PRMT1-mediated FLT3 arginine methylation promotes FLT3-ITD+ Acute Myeloid Leukemia maintenance
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE129754 | GEO | 2020/04/01
REPOSITORIES: GEO
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Project description:The presence of FLT3-ITD mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate FLT3-ITD+ leukemia stem cells (LSCs) which are potential sources of disease relapse, prompting us to ask whether FLT3-ITD protein regulates the AML LSCs survival through a kinase-independent mechanism. Here, we show that expression of PRMT1, the primary type I arginine methyltransferase, significantly increases in LSC-enriched CD34+CD38- populations relative to normal counterparts. Genetic PRMT1 depletion blocked AML CD34+ cell survival, and had more potent effects in AML cells from patients harboring FLT3-ITD. Our genome wide analysis of gene expression and PRMT1 conditional KO mouse study confirmed that PRMT1 preferentially cooperates with FLT3-ITD contributing to AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginines 972/973, and PRMT1 promoted leukemia cell growth in a FLT3 methylation-dependent manner. Moreover, effects of FLT3-ITD methylation in AML cells were in part due to crosstalk with FLT3-ITD phosphorylation at tyrosine 969 (Y969). Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following TKI (AC220) treatment, indicating that methylation occurs independently of kinase activity. Finally, in both patient-derived xenograft (PDX) and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes LSC activity and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to selectively target FLT3-ITD+ LSCs.
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Project description:Examination of gene expression patterns in lineage negative FLT3-ITD– and pMIG-transduced BM cells via microarray study. We performed a global mRNA profiling analysis of murine Lin– FLT3-ITD+ cells compared to empty-vector controls, purified 48h after transduction. We examined association patterns of FLT3-ITD and pMIG cells and identified a specific gene expression signature associated with FLT3-ITD signalling.
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Project description:Cooperative dependencies between mutant oncoproteins and wild-type proteins are critical in cancer pathogenesis and therapy resistance. Although spleen tyrosine kinase (SYK) has been implicated in hematologic malignancies, it is rarely mutated. We used kinase activity profiling to identify collaborators of SYK in acute myeloid leukemia (AML) and determined that FMS-like tyrosine kinase 3 (FLT3) is transactivated by SYK via direct binding. Highly activated SYK is predominantly found in FLT3-ITD positive AML and cooperates with FLT3-ITD to activate MYC transcriptional programs. FLT3-ITD AML cells are more vulnerable to SYK suppression than FLT3 wild-type counterparts. In a FLT3-ITD in vivo model, SYK is indispensable for myeloproliferative disease (MPD) development, and SYK overexpression promotes overt transformation to AML and resistance to FLT3-ITD-targeted therapy. HL-60, MOLM-14, and U937 cell lines were transduced in triplicate with a control luciferase-directed shRNA (target sequence CCTAAGGTTAAGTCGCCCTCG), and in duplicate with two SYK-directed shRNAs: shSYK_1 (clone ID TRCN0000197257, target sequence GCAGCAGAACAGACATGTCAA) and shSYK_2 (clone ID TRCN0000003163 , target sequence GCAGGCCATCATCAGTCAGAA), and were then selected with 1 µg/ml puromycin 48 hours post-infection. At day 5 post-infection, RNA was extracted and profiled using HT HG-U133A arrays (Affymetrix) at the Broad Institute (Cambridge, MA, USA). The computational analysis of the gene expression data was performed through the Genome Space bioinformatics platform (http://www.genomespace.org).
2014-01-15 | E-GEOD-54065 | biostudies-arrayexpress
Project description:FLT3 is the most frequently mutated gene in AML â up to 40% of AML harbor an activating mutation within FLT3 gene. Though AML is a relatively rare disease, such a high mutability rate, as observed with FLT3 gene, is striking. To elucidate the molecular background of this phenomenon, we have established nine unique FLT3/ITD - carrying 32D cell lines and a set of controls, and subjected them to whole genome expression analysis and 2DE LC/MS proteomics. Data obtained on this so far largest set of ITD mutants indicates that in comparison to the wild type FLT3 expressing 32D cells and transduction controls, FLT3/ITD positive cells exhibit less mature expression profiles resembling ST-HSC and MkEP/CMP/LMPP progenitors. We hypothesize that FLT3/ITD might contribute not only to the proliferative advantage of FLT3/ITD positive cells, but also to their reprogramming towards less differentiated stages, thus strengthening their malignant properties. This finding might explain the pronounced mutation rate of the aberrantly expressed FLT3 gene in AML, and, also, the inferior prognosis of FLT3/ITD positive AML patients. Moreover, the microarray data has revealed biological differences among individual ITD variants â a finding supporting the recent clinical data on the prognostic impact of the size of individual ITDs. Keywords: genetic modification Nine stable 32D cell lines harboring unique human FLT3/ITD mutants, two parental 32 cell lines, two 32D stable cell lines harboring cloning vector only, and two 32D cell lines stably expressing human wild type FLT3 were subjected to the microarray analysis.
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Project description:The expression level of microRNAs in FLT3-ITD+ AML is unknown. Using empty vector (EV) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing on small RNAs to determine microRNA expression level in these cells. We found a variety of evolutionarily conserved and non-conserved microRNAs expressed in our cells of interest.
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Project description:The expression profile in miR-155-/- FLT3-ITD+ AML is unknown. Using empty vector (EV) or two distinct miR-155 (S3 or S10) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing to determine the expression profile in these cells dependent on miR-155. We found a number of pathways dysregulated, including STAT5 activation.
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