Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney.

Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE: Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.

PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size.

Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity, and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C knockout cells, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C knockout cells. In regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control and thus the fidelity of cell division.

Developmental role of PHD2 in the pathogenesis of pseudohypoxic pheochromocytoma.

Despite a general role for the HIF hydroxylase system in cellular oxygen sensing and tumour hypoxia, cancer-associated mutations of genes in this pathway, including PHD2, PHD1, EPAS1 (encoding HIF-2α) are highly tissue-restricted, being observed in pseudohypoxic pheochromocytoma and paraganglioma (PPGL) but rarely, if ever, in other tumours. In an effort to understand that paradox and gain insights into the pathogenesis of pseudohypoxic PPGL, we constructed mice in which the principal HIF prolyl hydroxylase, Phd2, is inactivated in the adrenal medulla using TH-restricted Cre recombinase. Investigation of these animals revealed a gene expression pattern closely mimicking that of pseudohypoxic PPGL. Spatially resolved analyses demonstrated a binary distribution of two contrasting patterns of gene expression among adrenal medullary cells. Phd2 inactivation resulted in a marked shift in this distribution towards a Pnmt-/Hif-2α+/Rgs5+ population. This was associated with morphological abnormalities of adrenal development, including ectopic TH+ cells within the adrenal cortex and external to the adrenal gland. These changes were ablated by combined inactivation of Phd2 with Hif-2α, but not Hif-1α. However, they could not be reproduced by inactivation of Phd2 in adult life, suggesting that they arise from dysregulation of this pathway during adrenal development. Together with the clinical observation that pseudohypoxic PPGL manifests remarkably high heritability, our findings suggest that this type of tumour likely arises from dysregulation of a tissue-restricted action of the PHD2/HIF-2α pathway affecting adrenal development in early life and provides a model for the study of the relevant processes.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.