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Genome-wide CRISPR screen of MOLM13 cells after Idasanutlin treatment

ABSTRACT: Genome-wide CRISPR screen of MOLM13 cells after Idasanutlin treatment

PROVIDER: PRJNA1005974 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR25656540.fastq.gz	Fastqsanger.gz
	SRR25656541.fastq.gz	Fastqsanger.gz
	SRR25656542.fastq.gz	Fastqsanger.gz
	SRR25656543.fastq.gz	Fastqsanger.gz
	SRR25656544.fastq.gz	Fastqsanger.gz

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Genome-wide CRISPR screen of MOLM13 cells after Idasanutlin treatment

Project description:Targeting MDM2 is an attractive therapeutic approach for TP53 wild-type (WT) tumors, including the majority of de novo acute myeloid leukemia (AML) cases. However, patients with WT TP53 have shown variable responses to MDM2 inhibitors in clinical trials, highlighting the need to identify additional biomarkers to maximize the chances of clinical success. We performed CRISPR-Cas9 knockout screens to identify genes that confer resistance to an MDM2 inhibitor idasanutlin. We did not find recurrent sgRNA hits or mutations in p53 downstream targets, such as CDKN1A, BAX, PMIAP1, and BBC3, apart from TP53. This was consistent with the results of individual knockout validation experiments and exome sequencing data from idasanutlin resistant cell lines generated form long-term exposure to low doses of idasanutlin. RNA-seq differential expression analysis revealed that major p53 downstream targets were upregulated in both idasanutlin-sensitive MOLM13 and resistant OCIAML3 cell lines after idasanutlin treatment. These findings highlight the pleiotropic functions of TP53 and suggest that the loss of individual downstream targets of TP53 does not significantly contribute to MDM2 inhibitor resistance.

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Project description:MOLM13 cells were infected with CRISPR/Cas9 library, selected for puromycn resistance for integration events and exposed to Venetoclax or vehicle, DMSO, at 1microMolar concentrations. Cells were collected at time 0 (post puromycin selection), day 7 and 14 , DNA was extracted and sgRNA barcodes were amplified. PCR library was deep sequenced using highthroughput Illumina platform Hiseq using 6 samples per lane.

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Project description:MOLM13 or MV411 cells were generated to express Cas9, infected with CRISPR library (Tzelepis et al 2016) , selected for puromycin resistance and exposed to Sorafenib (50nM), Crenolanib (20nM) or vehicle, DMSO. Cells were collected at time 0 (post puromycin selection), day 7 and 14 for Sorafenib as well as 14 and 21 for Crenolanib. DNA was extracted and sgRNA barcodes were amplified. The resulting PCR library was deep sequenced using the Illumina HiSeq 2500 using 6 samples per lane.

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