKI proteins in these pathways has remained elusive. Here, by measuring activation of the MAPK pathway downstream of GPCR-Gαs-cAMP signaling, we show that the expression levels of PKI proteins can alter the balance of activation of two major cAMP targets: PKA and EPAC. Our results indicate that PKA maintains repressive control over MAPK signaling as well as a negative feedback on cAMP concentration. Overexpression of PKI and its subsequent repression of PKA dysregulates these signaling pathways, resulting in increased intracellular cAMP, and enhanced activation of EPAC and MAPK. We also find that amplifications of PKIA are common in prostate cancer and are associated with reduced progression free survival. Depletion of PKIA in prostate cancer cells leads to reduced migration, increased sensitivity to anoikis and reduced tumor growth. By altering PKA activity PKI can act as a molecular switch, driving GPCR-Gαs-cAMP signaling toward activation of EPAC-RAP1 and MAPK, ultimately modulating tumor growth.

Loss of myeloid-specific lamin A/C drives lung metastasis through Gfi-1 and C/EBPε-mediated granulocytic differentiation.

The immune-suppressive tumor microenvironment promotes metastatic spread and outgrowth. One of the major contributors is tumor-associated myeloid cells. However, the molecular mechanisms regulating their differentiation and function are not well understood. Here we report lamin A/C, a nuclear lamina protein associated with chromatin remodeling, was one of the critical regulators in cellular reprogramming of tumor-associated myeloid cells. Using myeloid-specific lamin A/C knockout mice and triple-negative breast cancer (TNBC) mouse models, we discovered that the loss of lamin A/C drives CD11b+ Ly6G+ granulocytic lineage differentiation, alters the production of inflammatory chemokines, decreases host antitumor immunity, and increases metastasis. The underlying mechanisms involve an increased H3K4me3 leading to the upregulation of transcription factors (TFs) Gfi-1 and C/EBPε. Decreased lamin A/C and increased Gfi-1 and C/EBPε were also found in the granulocytic subset in the peripheral blood of human cancer patients. Our data provide a mechanistic understanding of myeloid lineage differentiation and function in the immune-suppressive microenvironment in TNBC metastasis.

Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels.

Brain-derived neurotrophic factor (BDNF) is a potent modulator of brain synaptic plasticity. Signaling defects caused by dysregulation of its Ntrk2 (TrkB) kinase (TrkB.FL) and truncated receptors (TrkB.T1) have been linked to the pathophysiology of several neurological and neurodegenerative disorders. We found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of Rbfox1 in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by Rbfox1 deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes.