Performance comparison of high throughput single-cell RNA-Seq platforms in complex tissues.

Single-cell transcriptomics has emerged as the preferred tool to define cell identity through the analysis of gene expression signatures. However, there are limited studies that have comprehensively compared the performance of different scRNAseq systems in complex tissues. Here, we present a systematic comparison of two well-established high throughput 3'-scRNAseq platforms: 10× Chromium and BD Rhapsody, using tumours that present high cell diversity. Our experimental design includes both fresh and artificially damaged samples from the same tumours, which also provides a comparable dataset to examine their performance under challenging conditions. The performance metrics used in this study consist of gene sensitivity, mitochondrial content, reproducibility, clustering capabilities, cell type representation and ambient RNA contamination. These analyses showed that BD Rhapsody and 10× Chromium have similar gene sensitivity, while BD Rhapsody has the highest mitochondrial content. Interestingly, we found cell type detection biases between platforms, including a lower proportion of endothelial and myofibroblast cells in BD Rhapsody and lower gene sensitivity in granulocytes for 10× Chromium. Moreover, the source of the ambient noise was different between plate-based and droplet-based platforms. In conclusion, our reported platform differential performance should be considered for the selection of the scRNAseq method during the study experimental designs.

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

ALTEN: A High-Fidelity Primary Tissue-Engineering Platform to Assess Cellular Responses Ex Vivo.

To fully investigate cellular responses to stimuli and perturbations within tissues, it is essential to replicate the complex molecular interactions within the local microenvironment of cellular niches. Here, the authors introduce Alginate-based tissue engineering (ALTEN), a biomimetic tissue platform that allows ex vivo analysis of explanted tissue biopsies. This method preserves the original characteristics of the source tissue's cellular milieu, allowing multiple and diverse cell types to be maintained over an extended period of time. As a result, ALTEN enables rapid and faithful characterization of perturbations across specific cell types within a tissue. Importantly, using single-cell genomics, this approach provides integrated cellular responses at the resolution of individual cells. ALTEN is a powerful tool for the analysis of cellular responses upon exposure to cytotoxic agents and immunomodulators. Additionally, ALTEN's scalability using automated microfluidic devices for tissue encapsulation and subsequent transport, to enable centralized high-throughput analysis of samples gathered by large-scale multicenter studies, is shown.

The H2A.Z-nuclesome code in mammals: emerging functions.

H2A.Z is a histone variant that provides specific structural and docking-side properties to the nucleosome, resulting in diverse and specialised molecular and cellular functions. In this review, we discuss the latest studies uncovering new functional aspects of mammalian H2A.Z in gene transcription, including pausing and elongation of RNA polymerase II (RNAPII) and enhancer activity; DNA repair; DNA replication; and 3D chromatin structure. We also review the recently described role of H2A.Z in embryonic development, cell differentiation, neurodevelopment, and brain function. In conclusion, our cumulative knowledge of H2A.Z over the past 40 years, in combination with the implementation of novel molecular technologies, is unravelling an unexpected and complex role of histone variants in gene regulation and disease.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Cell Lineage Tracing Identifies Hormone-Regulated and Wnt-Responsive Vaginal Epithelial Stem Cells.

The intact vaginal epithelium is essential for women's reproductive health and provides protection against HIV and sexually transmitted infections. How this epithelium maintains itself remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) to define the diverse cell populations in the vaginal epithelium. We show that vaginal epithelial cell proliferation is limited to the basal compartment without any obvious label-retaining cells. Furthermore, we developed vaginal organoids and show that the basal cells have increased organoid forming efficiency. Importantly, Axin2 marks a self-renewing subpopulation of basal cells that gives rise to differentiated cells over time. These cells are ovariectomy-resistant stem cells as they proliferate even in the absence of hormones. Upon hormone supplementation, these cells expand and reconstitute the entire vaginal epithelium. Wnt/ß-catenin is essential for the proliferation and differentiation of vaginal stem cells. Together, these data define heterogeneity in vaginal epithelium and identify vaginal epithelial stem cells.

A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation.

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.