Project description:MLL-rearranged (MLL-r) leukemia, a particularly intractable disease, depends on DOT1L-mediated H3K79 methylation. Depletion of this transcriptionally activating mark by DOT1L loss-of-function or high concentrations of highly-specific inhibitor pinometostat (≥1 mM EPZ5676) leads to the downregulation of HOXA9 and MEIS1, and consequent reduction leukemia survival. . Yet, some MLL-r cell lines are inexplicably susceptible to low-dose pinometostat, below the threshold for downregulating these canonical oncogenic drivers. Here we define alternative pathways disrupted by low-dose pinometostat (10 nM), a concentration that reduces proliferation of the MLL-r MV4;11 cell line without affecting HOXA9 and MEIS1 expression and downregulates a subset of MLL-fusion targets including FLT3, one of the most commonly mutated genes in leukemia. Using quantitative ICeChIP-seq, we observe profound H3K79me2 depletion at downregulated MLL-r targets, with resulting increases in transcriptionally activating H3K4me3 at promoters and reductions in repressive H3K27me3. The presence of co-occurring activating FLT3 mutations, portend greater cytotoxicity to inhibitor treatment. Although downregulation of polycomb components modestly contributes to reductions in proliferation, overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, nearly completely rescues proliferation, accounting for the bulk of cytotoxicity from H3K79me2 depletion. We also observe a dependence of FLT3-STAT5A signaling on MLL function, suggesting that the FLT3 locus is exquisitely sensitivity to both H3K79me2 and H3K4me3 depletion and arguing that combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating the ~30% of all leukemias that carry FLT3 mutations.
Project description:Internal tandem duplications in the tyrosine kinase receptor FLT3 (FLT3-ITD) are among the most common lesions in acute myeloid leukemia (AML) and there exists a need for new forms of treatment. Using ex vivo drug sensitivity screening, we found that FLT3-ITD+ patient cells are particularly sensitive to HSP90 inhibitors. While it is well known that HSP90 is important for FLT3-ITD stability, we found that HSP90 family members play a much more complex role in FLT3-ITD signaling than previously appreciated. First, we found that FLT3-ITD activates the unfolded protein response (UPR), leading to increased expression of GRP94/HSP90B1. GRP94 rewires FLT3-ITD signaling by binding and retaining FLT3-ITD in the ER, where it aberrantly activates downstream signaling pathways. Second, HSP90 family proteins protect FLT3-ITD+ AML cells against apoptosis by alleviating proteotoxic stress, and treatment with HSP90 inhibitors results in proteotoxic overload that triggers UPR-induced apoptosis. Importantly, leukemic stem cells are strongly dependent upon HSP90 for their survival, and the HSP90 inhibitor ganetespib causes leukemic stem cell exhaustion in mouse PDX models. Taken together, our study reveals a molecular basis for HSP90 addiction of FLT3-ITD+ AML cells and provides a rationale for including HSP90 inhibitors in the treatment regime for FLT3-ITD+ AML.
Project description:ITD mutations in the FLT3 gene occur in the 30% of acute myeloid leukemia patients. The integration of ITD in the tyrosine kinase domain (TKD-ITD) of the FLT3 receptor has been shown to confer resistance to standard chemotherapy treatment. We applied state-of-the-art, high-sensitive, mass spectrometry (MS)-based (phospho)proteomics to investigate the molecular mechanisms underlying the sensitivity to cytarabine therapy in FLT3-ITD cells.
Project description:The miR-155-dependent differences in gene expression in the HSPC compartment of FLT3-ITD mice is unknown. In this experiment, we performed RNA sequencing on FLT3-ITD and FLT3-ITD miR-155-/- mouse LKS cells.
Project description:CircMYBL2 is more highly expressed in AML patients with FLT3-ITD mutations than in those without the FLT3-ITD mutation. We found that circMYBL2 knockdown specifically inhibits proliferation and promotes the differentiation of FLT3-ITD AML cells in vitro and in vivo. We used the ribosome profiling and RNA-seq libraries sequenced with Illumina HiSeq 2500 to identify the mRNA that circMYBL2 targeted. Interestingly, we found that circMYBL2 significantly influences the protein level of mutant FLT3 kinase, which contributes to the activation of FLT3-ITD-dependent signaling pathways. Mechanistically, circMYBL2 enhanced the translational efficiency of FLT3 kinase by increasing the binding of PTBP1 to FLT3 mRNA. Moreover, circMYBL2 knockdown impaired the cytoactivity of inhibitor-resistant FLT3-ITD-positive cells, with a significant decrease in FLT3 kinase expression, followed by the inactivation of its downstream pathways. In summary, we are the first to reveal a circRNA that specifically influences FLT3-ITD AML and regulates FLT3 kinase levels through translational regulation, suggesting that circMYBL2 may be a potential therapeutic target for FLT3-ITD AML.
Project description:We want to obtain FLT3-ITD gene signature. To do so, we transduced CB CD34+ cells with mock or FLT3-ITD vectors and performed RNA sequencing (RNA-Seq). Two Groups: Group1: CB CD34+ cells transduced with mock vector; Group2: CB CD34+ cells transduced with FLT3-ITD vector;
Project description:FLT3/ITD-SmoM2 mice developed rapidly fatal myeloid leukemia compared to FLT3/ITD only mice, suggesting that overactivation of the Hedgehog signaling pathway via SmoM2 can drive myeloid disease progression We used the Affymetrix Mouse 430_2.0 microarray to detail global gene expression responsible for disease progression in sorted bone marrow cells and found that the Hedgehog signaling pathway contributes to disease progression by enhancing FLT3 signaling
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.
Project description:mRNA expression regulated by FLT3/ITD and Cxcl12 were compared in the Ba/F3 cells expressing wild type FLT3 or FLT3/ITD and incubated with or without Cxcl12.