Spatial Transcriptomics Identifies Expression Signatures Specific to Lacrimal Gland Adenoid Cystic Carcinoma Cells.

Although primary tumors of the lacrimal gland are rare, adenoid cystic carcinoma (ACC) is the most common and lethal epithelial lacrimal gland malignancy. Traditional management of lacrimal gland adenoid cystic carcinoma (LGACC) involves the removal of the eye and surrounding socket contents, followed by chemoradiation. Even with this radical treatment, the 10-year survival rate for LGACC is 20% given the propensity for recurrence and metastasis. Due to the rarity of LGACC, its pathobiology is not well-understood, leading to difficulties in diagnosis, treatment, and effective management. Here, we integrate bulk RNA sequencing (RNA-seq) and spatial transcriptomics to identify a specific LGACC gene signature that can inform novel targeted therapies. Of the 3499 differentially expressed genes identified by bulk RNA-seq, the results of our spatial transcriptomic analysis reveal 15 upregulated and 12 downregulated genes that specifically arise from LGACC cells, whereas fibroblasts, reactive fibrotic tissue, and nervous and skeletal muscle account for the remaining bulk RNA-seq signature. In light of the analysis, we identified a transitional state cell or stem cell cluster. The results of the pathway analysis identified the upregulation of PI3K-Akt signaling, IL-17 signaling, and multiple other cancer pathways. This study provides insights into the molecular and cellular landscape of LGACC, which can inform new, targeted therapies to improve patient outcomes.

Early Mechanisms of Chemoresistance in Retinoblastoma.

Retinoblastoma is the most common eye cancer in children and is fatal if left untreated. Over the past three decades, chemotherapy has become the mainstay of eye-sparing treatment. Nevertheless, chemoresistance continues to represent a major challenge leading to ocular and systemic toxicity, vision loss, and treatment failure. Unfortunately, the mechanisms leading to chemoresistance remain incompletely understood. Here, we engineered low-passage human retinoblastoma cells to study the early molecular mechanisms leading to resistance to carboplatin, one of the most widely used agents for treating retinoblastoma. Using single-cell next-generation RNA sequencing (scRNA-seq) and single-cell barcoding technologies, we found that carboplatin induced rapid transcriptomic reprogramming associated with the upregulation of PI3K-AKT pathway targets, including ABC transporters and metabolic regulators. Several of these targets are amenable to pharmacologic inhibition, which may reduce the emergence of chemoresistance. We provide evidence to support this hypothesis using a third-generation inhibitor of the ABCB1 transporter.

Dual Screen for Efficacy and Toxicity Identifies HDAC Inhibitor with Distinctive Activity Spectrum for BAP1-Mutant Uveal Melanoma.

Drug screens leading to successful targeted therapies in cancer have been mainly based on cell viability assays identifying inhibitors of dominantly acting oncogenes. In contrast, there has been little success in discovering targeted therapies that reverse the effects of inactivating mutations in tumor-suppressor genes. BAP1 is one such tumor suppressor that is frequently inactivated in a variety of cancers, including uveal melanoma, renal cell carcinoma, and mesothelioma. Because BAP1 is an epigenetic transcriptional regulator of developmental genes, we designed a two-phase drug screen involving a cell-based rescue screen of transcriptional repression caused by BAP1 loss, followed by an in vivo screen of lead compounds for rescue of a BAP1-deficient phenotype with minimal toxicity in Xenopus embryos. The first screen identified 9 compounds, 8 of which were HDAC inhibitors. The second screen eliminated all except one compound due to inefficacy or toxicity. The resulting lead compound, quisinostat, has a distinctive activity spectrum, including high potency against HDAC4, which was recently shown to be a key target of BAP1. Quisinostat was further validated in a mouse model and found to prevent the growth of BAP1-mutant uveal melanomas. This innovative strategy demonstrates the potential for identifying therapeutic compounds that target tumor-suppressor mutations in cancer. IMPLICATIONS: Few drugs have been identified that target mutations in tumor suppressors. Using a novel 2-step screening approach, strategy, we identified quisinostat as a candidate for therapy in BAP1-mutant uveal melanoma. HDAC4 is implicated as a key target in uveal melanoma and perhaps other BAP1-mutant cancers.

BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

Methods for Isolating the Balbiani Body/Germplasm from Xenopus laevis Oocytes.

The Balbiani body (Bb) is a large membrane-less organelle, densely packed with mitochondria, endoplasmic reticulum, proteins, and RNA. The Bb is present in many vertebrate female gametes. In frogs, the Bb is established early during oogenesis and operates as a maternal inherited embryonic determinant that specifies germline identity through the formation of germplasm. We describe here two techniques to isolate the Bb/germplasm from Xenopus laevis primary oocytes.

Combined functions of two RRMs in Dead-end1 mimic helicase activity to promote nanos1 translation in the germline.

Dead-end1 (Dnd1) expression is restricted to the vertebrate germline where it is believed to activate translation of messenger RNAs (mRNAs) required to protect and promote that unique lineage. Nanos1 is one such germline mRNA whose translation is blocked by a secondary mRNA structure within the open reading frame (ORF). Dnd1 contains a canonical RNA recognition motif (RRM1) in its N-terminus but also contains a less conserved RRM2. Here we provide a mechanistic picture of the nanos1 mRNA-Dnd1 interaction in the Xenopus germline. We show that RRM1, but not RRM2, is required for binding nanos1. Similar to the zebrafish homolog, Xenopus Dnd1 possesses ATPase activity. Surprisingly, this activity appears to be within the RRM2, different from the C-terminal region where it is found in zebrafish. More importantly, we show that RRM2 is required for nanos1 translation and germline survival. Further, Dnd1 functions as a homodimer and binds nanos1 mRNA just downstream of the secondary structure required for nanos1 repression. We propose a model in which the RRM1 is required to bind nanos1 mRNA while the RRM2 is required to promote translation through the action of ATPase. Dnd1 appears to use RRMs to mimic the function of helicases.

A novel role for sox7 in Xenopus early primordial germ cell development: mining the PGC transcriptome.

Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation, and PGCs subsequently initiate zygotic transcription. However, the gene network(s) that operate to both preserve and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell mRNAs after lineage segregation. We identified 1865 transcripts enriched in PGCs compared with endoderm cells. We next compared the PGC-enriched transcripts with previously identified maternal, vegetally enriched transcripts and found that ∼38% of maternal transcripts were enriched in PGCs, including sox7 PGC-directed sox7 knockdown and overexpression studies revealed an early requirement for sox7 in germ plasm localization, zygotic transcription and PGC number. We identified pou5f3.3 as the most highly expressed and enriched POU5F1 homolog in PGCs. We compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of genes are conserved, underscoring the potential usefulness of Xenopus for understanding human germline specification.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration.

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.

Trends in Nanomaterial-Based Non-Invasive Diabetes Sensing Technologies.

Blood glucose monitoring is considered the gold standard for diabetes diagnostics and self-monitoring. However, the underlying process is invasive and highly uncomfortable for patients. Furthermore, the process must be completed several times a day to successfully manage the disease, which greatly contributes to the massive need for non-invasive monitoring options. Human serums, such as saliva, sweat, breath, urine and tears, contain traces of glucose and are easily accessible. Therefore, they allow minimal to non-invasive glucose monitoring, making them attractive alternatives to blood measurements. Numerous developments regarding noninvasive glucose detection techniques have taken place over the years, but recently, they have gained recognition as viable alternatives, due to the advent of nanotechnology-based sensors. Such sensors are optimal for testing the amount of glucose in serums other than blood thanks to their enhanced sensitivity and selectivity ranges, in addition to their size and compatibility with electronic circuitry. These nanotechnology approaches are rapidly evolving, and new techniques are constantly emerging. Hence, this manuscript aims to review current and future nanomaterial-based technologies utilizing saliva, sweat, breath and tears as a diagnostic medium for diabetes monitoring.