For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC, and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes.Consistent with the tumor suppressor function of UBR5 (Hyd) in Drosophila, Hyd suppressed dMyc-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were co-amplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC co-amplification. Further, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYCmediated apoptosis priming and in protection from drug-induced apoptosis. Significance:Findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells.Research.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

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Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering environmental causes of COPD and disease phenotypes, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts from ex-smokers and COPD patients. Epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair and matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes. Using phenotypic screens, we linked the function of multiple candidates to repair processes in the lung. Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across COPD stages. We reveal novel signatures associated with COPD onset and progression and identify candidate regulators involved in the pathogenesis of chronic respiratory diseases.

No abstract

MYC protein expression has to be tightly controlled to allow for maximal cell proliferation without inducing apoptosis. Here we discover UBR5 as a novel MYC ubiquitin ligase and demonstrate how it functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 effects on MYC protein stability are independent on N-terminal FBW7 degron of MYC. Endogenous UBR5 inhibition induces MYC protein expression and activates MYC target genes. Moreover, UBR5 governs MYC-dependent phenotypes in vivo in Drosophila. In cancer cells, UBR5-mediated MYC protein suppression diminishes cell killing activity of cancer therapeutics. Further, we demonstrate that UBR5 dominates MYC protein expression at the single-cell level in human basal-type breast cancer tissue. Myc and Ubr5 are co-amplified in MYC-driven human cancer types, and UBR5 controls MYC-mediated apoptotic threshold in co-amplified basal type breast cancer cells. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC protein expression in vivo. Clinically, expression of UBR5 may be important for protection of breast cancer cells from drug-induced, and MYCdependent, apoptosis.

<p>Includes description of materials and methods related to Western Blot, Quantitative RT-PCR, siRNA library and high-content screening, Analysis of mutual exclusivity of MYC and UBR5 on single cell level, Mouse experiments, RNA sequencing.</p>