Abstract Oxidative post-translational modifications on the sulfhydryl group of cysteines can occur spontaneously or enzymatically. The dioxygenation of N-terminal cysteines has emerged as a new oxygen sensing paradigm, catalysed by 2-aminoethanethiol dioxygenase (ADO) in mammals. Conflicting evidence has been reported in recent years on whether this reaction can occur in the absence of ADO. Here we sought to address whether physiological oxidative stress can interfere with ADO-catalysed N-terminal dioxygenation. Using a system to produce titratable intracellular levels of H 2 O 2 , we demonstrate that the stability of RGS4 and 5 is not affected by oxidative stress, whether ADO is present or not. However, cytotoxic levels of oxidative stress did induce an increase in RGS4/5 protein levels that occurred independently of the Cys N-degron pathway. This effect of tBHP was reduced by Fe 2+ chelation and perturbations of lysosomal function, suggesting the possible involvement of ferroptosis. We conclude that N-terminal cysteine dependent proteolysis of RGS4/5 is not sensitive to physiological oxidative stress, but these proteins can be stabilised during the process of oxidative stress-induced cell death through an N-terminal cysteine independent mechanism.

Adaptation to reduced levels of oxygen (hypoxia) is an essential feature of eukaryotic life. Within the animal kingdom, cellular responses are orchestrated by the transcription factor HIF (Hypoxia Inducible Factor) which is regulated by specific 2-oxoglutarate-dependent oxygenases. This family of enzymes also includes histone demethylases, and histone methylation has also been observed to increase in hypoxia. Since histone methylation is associated with gene expression, this has raised questions about whether this also contributes to transcriptional regulation in hypoxia. However, to date pangenomic studies have not been normalised in a way that preserves these bulk changes. Using drosophila chromatin spike-in normalisation, we have shown widespread increases in histone H3K4/9/27/36me3 in hypoxia at almost all gene loci that occur irrespective of whether gene expression is increased or reduced. However, methylation of H3K4me3 and H3K36me3 increases most at direct transcriptional targets of HIF and this is abrogated by inactivation of HIF. Taken together this suggests that global H3 trimethylation increases in hypoxia are widespread and not sufficient to predict transcriptional direction, whereas enhanced H3K4me3/H3K36me3 at direct HIF targets appears consequent to HIF binding and transcriptional engagement.

Cancers arising from dysregulation of generally operative signaling pathways are often tissue specific, but the mechanisms underlying this paradox are poorly understood. Based on striking cell-type specificity, we postulated that these mechanisms must operate early in cancer development and set out to study them in a model of von Hippel Lindau (VHL) disease. Biallelic mutation of the VHL ubiquitin ligase leads to constitutive activation of hypoxia inducible factors HIF1A and HIF2A and is generally a truncal event in clear cell renal carcinoma. We used an oncogenic tagging strategy in which VHL-mutant cells are marked by tdTomato, enabling their observation, retrieval, and analysis early after VHL-inactivation. Here, we reveal markedly different consequences of HIF1A and HIF2A activation, but that both contribute to renal cell-type specific consequences of VHL-inactivation in the kidney. Early involvement of HIF2A in promoting proliferation within the proximal tubular epithelium supports therapeutic targeting of HIF2A early in VHL disease.

Homeostatic control of cellular oxygen availability is a crucial feature of all eukaryotic life, and central to this process is the ability to sense oxygen across a broad range of concentrations and time scales. Much of our understanding of the molecular mechanisms underpinning oxygen sensing has been obtained using cell culture models, yet the biophysical properties of oxygen combined with the complex nature of cellular O2 consumption can make the interpretation of such data difficult. In this commentary, we have outlined some of the main problems encountered in measuring and manipulating cell monolayer oxygenation in vitro, and contextualised them using both historical and contemporary examples. This commentary outlines key challenges encountered in measuring and manipulating cell monolayer oxygenation in vitro and innovative approaches to overcome them.

Selected proteins containing an N-terminal cysteine (Nt-Cys) are subjected to rapid, O2-dependent proteolysis via the Cys/Arg-branch of the N-degron pathway. Cysteine dioxygenation is catalyzed in mammalian cells by 2-aminoethanethiol dioxygenase (ADO), an enzyme that manifests extreme O2 sensitivity. The canonical substrates of this pathway in mammalia are the regulators of G-protein signaling 4, 5, and 16, as well as interleukin-32. In addition to operating as an O2-sensing mechanism, this pathway has previously been described as a sensor of nitric oxide (NO), with robust effects on substrate stability upon modulation of NO bioavailability being widely demonstrated. Despite this, no mechanism to describe the action of NO on the Cys/Arg N-degron pathway has yet been substantiated. We demonstrate that NO can regulate the stability of Cys N-degron substrates indirectly via the regulation of ADO cosubstrate availability. Through competitive, O2-dependent inhibition of cytochrome C oxidase, NO can substantially modify cellular O2 consumption rate and, in doing so, alter the availability of O2 for Nt-Cys dioxygenation. We show that this increase in O2 availability in response to NO exposure is sufficient to alter both dynamic and steady-state ADO substrate levels. It is likely that this mechanism operates to couple O2 supply and mitochondrial respiration with responses to G-protein-coupled receptor stimulation.

Clear cell kidney cancers are characterized both by conserved oncogenic driver events and by marked intratumor genetic and phenotypic heterogeneity, which help drive tumor progression, metastasis, and resistance to therapy. How these are reflected in transcriptional programs within the cancer and stromal cell components remains an important question with the potential to drive novel therapeutic approaches to treating cancer. To better understand these programs, we perform single-cell transcriptomics on 75 multi-regional biopsies from kidney tumors and normal kidney. We identify conserved patterns of transcriptional dysregulation and their upstream regulators within the tumor and associated vasculature. We describe recurrent subclonal transcriptional consequences of Chr14q loss linked to metastatic potential. We identify prognostically significant conserved patterns of intratumor transcriptional heterogeneity. These reflect co-existing cell states found in both cancer cells and normal kidney cells, indicating that rather than arising from genetic heterogeneity they are a consequence of lineage plasticity.

More than 100 years after the original descriptions of altitude adaptation, it is now clear that many of these responses are mediated by a specific isoform of the transcription factor hypoxia-inducible factor (HIF-2α). Here, we review this work, including connectivity with the oxygen chemosensitive response itself, and with paraganglioma, a tumour often affecting chemosensitive tissues.

More than 100 years after the original descriptions of altitude adaptation, it is now clear that many of these responses are mediated by a specific isoform of the transcription factor hypoxia-inducible factor (HIF)-2α. Here, we review this work, including connectivity with the oxygen chemosensitive response itself and with paraganglioma, a tumor often affecting chemosensitive tissues.

Abstract 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.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.

Vhl-null cells specifically undergo time-dependent alterations in gene expression

scRNA-seq on flow-sorted renal cells. A, UMAP plot of tdTomato-negative (left) or tdTomato-positive cells (right) from kidneys of Control mice harvested at the early time point. Cells are colored by inferred cell type. LoH, loop of Henle; DCT, distal convoluted tubule; CD, collecting duct; PC, principal cell; IC, intercalated; PEC, parietal epithelial cell; VSMC, vascular smooth muscle cell; NK, natural killer cell. B, Proportion of sequenced cells inferred to be of each cell type in tdTomato-positive or tdTomato-negative populations from kidneys of Control mice harvested at the early time point. Median and interquartile range plotted. C, UMAP plot depicting expression of PT Module A (left) and PT Module B (right) genes in PT cells from Control mice. D, Representative in situ RNA hybridization exhibiting spatially distinct expression of Neat1 (blue) and Fxyd2 (red) mRNA in FFPE kidney cortex from Control mice harvested at the early time point. Scale bar, 10 μm. Magnification, ×40. E, UMAP plot depicting cells from Control mice at the early time point. Cells are colored by assigned PT Class. A–E, scRNA-seq data shown for n = 3 female (3F) and n = 1 male (1M) Control mice.

Abstract Characterizing the early events preceding tumorigenesis is a major challenge in understanding cancer. To address this, we are investigating the paradigm of clear cell renal cell carcinoma (ccRCC) that is driven by the biallelic loss of the von Hippel Lindau (VHL) tumor suppressor gene. Though VHL loss is required, additional mutations have been found in the evolution of the cancer, most commonly in Polybromo-1 (PBRM1), a subunit of the PBAF SWI/SNF chromatin remodeling complex. Concomitant Vhl and Pbrm1 deletion, but not Vhl deletion alone, in the renal tubular epithelium (RTE) has been reported to be sufficient to drive the formation of renal tumors in mice. However, the early in vivo consequences of Vhl loss, and the exact requirement for Pbrm1 loss to tumorigenesis remain poorly studied. We have previously reported the development of a novel lineage-tracing model of Vhl deletion that directly couples loss of a conditional Vhl allele to the expression of a tdTomato reporter and allows accurate identification and retrieval of marked Vhl-null cells. In the work reported here, we have combined this system with conditional Pbrm1 deletion using an RTE-restricted Pax8-CreERT2 to precisely identify and assay Vhl/Pbrm1-null cells. We describe the histological abnormalities specifically comprising Vhl/Pbrm1-null cells observed at early (1-3 weeks), intermediate (10 months), and late (17 months) intervals post recombination. We found that Vhl/Pbrm1-null cells formed extensive ‘tumorlets’, disorganized, multilayered tubules, and cystic dilated tubules specifically in the renal cortex at 17 months post recombination. Immunohistochemistry confirmed that all epithelial cells in the lesions were Vhl/Pbrm1 null and expressed HIF1 and HIF2. These lesions contained cycling cells as identified by dual tdTomato/Ki67 staining, foci of immune cells and were strikingly surrounded by cells expressing high levels of p-ERK. We then traced these lesions to earlier timepoints, and observed Vhl/Pbrm1-null lesions resembling ‘tumorlet’ precursors and disorganized tubules at 10 months post recombination specifically in the renal cortex. Interestingly, these precursors were encompassed by macrophages and could be discerned in situ by a ring of p-ERK-expressing cells surrounding these lesions. No similar lesions were detected at 3 weeks after recombination. Interestingly, the presence of cortical lesions was associated with a region-specific expansion of the total Vhl/Pbrm1-null population over time. Our data suggest that morphological abnormalities arise after a general proliferation of Vhl/Pbrm1-null cells in the renal cortex and from a subset of cells expressing specific markers. We are now comparing the differential gene expression patterns of Vhl-null cells and Vhl/Pbrm1-null normal and transformed cells at single cell resolution to expand on these observations. Citation Format: Joanna Lima, Samvid Kurlekar, Norma Masson, Ayslan B. Barros, Maria Fernanda Soares, Christopher W. Pugh, Julie Adam, Peter J. Ratcliffe. Identification of early oncogenic lesions following concomitant Vhl and Pbrm1 loss in the murine kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6622.

Renal cortex, but not the papilla, is permissive to long-term survival of Vhl-null cells. A, Representative tdTomato IHC counterstained with hematoxylin in different renal anatomical regions of Control or KO mice harvested at the early or late time points. Scale bar, 100 μm. Magnification, ×20. B, Proportion of cells that are tdTomato-positive in different regions of the kidney as quantified by tdTomato IHC in kidneys from Control or KO mice harvested at different intervals after recombination. n = 7F, 16M for all regions for KO; n = 9F, 15M; 9F, 15M; 9F, 14M; 8F, 14M for cortex, outer medulla, inner medulla, and papilla, respectively, for Control. Line denotes linear regression. Significance testing performed for slope and intercept of linear regression by t test.

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6944.

Vhl-null cells specifically undergo time-dependent alterations in gene expression. A, Density plot depicting UMAP distribution of tdTomato-positive cells from kidneys of Control and KO mice harvested at the early or late time points. B, Left, UMAP plot depicting tdTomato-positive cells from Control and KO mice harvested at the early and late time points colored by UMAP cluster. Right, proportion of cells from each condition belonging to any cluster. C, Scatter plot depicting frequency of expression in tdTomato-positive cells from KO mice at the late time point against log2-fold change (log2FC) between cells from KO mice at the late versus early time points for all genes. Orange, significantly regulated genes. Genes explicitly mentioned in the main text are labeled. D, Gene set enrichment plots depicting upregulation of genes regulated early after Vhl inactivation (left) or genes known to be HIF targets (right) in Vhl-null cells at the late versus early time points. NES, normalized enrichment score. P value adjusted by Bonferroni correction for multiple testing. E, UMAP plot depicting “PT like” cells among tdTomato-positive cells from Control and KO mice harvested at the early and late time points. Black, PT-like cells. F, Proportion of cells inferred to be “PT like” within tdTomato-positive (top) or tdTomato-negative (bottom) cells across conditions. Median and interquartile range plotted. Pairwise comparisons tested by one-way ANOVA with Holm–Šídák correction. G, Representative CD45 IHC on kidneys from Control (n = 1F, 4M) and KO (n = 6M) mice harvested at the late time point. Scale bar, 50 μm. Magnification, ×40. A–F, scRNA-seq data shown for n = 3F, 1M mice for Control early and Control late samples; n = 2F, 2M mice for KO early and KO late samples.

Biallelic Vhl loss entrains early cell-specific transcriptomic changes in renal tubular cells

scRNA-seq metrics, cell type markers, and lists of differentially expressed genes