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Genetic and proteomic screens reveal perturbed nucleotide metabolism and DNA-protein crosslink formation as central mediators of decitabine cytotoxicity

ABSTRACT: Decitabine (5-aza-dC) is a DNMT1-DPC inducing agent used to treat several hematological cancers. However, response rates vary, relapse is common, and predictive biomarkers are unknown. Here, we develop an adaptation of identification of proteins on nascent DNA (iPOND) by combining EdU with 5-aza-dC incorporation by itself or the presence of either ubiquitin E1 or SUMO E1 inhibitor which we term iPOND-DPC. The purpose of this experiments is to identify proteins that are recruited to DNMT1-DPCs and categorize whether their recruitment is dependent on SUMOylation or ubiquitylation.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Petra Beli

LAB HEAD: Petra Beli

PROVIDER: PXD045071 | Pride | 2024-04-18

REPOSITORIES: Pride

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			Action	DRS
	20220712_CB_Stingele_Goetz_iDPC_TMT16_PRIDE.zip	Other
	20220712_SFB1_PBE_CB_Stingele_Goetz_iDPC_TMT16_R1.raw	Raw
	20220712_SFB1_PBE_CB_Stingele_Goetz_iDPC_TMT16_R2.raw	Raw
	20220712_SFB1_PBE_CB_Stingele_Goetz_iDPC_TMT16_R3.raw	Raw
	checksum.txt	Txt

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Project description:Esophageal cancers (ECs) are highly aggressive tumors with poor prognosis and few treatment options. This study investigated the possibility of treating esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells by inhibitors of broad and specific histone deacetylases (HDACi; SAHA, MS-275, FK228) and/or of DNMT (Azacytidine, AZA). Drug targets (HDAC1,2,3 and DNMT1) were present in non-neoplastic (HET-1A), ESCC (OE21) and EAC (OE33) cell lines. All cell lines responded to HDACi by reduced HDAC activity and increased histone acetylation as well as to AZA by up-regulation of p21. Expression of drug targets remained largely unaffected by HDACi and AZA treatment. Importantly, cell viability, apoptosis, cell cycle dynamics and DNA damage were only affected by HDACi and/or AZA in ESCC and EAC, but not the non-neoplastic cells. This was specifically seen for the combination of MS-275 and AZA, leading to enhanced cancer cell selectivity and drug efficiency. By transcriptome analyses of MS-275, AZA and MS-275/AZA treated cells, known (e.g. p21) as well as novel regulated genes significantly associated with the cellular effects post HDACi and/or AZA treatment in ESCC and EAC cells were identified. Finally, human EC tissue specimens frequently expressed the actionable drug targets HDAC1/2/3 and DNMT1. In summary, a combined HDACi (MS-275)/AZA treatment is cancer cell selective and efficient in vitro. Since the majority of ECs express the drug targets in situ, this paves the way for further investigations of HDACi/AZA treatment in esophageal cancer cells and their translation into a clinico-pathological setting. To elucidate the transcriptome response to HDAC inhibitors of normal esophageal cells and esophageal tumor cells, total RNA was isolated from non-neoplastic esophageal epithelial cells (Het1A cells) a well as from two esophageal tumor cell lines (OE21 and OE33), respectively. Cells were treated with either MS-275, Azacytidine (AZA) or in combination of both. DMSO treatment was used as control in each case. Total RNA was isolated from cells 24 h after treatment and experiments were performed in biological triplicates.

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Genetic and proteomic screens reveal perturbed nucleotide metabolism and DNA-protein crosslink formation as central mediators of decitabine cytotoxicity

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