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1.
Eng Life Sci ; 22(2): 100-114, 2022 Feb.
Article in English | MEDLINE | ID: mdl-35140557

ABSTRACT

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

2.
Mol Cancer Ther ; 20(1): 50-63, 2021 01.
Article in English | MEDLINE | ID: mdl-33203732

ABSTRACT

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis-addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug-target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis-addicted cancers.


Subject(s)
Antidepressive Agents/pharmacology , Breast Neoplasms/pathology , Drug Repositioning , Glycine Hydroxymethyltransferase/antagonists & inhibitors , Glycine/biosynthesis , Serine/blood , Sertraline/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Glycine Hydroxymethyltransferase/metabolism , Humans , Mice, Inbred NOD , Mice, SCID , Molecular Docking Simulation , Phosphoglycerate Dehydrogenase/metabolism , Thimerosal/pharmacology
3.
Nat Commun ; 10(1): 2542, 2019 06 11.
Article in English | MEDLINE | ID: mdl-31186416

ABSTRACT

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.


Subject(s)
Glycine/metabolism , Mutation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Serine/metabolism , Animals , Cell Line , Gene Expression Profiling , Mice , Phosphoric Monoester Hydrolases , Polyribosomes/genetics , Polyribosomes/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , Ribosomal Protein L10 , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Sequence Analysis, RNA
4.
Leukemia ; 33(4): 1055-1062, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30850735

ABSTRACT

Following the publication of this article, the authors noted that Dr Laura Fancello was not listed among the authors. The corrected author list is given below. Additionally, the following was not included in the author contribution statement: 'L.F. analyzed RNA sequencing data'.

5.
Leukemia ; 33(2): 319-332, 2019 02.
Article in English | MEDLINE | ID: mdl-29930300

ABSTRACT

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL.


Subject(s)
Internal Ribosome Entry Sites , Mutation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Biosynthesis , Proto-Oncogene Proteins c-bcl-2/metabolism , Ribosomal Proteins/genetics , Ribosomes/metabolism , Animals , Gene Expression Regulation, Leukemic , Humans , Male , Mice , Mice, Inbred NOD , Oxidative Stress/drug effects , Phosphorylation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Ribosomal Protein L10 , Ribosomal Proteins/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
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