{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":[null],"organism":["Homo sapiens"],"software":["R v4.0, Seurat v3","CellRanger v3.0.2 CITE-seq-count v1.4.3"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-9803"],"description":["Loss of GADD45A expression is implicated in poor patient outcomes in acute myeloid leukemia (AML) but its role in leukemia stem cells (LSCs) and disease pathogenesis remains largely unknown. Here we report that GADD45A loss is a critical determinant of high self-renewal potential and knockout of GADD45A supports long-term self-renewal and promotes LSC quiescence, accompanied by the acquisition of an increasingly aggressive phenotype upon serial transplantation in mice. The enhanced LSC characteristics are associated with GADD45A deletion-induced increase in key WNT/self-renewal-related genes. GADD45A knockout promotes engraftment of patient-derived xenograft (PDX) of relapsed AML in mice. Single cell RNA-seq on primary LSCs of AML PDXs and subsequent functional studies show that low expression of GADD45A, an important sensor of oxidative stress, confers ferroptosis resistance through upregulation of genes involved in detoxification of excess iron and reactive oxygen species (ROS), revealing a mechanism of drug resistance in primary AML with unfavorable cytogenetics."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - All samples were from one PDX derived from a patient sample with relapsed AML. GADD45A gene was deleted using CRISPR/Cas9 technology in PDX cells (PDX42_GADD45A_Knockout), compared to CRISPR control (PDX42_GADD45A_Control). PDX cells were harvested from the bone marrow of transplanted NSG mice using standard protocols. Mouse cells were depleted using the AutoMACS separator in conjunction with the mouse cell depletion kit. Remaining human PDX cells were stained with antibody-oligo complexes (i.e. CD34, CD90, CD45RA TotalSeq-A antibodies) using standard Biolegend CITE-seq protocols. The stained cells were resuspended as single-cell suspensions and then counted using Countess FL2 after trypan blue staining.","Sequencing - Libraries were multiplexed and sequenced using NovaSeq 6000 S1 (100 cycle kit), with the following cycling protocol, Read 1 = 28, Index Read = 8, Read 2 = 91.","Nucleic Acid Extraction - Cells were subject to the 10x genomics single cell 3' v3 workflow. Cells were loaded onto the chromium controller as per manufacturer instructions with a target of 5000 cells and subsequent RT-PCR was performed.","Library Construction - Library construction for mRNA was conducted as per 10x genomics 3'v3 instructions, where cDNA amplification was undertaken for 12 cycles and then SI-PCR was undertaken for another 12 cycles. CITE-seq libraries were prepared using standard Biolegend protocols, wherein size selected fragments after cDNA amplification from the 10x mRNA protocol (~150bp) were subject to a further 11 cycles of CITE amplification.  The ADT library includes a mixture of 3 antibodies including human CD34 (barcode: 0054 - GCAGAAATCTCCCTT), human CD90 (barcode: 0060 - GCATTGTACGATTCA) and human CD45RA (barcode: 0063 - TCAATCCTTCCGCTT)."],"figure_sub":["MINSEQE Score","Assays and Data","Processed Data","organisation","MAGE-TAB Files"],"data_protocol":["Data Transformation - UMI count matrices were imported into R v4.0 via the Seuratv3 package. Cells with more than 15% of genes mapping to mitochondrial genome and inappropriate total transcript counts (nFeatures < 500 or nFeatures > 7,000) were removed from the analysis, yielding a total of 14,634 cells (6,227 cells from CRISPR Ctr experiment and 8,407 cells from GADD45A KO experiment). Ribosomal genes were regressed out from the analysis. Normalization was then performed using log-normalization for RNA assay and centered log-ratio normalization for ADT assay on each experiment as per developers’ instructions. Both datasets were then integrated. It resulted in a unique Seurat object that was scaled and standard workflow for CITE-seq data analysis was performed according to Seurat tutorial.","Sequence Alignment - Data were processed with Cell Ranger v.3.0.2. Raw bcl file hierarchies were first processed with cellranger mkfastq for demultiplexing using Illumina unique index built for our libraries. Resultant fastq files were aligned to human genome GRCh38 using cellranger_count. Python-based CITEseq Count tools was also used on fastq files to align and extract antibody-derived tag (ADT) counts belonging to each cell."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"additional_accession":["ERP125424"],"pubmed_authors":["Jenny Wang"]},"is_claimable":false,"name":"GADD45A loss enhances leukemia-initiating cell self-renewal and ferroptosis resistance in AML","description":"Loss of GADD45A expression is implicated in poor patient outcomes in acute myeloid leukemia (AML) but its role in leukemia stem cells (LSCs) and disease pathogenesis remains largely unknown. Here we report that GADD45A loss is a critical determinant of high self-renewal potential and knockout of GADD45A supports long-term self-renewal and promotes LSC quiescence, accompanied by the acquisition of an increasingly aggressive phenotype upon serial transplantation in mice. The enhanced LSC characteristics are associated with GADD45A deletion-induced increase in key WNT/self-renewal-related genes. GADD45A knockout promotes engraftment of patient-derived xenograft (PDX) of relapsed AML in mice. Single cell RNA-seq on primary LSCs of AML PDXs and subsequent functional studies show that low expression of GADD45A, an important sensor of oxidative stress, confers ferroptosis resistance through upregulation of genes involved in detoxification of excess iron and reactive oxygen species (ROS), revealing a mechanism of drug resistance in primary AML with unfavorable cytogenetics.","dates":{"release":"2025-08-29T00:00:00Z","modification":"2025-08-29T01:02:58.966Z","creation":"2022-02-03T14:56:44.725Z"},"accession":"E-MTAB-9803","cross_references":{"ENA":["ERP125424"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0003816","EFO_0005518","EFO_0004184"]}}