Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation

Fioretti, Tiziana; Cevenini, Armando; Zanobio, Mariateresa; Raia, Maddalena; Sarnataro, Daniela; Salvatore, Francesco; Esposito, Gabriella

doi:10.1007/s13402-019-00468-6

Cited by 7 publications

(31 citation statements)

References 55 publications

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“…In addition, our results suggest that inhibition of the SEC by avopiridol could reduce the sphere-forming ability and proliferating cell number of Lgr5+ progenitors in the differentiation assay, which is consistent with previous reports that AFF1 promotes leukemia cell proliferation 42 , that AFF4 enhances the sphereforming capacity and tumor-initiation capacity in head and neck squamous cell carcinoma 43 , and that ELL3 stimulates the proliferation and stem cell properties of breast cancer cells 44 .…”

Section: Discussionsupporting

confidence: 92%

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

Chen

Qiang²,

Zhang³

et al. 2021

Preprint

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Background: The super elongation complex (SEC) has been reported to play a key role in the proliferation and differentiation of mouse embryonic stem cells. However, the expression pattern and function of the SEC in the inner ear has not been investigated.Methods: Here, we measured the expression of the three key SEC subunits AFF1, AFF4, and ELL3. We also used Lgr5-EGFP-Ires-CreERT2 mice to isolate the Lgr5+ progenitors via flow cytometry to evaluate the effect of the SEC on the proliferation and differentiation ability of Lgr5+ progenitors.Results: We studied the inner ear expression pattern of three key SEC components, AFF1, AFF4 and ELL3, and found that these three proteins are all expressed in both cochlear hair cells and supporting cells. We also cultured Lgr5+ inner ear progenitors in vitro for sphere-forming assays and differentiation assays in the presence of the SEC inhibitor flavopiridol. We found that flavopiridol treatment decreased the proliferation ability of Lgr5+ progenitors, while the differentiation ability of Lgr5+ progenitors was not affected.Conclusions: Our results suggest that the SEC might play important roles in regulating inner ear progenitors and thus regulating hair cell regeneration. Therefore, it will be very meaningful to further investigate the detailed roles of the SEC signaling pathway in the inner ear in vivo in order to develop effective treatments for sensorineural hearing loss.

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Section: Discussionsupporting

confidence: 92%

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

Chen

Qiang²,

Zhang³

et al. 2021

Preprint

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“…Then, qRT-PCR was carried out in a 7500 Real-Time PCR System PCR Thermal Cycler with appropriate primers using a SYBR ® Select Master Mix (Applied Biosystems, Monza, Italy). Relative gene expression levels of VIM, EXT2, SDC2, CDH1, and GLUL were normalized to RNA polymerase II (POLR2A), α-tubulin (TUBA1A), and β-actin (ACTB) and calculated using the 2 −ΔΔCt method, as reported elsewhere [ 52 , 53 ]. Average values from at least three independent experiments were graphically reported as relative units.…”

Section: Methodsmentioning

confidence: 99%

Proteomics Reveals that Methylmalonyl-CoA Mutase Modulates Cell Architecture and Increases Susceptibility to Stress

Costanzo

Caterino

Cevenini

et al. 2020

IJMS

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Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H2O2-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes.

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“…It is widely proven that MLL-AF4 binds to the HOXA9 promoter and activates the transcription of this gene in MLL-related leukemia [ 17 , 35 ]. Since MLL-AF4 is a protein interactor of FGFR2, and FGFR2 silencing decreased expression of HOXA9, we evaluated the binding efficiency of MLL-AF4 to the HOXA9 promoter ( HOXA9 pr) in FGFR2-silenced RS4;11 cells.…”

Section: Resultsmentioning

confidence: 99%

“…Immunoprecipitates were dissolved in elution buffer (0.5 M EDTA, 1 M Tris-HCl pH 8.0) and DNA was isolated by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Real time quantitative PCR (RT-qPCR) was performed with 1 µL of DNA using a custom-made primer set [17] One million RS4;11 cells were plated in serum-free medium for 12 h and then stimulated with various concentrations of FGF2 (0.05-0.1-0.5 ng/mL). 50 µg of nuclear protein were loaded onto 10% SDS-PAGE for the western blot, performed with an anti-phospho-FGFR2 (P-FGFR2) or an anti-FGFR2 (FGFR2) antibody [47].…”

Section: Chromatin Immunoprecipitation (Chip) Assaymentioning

confidence: 99%

“…In addition to AF4, the AEP complex is formed by the transcriptional activators ENL, ELL, and AF5q—which are also common MLL fusion partners in human leukemia—as well as the positive elongation factor (P-TEFb) [ 16 ]. Through the direct interaction with the scaffold protein 14-3-3θ, AF4 and/or AF5q heterodimerize with MLL-AF4, thereby promoting the constitutive assembly of the AEP complex and the subsequent retrieval of RNA Pol II on target-gene promoters [ 17 , 18 ]. Moreover, MLL-AF4 forms a complex with DOT1L, a histone H3 lysine-79 (H3K79) methyltransferase, and other MLL fusion partners, such as AF9 and AF10 [ 11 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates HOXA9 Gene Expression in t(4;11) Leukemia Cells

Fioretti

Cevenini

Zanobio

et al. 2021

IJMS

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The chromosomal translocation t(4;11) marks an infant acute lymphoblastic leukemia associated with dismal prognosis. This rearrangement leads to the synthesis of the MLL-AF4 chimera, which exerts its oncogenic activity by upregulating transcription of genes involved in hematopoietic differentiation. Crucial for chimera’s aberrant activity is the recruitment of the AF4/ENL/P-TEFb protein complex. Interestingly, a molecular interactor of AF4 is fibroblast growth factor receptor 2 (FGFR2). We herein analyze the role of FGFR2 in the context of leukemia using t(4;11) leukemia cell lines. We revealed the interaction between MLL-AF4 and FGFR2 by immunoprecipitation, western blot, and immunofluorescence experiments; we also tested the effects of FGFR2 knockdown, FGFR2 inhibition, and FGFR2 stimulation on the expression of the main MLL-AF4 target genes, i.e., HOXA9 and MEIS1. Our results show that FGFR2 and MLL-AF4 interact in the nucleus of leukemia cells and that FGFR2 knockdown, which is associated with decreased expression of HOXA9 and MEIS1, impairs the binding of MLL-AF4 to the HOXA9 promoter. We also show that stimulation of leukemia cells with FGF2 increases nuclear level of FGFR2 in its phosphorylated form, as well as HOXA9 and MEIS1 expression. In contrast, preincubation with the ATP-mimetic inhibitor PD173074, before FGF2 stimulation, reduced FGFR2 nuclear amount and HOXA9 and MEIS1 transcript level, thereby indicating that MLL-AF4 aberrant activity depends on the nuclear availability of FGFR2. Overall, our study identifies FGFR2 as a new and promising therapeutic target in t(4;11) leukemia.

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Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation

Cited by 7 publications

References 55 publications

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

Proteomics Reveals that Methylmalonyl-CoA Mutase Modulates Cell Architecture and Increases Susceptibility to Stress

Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates HOXA9 Gene Expression in t(4;11) Leukemia Cells

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