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Targeting PCNA in a subgroup of Multiple Myeloma cells inhibits the glycolysis and pentose phosphate pathway (batch TK9)

ABSTRACT: PCNA is an essential protein in all cells with multiple well-known function in DNA replication and DNA repair. Recently, PCNA was shown to be involved in regulation of cellular signalling and central carbon metabolism via stabilizing the metabolic enzymes ENO1 and 6PGD. Furthermore, ATX-101, a PCNA targeting peptide currently in Phase II, was shown to reduce metabolite pools in the glycolysis and the pentose phosphate pathway (PPP) in multiple haematological cells. Here integrated analysis of signalome and metabolome data from multiple myeloma (MM) cells reveals large effects of ATX-101 on signalling, metabolism and red-ox regulation. The sensitivity to ATX-101 varied between the eight MM cell lines studied and a metabolic shift was detected in the sensitive cell lines. These cell lines were characterized by low endogenous levels of the redox metabolites NAD+, NADH and GSH. Interestingly, 11 proteins were pulled down from untreated cells from the sensitive cell lines while not from 9 other cancer cell lines or primary monocytes from three donors. These proteins could be potential biomarkers for MM cells with low red-ox capacity and hypersensitivity for inhibitors of glycolysis or the PPP.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Animesh Sharma

LAB HEAD: Marit Otterlei

PROVIDER: PXD033531 | Pride | 2024-11-25

REPOSITORIES: Pride

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			Action	DRS
	191107_SIRI_10_TK9_combi_1.raw	Raw
	191107_SIRI_11_TK9_combi_2.raw	Raw
	191107_SIRI_12_TK9_combi_3.raw	Raw
	191107_SIRI_13_TK9_ctr_poolet.raw	Raw
	191107_SIRI_14_TK9_apim_poolet.raw	Raw

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Similar Datasets

Targeting PCNA in a subgroup of Multiple Myeloma cells inhibits the glycolysis and pentose phosphate pathway

Project description:PCNA is an essential protein in all cells with multiple well-known function in DNA replication and DNA repair. Recently, PCNA was shown to be involved in regulation of cellular signalling and central carbon metabolism via stabilizing the metabolic enzymes ENO1 and 6PGD. Furthermore, ATX-101, a PCNA targeting peptide currently in Phase II, was shown to reduce metabolite pools in the glycolysis and the pentose phosphate pathway (PPP) in multiple haematological cells. Here integrated analysis of signalome and metabolome data from multiple myeloma (MM) cells reveals large effects of ATX-101 on signalling, metabolism and red-ox regulation. The sensitivity to ATX-101 varied between the eight MM cell lines studied and a metabolic shift was detected in the sensitive cell lines. These cell lines were characterized by low endogenous levels of the redox metabolites NAD+, NADH and GSH. Interestingly, 11 proteins were pulled down from untreated cells from the sensitive cell lines while not from 9 other cancer cell lines or primary monocytes from three donors. These proteins could be potential biomarkers for MM cells with low red-ox capacity and hypersensitivity for inhibitors of glycolysis or the PPP.

2024-11-25 | PXD033510 | Pride

PCNA regulates metabolism by stabilizing ENO1 and 6PGD

Project description:The essential roles of PCNA as a scaffold protein in DNA replication and repair are well established, while its more recently discovered cytosolic roles are less explored. Alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), important proteins in glycolysis and the Pentose Phosphate Pathway (PPP), respectively, both contain PCNA interacting motifs. Here we show reduced binding to PCNA when the PCNA interacting motif APIM in ENO1 is mutated. Mutant cells have lower ENO1 protein levels, have reduced glucose consumption, altered glycolytic metabolite and nucleoside phosphate pools and increased activation of the citric acid cycle (TCA) compared to parental cells. Treatment of a panel of haematological cell lines with a cell penetrating peptide containing APIM (ATX-101), lead to a metabolic shift with reduction in glycolytic metabolite and nucleoside phosphate pools as well reduced levels of ENO1 and 6PGD proteins. These results suggests that PCNA stabilize ENO1 and 6PGD, and that targeting PCNA reduce the glycolysis, PPP and nucleoside phosphate pools via destabilization of these proteins.

2023-03-10 | PXD028314 | Pride

PCNA has specific functions in regulation of metabolism in haematological cells

Project description:PCNA´s essential roles in DNA replication and repair are well established, while it’s recently discovered non-canonical roles in cellular signalling and regulation of metabolism are less explored. Recent data has indicated that PCNA has a specific non-canonical role in cells of haematological origin. In the present study we have explored this further at the system level by extensive protein-, gene expression, and metabolite profiling combined with integrated pathway analysis and by 13C label experimentation, Here we show that impairing PCNA´s scaffold functions led to massive changes in cellular signalling, but more importantly, a strong reduction in glycolytic metabolites and nucleoside phosphate pools in haematological cancer cell lines was detected, including multiple myeloma cells and acute myelogen leukaemia cells. This was not observed in cell lines from solid tissues such as kidney, bladder and prostate, nor in primary monocytes. Lipopolysaccharide (LPS) stimulated monocytes on the other hand, responded to treatment similarly to the haematological cancer cells, suggesting that cellular stress is an important factor for the response to the APIM-peptide in haematological cells. Integrated Pathway analysis of the metabolite and protein datasets revealed that protein ubiquitination and antigen presentation pathways are affected in haematopoietic cancer cells upon treatment. Focusing on one multiple myeloma cell line we further verified these trends by gene-expression and show that the consequences of reduced central carbon metabolite pools and energy charge is impaired redox and HIF1A regulation. Altogether, our data suggests that PCNA has an important role in regulation of cytoplasmic stress via regulation of central carbon metabolism in haematological cells that can be targeted in cancer treatment.

2023-03-10 | PXD017474 | Pride

Nucleoside Analogues are Potent Inducers of Bacterial Mutagenesis

Project description:Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli using the Rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we appliedanalyzed metabolome and proteome of E.coli deletion mutants., and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR.

2021-07-15 | PXD025370 | Pride

Differential protein expression between Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) multiple myeloma cells

Project description:Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) MM cells were grown in RPMI-1640 medium supplemented with 10 % FBS and 0.68 mM L-glutamine, in a humidified incubator at 5% CO2 and 37 °C. The AMO1-CFZ medium was additionally added 90 nM CFZ, while the CFZ sensitive AMO1 cells were added vehicle (0.009 % DMSO) only. AMO1-CFZ was either harvested directly or grown for 1 week in CFZ free medium (vehicle only) prior to harvest. AMO1 and carfilzomib-resistant AMO1-CFZ cells were resuspended in 100 µl 1% sodium deoxycholate, 100 mM Tris-HCl pH 8.5, 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide (CAA), heated at 90 °C for 45 min and sonicated for 10 cycles (30 s ON/30 s OFF) using a Bioruptor pico sonicator. After centrifugation at 16000 × g for 10 min, 50 µg soluble protein from each sample was added 100 µl 0.1M ammonium bicarbonate, 0.5 µg trypsin and digested overnight at 37°C. Peptides were desalted using C18 spin columns, dried in a speedvac centrifuge and resuspended in 0.1% formic acid prior to MS analysis. Label-free quantitatative (LFQ) LC-MS/MS was performed on a timsTOF Pro (Bruker Daltonics) connected to a nanoElute (Bruker Daltonics) HPLC. Peptides were separated over a Bruker PepSep C18 (75 µm × 15 cm) column with running buffers A (0.1 % formic acid) and B (0.1 % formic acid in acetonitrile) using a 100 min gradient from 2 % B to 40 % B. The timsTof instrument was operated in the DDA PASEF mode with 10 PASEF scans per acquisition cycle and accumulation and ramp times of 100 ms each. The ‘target value’ was set to 20000 and dynamic exclusion was activated and set to 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 800.

2024-06-07 | PXD045439 | Pride

PCNA-targeting peptide modifies and increases the response to EGFR/HER2/VEGF inhibition in breast cancer cells

Project description:To combine inhibition of EGFR/HER2 and targeting of the cytosolic roles of PCNA using the APIM-peptide, and study the anti-cancer effects of this combinatory therapy in in vitro and in vivo breast cancer models.

2020-01-15 | PXD011044 | Pride

Profiling uterine fibroid specimens with an in-house array representing all 90 human tyrosine kinases and their ligands

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