Detection of phenotype-specific therapeutic vulnerabilities in breast cells using a CRISPR loss-of-function screen

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O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes

Post-translational modifcation of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the signifcance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells

O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes

Post-translational modifcation of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the signifcance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer

Introduction Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. Experimental MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). Results Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. Conclusion Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer