Effectors Enabling Adaptation to Mitochondrial Complex I Loss in Hürthle Cell Carcinoma.

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.

Comparative Transcriptomic Analysis of Archival Human Vestibular Schwannoma Tissue from Patients with and without Tinnitus.

Vestibular schwannoma (VS) is an intracranial tumor that commonly presents with tinnitus and hearing loss. To uncover the molecular mechanisms underlying VS-associated tinnitus, we applied next-generation sequencing (Illumina HiSeq) to formalin-fixed paraffin-embedded archival VS samples from nine patients with tinnitus (VS-Tin) and seven patients without tinnitus (VS-NoTin). Bioinformatic analysis was used to detect differentially expressed genes (DEG; i.e., ≥two-fold change [FC]) while correcting for multiple comparisons. Using RNA-seq analysis, VS-Tin had significantly lower expression of GFAP (logFC = -3.04), APLNR (logFC = -2.95), PREX2 (logFC = -1.44), and PLVAP (logFC = -1.04; all p < 0.01) vs. VS-NoTin. These trends were validated by using real-time RT-qPCR. At the protein level, immunohistochemistry revealed a trend for less PREX2 and apelin expression and greater expression of NLRP3 inflammasome and CD68-positive macrophages in VS-Tin than in VS-NoTin, suggesting the activation of inflammatory processes in VS-Tin. Functional enrichment analysis revealed that the top three protein categories-glycoproteins, signal peptides, and secreted proteins-were significantly enriched in VS-Tin in comparison with VS-NoTin. In a gene set enrichment analysis, the top pathway was allograft rejection, an inflammatory pathway that includes the MMP9, CXCL9, IL16, PF4, ITK, and ACVR2A genes. Future studies are needed to examine the importance of these candidates and of inflammation in VS-associated tinnitus.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.

Transcriptomic signature of painful human neurofibromatosis type 2 schwannomas.

Schwannomas are benign neoplasms that can cause gain- and loss-of-function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma-associated pain we compared the RNA sequencing profile of painful and non-painful schwannomas from NF2 patients. Distinct segregation of painful and non-painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2-schwannoma model in nude mice. In this model, over-expression of FGF7 in intra-sciatically implanted NF2 tumor cells generated pain behavior compared with controls.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Distinct functions of tissue-resident and circulating memory Th2 cells in allergic airway disease.

Memory CD4+ T helper type 2 (Th2) cells drive allergic asthma, yet the mechanisms whereby tissue-resident memory Th2 (Th2 Trm) cells and circulating memory Th2 cells collaborate in vivo remain unclear. Using a house dust mite (HDM) model of allergic asthma and parabiosis, we demonstrate that Th2 Trm cells and circulating memory Th2 cells perform nonredundant functions. Upon HDM rechallenge, circulating memory Th2 cells trafficked into the lung parenchyma and ignited perivascular inflammation to promote eosinophil and CD4+ T cell recruitment. In contrast, Th2 Trm cells proliferated near airways and induced mucus metaplasia, airway hyperresponsiveness, and airway eosinophil activation. Transcriptional analysis revealed that Th2 Trm cells and circulating memory Th2 cells share a core Th2 gene signature but also exhibit distinct transcriptional profiles. Th2 Trm cells express a tissue-adaptation signature, including genes involved in regulating and interacting with extracellular matrix. Our findings demonstrate that Th2 Trm cells and circulating memory Th2 cells are functionally and transcriptionally distinct subsets with unique roles in promoting allergic airway disease.