RNA-seq analysis of NRAS-mutant MV-4-11 AML cells treated with gilteritinib, clofarabine, or combined gilteritinib and clofarabine
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE339322 | GEO | 2026/09/25
REPOSITORIES: GEO
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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.
2022-07-07 | GSE199333 | GEO
Project description:While the FLT3 inhibitor gilteritinib is initially effective in patients with FLT3-mutated acute myeloid leukemia (AML), patients invariably relapse within months of treatment. Gilteritinib resistance is commonly driven by the emergence of NRAS mutations and a shift towards a more monocytic cell state. We hypothesized that directly targeting and depleting NRAS protein would reverse these adaptive differentiation changes and restore therapeutic sensitivity. To test this, we utilized a mutation-agnostic antisense oligonucleotide (ASO) to selectively knock down NRAS expression across gilteritinib-resistant AML cell lines, in vivo cell-line-derived xenografts, and primary patient samples. NRAS ASO successfully resensitized gilteritinib resistant cells with multiple distinct NRAS mutations, displaying superior efficacy compared to downstream MEK inhibition. This therapeutic efficacy was independent of NRAS mutant variant allele frequency, suggesting that wild-type NRAS may also contribute to resistance. Comprehensive multi-omic profiling (transcriptomics, proteomics, phosphoproteomics) revealed that NRAS knockdown consistently reversed monocytic phenotype, shifting cells back toward a more primitive cell state. Monocytic differentiation and NRAS mutations are established drivers of resistance to diverse targeted regimens in AML, including FLT3, IDH, and BCL2 inhibitors, so we also tested venetoclax resistant primary cells with NRAS mutations. Resistant cells were resensitized to venetoclax after NRAS knockdown, suggesting that NRAS knockdown may be more broadly applicable in overcoming monocytic cell state and drug resistance in AML.
2026-07-13 | GSE338135 | GEO
Project description:While the FLT3 inhibitor gilteritinib is initially effective in patients with FLT3-mutated acute myeloid leukemia (AML), patients invariably relapse within months of treatment. Gilteritinib resistance is commonly driven by the emergence of NRAS mutations and a shift towards a more monocytic cell state. We hypothesized that directly targeting and depleting NRAS protein would reverse these adaptive differentiation changes and restore therapeutic sensitivity. To test this, we utilized a mutation-agnostic antisense oligonucleotide (ASO) to selectively knock down NRAS expression across gilteritinib-resistant AML cell lines, in vivo cell-line-derived xenografts, and primary patient samples. NRAS ASO successfully resensitized gilteritinib resistant cells with multiple distinct NRAS mutations, displaying superior efficacy compared to downstream MEK inhibition. This therapeutic efficacy was independent of NRAS mutant variant allele frequency, suggesting that wild-type NRAS may also contribute to resistance. Comprehensive multi-omic profiling (transcriptomics, proteomics, phosphoproteomics) revealed that NRAS knockdown consistently reversed monocytic phenotype, shifting cells back toward a more primitive cell state. Monocytic differentiation and NRAS mutations are established drivers of resistance to diverse targeted regimens in AML, including FLT3, IDH, and BCL2 inhibitors, so we also tested venetoclax resistant primary cells with NRAS mutations. Resistant cells were resensitized to venetoclax after NRAS knockdown, suggesting that NRAS knockdown may be more broadly applicable in overcoming monocytic cell state and drug resistance in AML.
2026-08-17 | GSE341909 | GEO
Project description:FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML), and is associated with a poor prognosis and high relapse rate. Gilteritinib, as a second-generation FLT3 inhibitor, is an important target drug in the treatment of FLT3-ITD AML patients, but it still faces the problems of drug resistance and reduced potency. In this study, we found that mitoxantrone-liposome can sensensitized gilteritinib and enhanced the therapeutic effect in vivo and in vitro. RNA-sequencing revealed that the combination treatment resulted in specific changes in gene expression, reduced drug resistance and the mechanism. Primary AML cells harvested from patients with FLT3-ITD AML showed a significant response to combination treatment in vitro. Our data suggest represents a novel and promising therapeutic strategy for FLT3-ITD AML patients and relapsed/refractory(R/R) AML.
2025-03-19 | GSE276384 | GEO
Project description:While the FLT3 inhibitor gilteritinib is initially effective in patients with FLT3-mutated acute myeloid leukemia (AML), patients invariably relapse within months of treatment. Gilteritinib resistance is commonly driven by the emergence of NRAS mutations and a shift towards a more monocytic cell state. We hypothesized that directly targeting and depleting NRAS protein would reverse these adaptive differentiation changes and restore therapeutic sensitivity. To test this, we utilized a mutation-agnostic antisense oligonucleotide (ASO) to selectively knock down NRAS expression across gilteritinib-resistant AML cell lines, in vivo cell-line-derived xenografts, and primary patient samples. NRAS ASO successfully resensitized gilteritinib resistant cells with multiple distinct NRAS mutations, displaying superior efficacy compared to downstream MEK inhibition. This therapeutic efficacy was independent of NRAS mutant variant allele frequency, suggesting that wild-type NRAS may also contribute to resistance. Comprehensive multi-omic profiling (transcriptomics, proteomics, phosphoproteomics) revealed that NRAS knockdown consistently reversed monocytic phenotype, shifting cells back toward a more primitive cell state. Monocytic differentiation and NRAS mutations are established drivers of resistance to diverse targeted regimens in AML, including FLT3, IDH, and BCL2 inhibitors, so we also tested venetoclax resistant primary cells with NRAS mutations. Resistant cells were resensitized to venetoclax after NRAS knockdown, suggesting that NRAS knockdown may be more broadly applicable in overcoming monocytic cell state and drug resistance in AML.
2026-08-17 | GSE341910 | GEO
Project description:Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25% of acute myeloid leukemia (AML) patients and lead to constitutive activation of FLT3, driving leukemia cell survival and proliferation. Quizartinib, crenolanib and gilteritinib are second-generation FLT3 inhibitors (FLT3i) in phase III trials or clinical use for the targeted treatment of FLT3-ITD+ AML. However, they demonstrated only limited benefit and were not curative. A full understanding of cellular resistance factors contributing to this poor response is lacking. Here, we examined cell-autonomous pathways modulated by FLT3i using global translatome and phosphoproteome proteomics to identify non-genetic resistance mechanisms.
2022-08-25 | PXD023123 | Pride
Project description:Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25% of acute myeloid leukemia (AML) patients and lead to constitutive activation of FLT3, driving leukemia cell survival and proliferation. Quizartinib, crenolanib and gilteritinib are second-generation FLT3 inhibitors (FLT3i) in phase III trials or clinical use for the targeted treatment of FLT3-ITD+ AML. However, they demonstrated only limited benefit and were not curative. A full understanding of cellular resistance factors contributing to this poor response is lacking. Here, we examined cell-autonomous pathways modulated by FLT3i using global translatome and phosphoproteome proteomics to identify non-genetic resistance mechanisms.
2022-08-25 | PXD023125 | Pride
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.
2022-09-21 | GSE185519 | GEO
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.
2024-06-14 | GSE244855 | GEO
Project description:FLT3-ITD mutations are among the most frequent mutations in acute myeloid leukemia (AML) and are associated with poor prognosis and high relapse rates. Despite the extensive use of existing FLT3 inhibitors, resistance caused by secondary TKD mutations remains a major challenge. Therefore, the development of next-generation FLT3 inhibitors with broad-spectrum activity and sensitivity to drug-resistant mutations is of considerable clinical value. In this study, we systematically evaluated the antileukemic activity of a novel type I FLT3 inhibitor, SGI-7079, within the context of FLT3-ITD mutations and drug-resistant secondary mutations. SGI-7079 notably inhibited the proliferation of AML cell lines harboring FLT3-ITD mutations and multiple drug-resistant secondary mutations, such as D835Y/V/F, Y842C, and F691L, in vitro. Mechanistically, SGI-7079 was bound stably to the FLT3 protein, as indicated by molecular docking and CETSA analyses; inhibited FLT3 phosphorylation and downstream STAT5, AKT, and ERK signaling; and induced G1-phase arrest and apoptosis. In mouse models harboring FLT3-ITD mutations, SGI-7079 greatly reduced leukemia burdens, mitigated histopathological abnormalities, and prolonged median survival. Notably, SGI-7079 effectively overcame gilteritinib resistance driven by FLT3-ITD-F691L mutations in vivo and exhibited effectiveness against quizartinib-resistant FLT3-ITD-D835Y mutations. Importantly, SGI-7079 demonstrated notable inhibitory activity in primary AML cells from FLT3-ITD-positive patients, with effectiveness surpassing that of approved agents gilteritinib and quizartinib. In summary, this study is the first to demonstrate that SGI-7079 provides broad-spectrum and potent inhibitory activity across diverse FLT3 mutation contexts, showing remarkable advantages against drug-resistant mutations, especially F691L, and exhibits high potential for clinical translation.
2026-03-03 | GSE322773 | GEO