Genome-wide CRISPR rensensitization screens in FGF2 and FL-derived Early and Late Gilteritinib Resistant MOLM14 cultures using Y. Kosuke library
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PROVIDER: PRJNA666344 | ENA |
REPOSITORIES: ENA
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Project description:Early and late gilteritinib resistant MOLM14 cells were transduced with Cas9-blasticidin lentiviral vector to generate Cas9+-expressing cells. These cells were then infected with the Yusa CRISPR library consisting of an average of 5 single-guide RNAs (sgRNAs) per gene for 18,010 genes. After transduction, cells were selected with puromycin for 5 days to ensure stable guide integration and subjected to treatment with 100 nM of gilteritinib or vehicle (DMSO). Early gilteritinib screen cultures were maintained in 10 ng/mL of FGF2 or FL, respectively. DNA was harvested from 0-21-day cultures to amplify library of sgRNA barcodes for deep sequencing analyses. Deep sequencing of early and late resistant screens was performed on the NovaSeq and HiSeq 2500, respectively (Illumina Inc.). Genes that were significantly depleted from gilteritinib-treated cultures relative to controls were deemed essential for the survival of gilteritinib resistant cells.
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Project description:While clinical benefit has been observed with gilteritinib, most patients relapse through unknown mechanisms. To investigate mechanisms of gilteritinib resistance, we performed targeted genomic sequencing and single cell (sc) RNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common. In gilteritinib-unresponsive patients, increased expression of bone marrow-derived hematopoietic cytokines and chemokines was observed after treatment compared to gilteritinib-sensitive patients. Expression of the TEK-family kinase, BMX, was higher in gilteritinib-unresponsive patients after treatment compared to gilteritinib-sensitive patients. BMX contributed to gilteritinib resistance in FLT3-mutant cells lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, which was reversed with pharmacological inhibition and genetic knockout. In primary FLT3-mutated AML samples, pharmacological inhibition of BMX enhanced the antileukemic activity of gilteritinib and decreased chemokine expression. Gene module analysis associated gilteritinib responsiveness with lymphocyte differentiation and myeloid leukocyte activation. By contrast, unresponsiveness to gilteritinib associated with upregulation of cell-cycle, DNA/RNA metabolic processes, and protein translation. Together, these data support a role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and gilteritinib resistance. This analysis provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity.
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Project description:Purpose: Compare transcriptional changes between drug treated (gilteritinib, TP-0903) and vehicle in acute myeloid leukemia xenografts. Methods: FLT3-ITD+ MOLM13-RES-Luc+ (MOLM13 cells with a D835Y mutation generating a resistant phenotype) AML cells were xenografted into NSG mice. Mice were randomized according to bioluminescence imaging, and treatment of FLT3 tyrosine kinase inhibitor (gilteritinib, 30 mg/kg, once daily for 5 days/week) was started on day 13 post-tail vein injection. At study end point, mice were humanely euthanized and bone marrow was collected. AML cells (human CD45+) were isolated, and ATAC-seq was performed on HiSeq 4000, total RNA. Sequencing reads were aligned and analyzed for differential gene expression of treatment vs vehicle cohorts. Results: The analysis revealed the chromatin accessibility changes induced by gilteritinib in MOLM13-RES AML xenograft model.
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Project description:Acquisition of an internal tandem duplication of the juxtamembrane region of FLT3 (FLT3-ITD) is a common event in Acute myeloid leukemia (AML) patients and is associated with poor outcomes at high allelic frequency. Although targeted therapies against FLT3-ITD exist, narrow therapeutic index as monotherapies and failure to achieve complete remission limits their clinical application. Using a genome-wide CRISPR knockout screen, we identified cyclin-dependent kinase (CDK9), protein arginine methyltransferase (PRMT5) and dihydroorotate dehydrogenase (DHODH) as novel synthetic lethal partners with gilteritinib treatment. We demonstrated that genetic or pharmacologic inhibition of these targets in combination with gilteritinib synergistically kill AML cells. The presence of FLT3-ITD was shown to cause a significant increase in aerobic glycolysis, rending the leukemia cells highly sensitive to pharmacological inhibition of glycolytic activity. Supportive of this, our screen shows that sgRNAs targeting ~28 genes in the glycolysis pathway are enriched in cells treated with gilteritinib, suggesting that switching to oxidative phosphorylation from aerobic glycolysis may represent a metabolic adaption of the leukemic cells to resistance to FLT3-targeted therapy. Interestingly, the knockdown of CDK9, PRMT5 or DHODH in presence of gilteritinib cooperatively shuts down oxidative phosphorylation and associated purine biosynthesis and mevalonate pathway, implying inhibition of these genes along with proteins involved in the mitochondria respiratory chain complexes and energy metabolism may represent an attractive strategy to resensitize leukemic cells to gilteritinib treatment. Overall, these findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and provide a rationale for a clinical trial of these novel combinatorial therapies.
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Project description:Purpose: Compare transcriptional changes between drug treated (gilteritinib, TP-0903) and vehicle in acute myeloid leukemia xenografts. Methods: FLT3-ITD+ MOLM13-Luc+ AML cells were xenografted into NSG mice. Mice were randomized according to bioluminescence imaging, and treatment of FLT3 tyrosine kinase inhibitors (TP-0903, 60 mg/kg, once daily for 5 days/week; gilteritinib, 30 mg/kg, once daily for 5 days/week) was started on day 7 post-tail vein injection. At study end point, mice were humanely euthanized and bone marrow was collected. AML cells (human CD45+) were isolated, and RNA-seq was performed on total RNA. Sequencing reads were aligned and analyzed for differential gene expression of treatment vs vehicle cohorts. Results: The analysis revealed the transcriptional changes induced by TP-0903 or gilteritinib in MOLM13 AML xenograft model.
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Project description:Purpose: Compare transcriptional changes between drug treated (gilteritinib, TP-0903) and vehicle in acute myeloid leukemia xenografts. Methods: FLT3-ITD+ MOLM13-RES-Luc+ (MOLM13 cells with a D835Y mutation generating a resistant phenotype) AML cells were xenografted into NSG mice. Mice were randomized according to bioluminescence imaging, and treatment of FLT3 tyrosine kinase inhibitors (TP-0903, 60 mg/kg, once daily for 5 days/week; gilteritinib, 30 mg/kg, once daily for 5 days/week) was started on day 13 post-tail vein injection. At study end point, mice were humanely euthanized and bone marrow was collected. AML cells (human CD45+) were isolated, and RNA-seq was performed on total RNA. Sequencing reads were aligned and analyzed for differential gene expression of treatment vs vehicle cohorts. Results: The analysis revealed the transcriptional changes induced by TP-0903 or gilteritinib in MOLM13-RES AML xenograft model.
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