Mechanistic study and precision treatment of primary liver cancer (PLC) are hindered by marked heterogeneity, which is challenging to recapitulate in any given liver cancer mouse model. Here, we report the generation of 25 mouse models of PLC by in situ genome editing of hepatocytes recapitulating 25 single or combinations of human cancer driver genes. These mouse tumors represent major histopathological types of human PLCs and could be divided into three human-matched molecular subtypes based on transcriptomic and proteomic profiles. Phenotypical characterization identified subtype- or genotype-specific alterations in immune microenvironment, metabolic reprogramming, cell proliferation, and expression of drug targets. Furthermore, single-cell analysis and expression tracing revealed spatial and temporal dynamics in expression of pyruvate kinase M2 ( Pkm2 ). Tumor-specific knockdown of Pkm2 by multiplexed genome editing reversed the Warburg effect and suppressed tumorigenesis in a genotype-specific manner. Our study provides mouse PLC models with defined genetic drivers and characterized phenotypical heterogeneity suitable for mechanistic investigation and preclinical testing.
Significance Epidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.
O‐linked N‐acetylglucosamine (O‐GlcNAcylation) is a ubiquitous post‐translational modification of proteins that is essential for cell function. Perturbation of O‐GlcNAcylation leads to altered cell‐cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one‐step enzymatic strategy for capture and profiling O‐GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O‐GlcNAcylation. FEN1 O‐GlcNAcylation is dynamically regulated during the cell cycle. O‐GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O‐GlcNAcylated proteins, and reveals an unknown mechanism of O‐GlcNAcylation in regulating cell cycle progression and DNA damage response.
Mouse embryonic stem cells (mESCs) can self‐renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR inhibition‐induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3 and MEK inhibitors and LIF). We also show that mTORC1 regulates self‐renewal in both conditions mainly through eIF4F‐mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1‐independent but MEK/ERK‐dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self‐renewal in mESCs and uncovers a context‐dependent function of eIF4F in pluripotency regulation.
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