BMX Kinase Mediates Gilteritinib Resistance in FLT3-mutated AML through Microenvironmental Factors
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ABSTRACT:
PROVIDER: PRJNA819410 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18464727_1.fastq.gz | Fastqsanger.gz | |||
| SRR18464727_2.fastq.gz | Fastqsanger.gz | |||
| SRR18464728_1.fastq.gz | Fastqsanger.gz | |||
| SRR18464728_2.fastq.gz | Fastqsanger.gz | |||
| SRR18464729_1.fastq.gz | Fastqsanger.gz |
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Project description:Here, we report that Metformin shows a striking synergistic effect with Gilteritinib in suppressing cell proliferation and promoting apoptosis and cell cycle arrest in multiple FLT3-ITD AML cell lines, including FLT3 TKI-resistant MOLM13 cells. Mechanistically, the combinational treatment synergistically suppresses Polo-like kinase 1 (PLK1) expression and phosphorylation of FLT3/STAT5/ERK/mTOR in MOLM13-RES cells. Intriguingly, our retrospective clinical analysis has unveiled a significant correlation between Metformin intake and improved survival rates among FLT3-ITD AML patients. Collectively, the cotreatment of Metformin and Gilteritinib shows robustly enhanced therapeutic efficacy in treating FLT3-mutated AML by synergistically suppressing PLK1 expression and phosphorylation of FLT3/STAT5/ERK/mTOR.
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Project description:Oncogenic addiction to FLT3 kinase signaling is a hallmark of FLT3-ITD+ acute myeloid leukemia (AML). While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, utilizing RNA-Seq based analysis of patient leukemic cells, we found significant up-regulation of the Tec-family kinase BMX during sorafenib resistance. BMX upregulation was recapitulated in an in vivo FLT3-ITD+ model of sorafenib resistance. Mechanistically, we found that the anti-angiogenic effects of sorafenib led to increased bone-marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX up-regulation was observed in both AML and non-AML cell lines. Functional studies in FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Our results demonstrate that hypoxia-dependent up-regulation of BMX contributes to therapeutic resistance through a compensatory pro-survival signaling mechanism. These results also reveal the role of ‘off-target’ drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anti-cancer therapeutics, resulting in drug resistance through cell non-autonomous microenvironment-dependent effects.
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Project description:Primary human AML cells (newly diagnosed, prior to treatment initation) were obtained from donor after consent and AML blasts were isolated by standard Ficoll centrifugation. AML blasts were treated with FLT3 inhibitor gilteritinib (or DMSO vehicle control) at 10 nM concentration for 6 h, followed by harvest for LC/MS.
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Project description:Kinase hyperactivity is a common driver of acute myeloid leukemia (AML) and serves as a therapeutic target. The most frequent genetic aberration leading to hyperactive kinase signaling and poor prognosis is internal tandem duplication (ITD) of the FMS-tyrosine-like Kinase 3-gene (FLT3). FLT3-ITD induces ligand independent activation of FLT3 and downstream pathways leading to proliferation, decreased apoptosis and partial differentiation block. Combined with chemotherapy, FLT3-Tyrosine Kinase Inhibitor (FLT3-TKI) midostaurin improves overall survival (OS) of newly diagnosed FLT3-mutated AML patients, whereas single agent gilteritinib proved superior to chemotherapy in relapsed/refractory FLT3-mutated AML patients . As the primary targets of currently approved FLT3-TKIs are tyrosine (Y) kinases, we hypothesized that direct evaluation of tyrosine phosphorylation status could reveal pY phosphorylation profiles associated with FLT3-TKI response. Therefore, we used label-free pTyr-based phosphoproteomics in 35 primary AML samples (18 FLT3-WT, 17 FLT3-ITD), to identify differentially phosphorylated proteins underlying response to the FLT3-TKIs gilteritinib and midostaurin. We identified a total of 3024 unique phosphosites (median 1299 per sample, range 286 – 1612, pS:pT:pY 11.9%:9.5%:78.6%). Due to low number of identified phosphosites, we excluded two samples from further analyses. On the remaining 33 samples, we additionally performed IMAC global phosphoproteomics and on 17 samples protein expression analysis.
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Project description:Gilteritinib is a FLT3 inhibitor that has been approved for relapsed acute myeloid leukemia (AML) carrying FLT3 mutations. However, the effects of this drug on chromatin remodeling of AML cells remains unknown. We tested the state of chromatin openness of AML cells obtained from a patient before and after treatment with gilteritinib using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq).
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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-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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