Margaret Ashcroft

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here, we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions for an MYC-driven murine liver cancer model. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFβ-SMAD, which is upregulated in 3D culture, specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3, and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic, and organogenesis signaling dependencies under different cellular settings.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions for a MYC-driven murine liver cancer model. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFb-SMAD, which is upregulated in 3D culture, specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mett13 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions for a MYC-driven murine liver cancer model. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFb-SMAD, which is upregulated in 3D culture, specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

Mitochondria are key organelles in eukaryotic evolution that perform crucial roles as metabolic and cellular signaling hubs. Mitochondrial function and dysfunction are associated with a range of diseases, including cancer. Mitochondria support cancer cell proliferation through biosynthetic reactions and their role in signaling, and can also promote tumorigenesis via processes such as the production of reactive oxygen species (ROS). The advent of (nuclear) genome-wide CRISPR-Cas9 deletion screens has provided gene-level resolution of the requirement of nuclear-encoded mitochondrial genes (NEMGs) for cancer cell viability (essentiality). More recently, it has become apparent that the essentiality of NEMGs is highly dependent on the cancer cell context. In particular, key tumor microenvironmental factors such as hypoxia, and changes in nutrient (e.g., glucose) availability, significantly influence the essentiality of NEMGs. In this mini-review we will discuss recent advances in our understanding of the contribution of NEMGs to cancer from CRISPR-Cas9 deletion screens, and discuss emerging concepts surrounding the context-dependent nature of mitochondrial gene essentiality.

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

Mitochondria are pivotal for normal cellular physiology, as they perform a crucial role in diverse cellular functions and processes, including respiration and the regulation of bioenergetic and biosynthetic pathways, as well as regulating cellular signalling and transcriptional networks. In this way, mitochondria are central to the cell's homeostatic machinery, and as such mitochondrial dysfunction underlies the pathology of a diverse range of diseases including mitochondrial disease and cancer. Mitochondrial import pathways and targeting mechanisms provide the means to transport into mitochondria the hundreds of nuclear-encoded mitochondrial proteins that are critical for the organelle's many functions. One such import pathway is the highly evolutionarily conserved disulfide relay system (DRS) within the mitochondrial intermembrane space (IMS), whereby proteins undergo a form of oxidation-dependent protein import. A central component of the DRS is the oxidoreductase coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing protein 4 (CHCHD4, also known as MIA40), the human homologue of yeast Mia40. Here, we summarise the recent advances made to our understanding of the role of CHCHD4 and the DRS in physiology and disease, with a specific focus on the emerging importance of CHCHD4 in regulating the cellular response to low oxygen (hypoxia) and metabolism in cancer.

Viral replication is defined by the cellular microenvironment and one key factor is local oxygen tension, where hypoxia inducible factors (HIFs) regulate the cellular response to oxygen. Human immunodeficiency virus (HIV) infected cells within secondary lymphoid tissues exist in a low-oxygen or hypoxic environment in vivo. However, the majority of studies on HIV replication and latency are performed under laboratory conditions where HIFs are inactive. We show a role for HIF-2α in restricting HIV transcription via direct binding to the viral promoter. Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin, mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. Our data support a model where the low-oxygen environment of the lymph node may suppress HIV replication and promote latency. We identify a mechanism that may contribute to the limited efficacy of latency reversing agents in reactivating HIV and suggest new strategies to control latent HIV-1.

Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.