Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: KMT2D p.(Arg191Trp) variant acquisition and expression in imatinib resistance. (A) Variant allele frequencies (VAF) of the KMT2D p. (Arg191Trp) variant in imatinib-resistant cell lines determined by exome or in-depth sequencing of K-562 cells resistant against 0.5 or 2 µM imatinib. (B) KMT2D mRNA expression in imatinib-resistant cells harboring KMT2D p. (Arg191Trp) measured by RT-qPCR and normalized to TBP , GAPDH and 18 S and treatment-naïve cells. (C) Protein levels of H3K4me1, Histon 3 (H3) and HSP90 in treatment-naïve and imatinib-resistant cells harboring KMT2D p. (Arg191Trp). Statistical analyses were performed using One-way ANOVA followed by Dunnett’s tests. Error bars indicate standard deviation. N = 3. *: p < 0.05, ***: p < 0.001. IM, imatinib; ND, no data; R, resistant.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Variant Assay, Expressing, Sequencing, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: Knockdown of KMT2D hampers the response to imatinib treatment. (A) KMT2D mRNA expression after transfection with an KMT2D -targeting siRNA or siRNA negative control (NC) analyzed by RT-qPCR and normalized to TBP , GAPDH , 18 S and NC. (B–D) Cell fitness after KMT2D knockdown under treatment with 2 µM imatinib analyzed on the level of (B) cell numbers (C) metabolic activity and (D) Ki-67 expression. (E) Protein levels of H3K4me1, histone H3 and HSP90 after knockdown of KMT2D . N = 3. Statistical analyses were performed using student’s tests. Error bars indicate standard deviation. N = 3. *: p < 0.05, ***: p < 0.001.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Activity Assay, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: Restoration of KMT2D expression improves the response to imatinib in imatinib-resistant cells. (A) KMT2D mRNA expression after rescue of KMT2D in two imatinib-resistant cell lines harboring KMT2D p. (Arg191Trp) analyzed by RT-qPCR. Data was normalized to TBP , GAPDH , 18 S and the respective empty vector negative control transfection (NC). (B–D) Cell fitness after rescue of KMT2D expression analyzed by (B) cell numbers, (C) metabolic activity and (D) Ki-67 expression after imatinib exposure. (E) Protein levels of H3K4me1 and histone H3 compared to HSP90 after restoration of KMT2D expression. Statistical analyses were performed using student’s tests. Error bars indicate standard deviation. N = 3. *: p < 0.05, ***: p < 0.001. NC: negative control.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Negative Control, Transfection, Activity Assay, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: The presence of the KMT2D p.(Arg191Trp) variant promotes imatinib resistance. Transfection of treatment-naïve K-562 cells with KMT2D wild-type (WT) or p. (Arg191Trp). (A) RT-qPCR of KMT2D compared to TBP , GAPDH , 18 S and normalized to the respective empty vector negative control (NC). (B) Protein levels of H3K4me1 and histone H3 compared to HSP90. (C–E) Cell fitness after transfection of KMT2D WT and p. (Arg191Trp) under imatinib treatment. (C) Cell numbers, (D) metabolic activity and (D) Ki-67 expression after exposure to 2 µM imatinib. Statistical analyses were performed using student’s tests. Error bars indicate standard deviation. N = 3. *: p < 0.05, **: p < 0.01, ***: p < 0.001. NC: negative control, pKMT2D : plasmid encoding KMT2D wild-type or p. (Arg191Trp).

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Variant Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, Negative Control, Activity Assay, Expressing, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: Sustained methylation of histone 3 compensates KMT2D p.(Arg191Trp)-mediated decrease in imatinib response. (A) Protein levels of H3K4me1 and histone H3 compared to HSP90. Depicted is one blot out of N = 3. (B) Cell fitness under imatinib treatment (2 µM) after transfection of KMT2D p. (Arg191Trp) in the presence of the HDMT inhibitor LSD1 inhibitor II (LSD1 inhibitor) on the level of cell numbers, metabolic activity and Ki-67 expression compared to solvent controls and normalized to KMT2D wild-type (WT). (C) IC50 values determined by metabolic activities of imatinib-resistant cells harboring KMT2D WT (black) or p. (Arg191Trp) (grey) analyzing two biological replicates, respectively. IC50 values were determined by non-linear regression with variable slope. Statistical analyses were performed using student’s tests. Error bars indicate standard deviation. N = 3. **: p < 0.01, ***: p < 0.001.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Methylation, Transfection, Activity Assay, Expressing, Solvent, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: Identification of putative KMT2D target genes. (A–C) Genome-wide expression data from imatinib-resistant K-562 cells harboring KMT2D wild-type (WT) or p. (Arg191Trp) were compared to the KMT2D essentiality network (1954 genes) obtained from . (A) Overlap of differentially expressed genes in imatinib-resistant cells harboring KMT2D WT or p. (Arg191Trp). (B) KEGG pathway cluster and (C) STRING analysis of the 197 genes differentially expressed in KMT2D p. (Arg191Trp) belonging to the KMT2D essentiality network (including KMT2D , high confidence settings). Pink: experimentally validated, turquoise: from curated database, green: gene neighborhood, red: gene fusions, blue: gene co-occurrence, yellow: text mining, black: co-expression, light blue: protein homology. (D) mRNA expression of CCND3 , CCNE2 and CDK4 in imatinib-resistant cells harboring KMT2D p. (Arg191Trp) compared to treatment-naïve cells analyzed by RT-qPCR normalized to the housekeeping genes TBP , GAPDH , and 18 S. N = 3. ***: p < 0.001.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Genome Wide, Expressing, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: The role of the lysine histone methylase KMT2D in chronic myeloid leukemia

doi: 10.3389/fphar.2025.1652373

Figure Lengend Snippet: Epigenetic modifiers and their inhibition in imatinib-resistant cells harboring KMT2D wild-type or p.(Arg191Trp). (A) Pie charts of differentially expressed epigenetic modifiers and histones in imatinib-resistant K-562 cells harboring KMT2D wild-type (WT) or p. (Arg191Trp). Blue: DNA-Methyltransferases, Red: Lysine methyltransferases, Green: Lysine demethylases, Orange: MLLT, Black: Histone family, Brown: Histone chaperone, Yellow: Histone deacetylase, purple: Histones. (B–C) Metabolic activity of imatinib-resistant cell lines harboring KMT2D WT or p. (Arg191Trp) in the presence of (B) 5′-azacytidine or (C) vorinostat with the respective IC50 values. Data were normalized to the respective solvent control. IC50 values were determined by non-linear fit. Error bars indicate standard deviation. N = 3.

Article Snippet: Samples were examined in triplicates using the TaqMan Universal Master Mix without UNG (Thermo Fisher Scientific) and the following TaqMan assays obtained from Thermo Fisher Scientific: KMT2D (Hs00912419_m1), CDK4 (Hs00364847_m1), CCND3 (Hs00236949_m1), CCNE2 (Hs00180319_m1), TBP (Hs00427620_m1), GAPDH (Hs02786624_g1), 18S (Hs99999901_s1).

Techniques: Inhibition, Histone Deacetylase Assay, Activity Assay, Solvent, Control, Standard Deviation

Journal: Molecular cancer therapeutics

Article Title: MicroRNA-203 inhibits long noncoding RNA HOTAIR and regulates tumorigenesis through epithelial-to-mesenchymal transition pathway in renal cell carcinoma

doi: 10.1158/1535-7163.MCT-17-0925

Figure Lengend Snippet: (A–D) qRT-PCR analysis showing significant increase in the mRNA levels of E-cadherin, claudin and decrease in mRNA levels of vimentin. (E) Western blot analysis showing protein levels of E-cadherin, claudin, vimentin and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203. (F) E-cadherin (red) and vimentin (green) immunostaining counterstained with DAPI (blue) in ACHN and Caki1 cells after transfections with miR-Control or miR-203 mimic, scale bar: 500 μm (right bottom). (G) Western blot analysis showing expression of stemness marker protein levels of KLF4, nanog and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203.

Article Snippet: The following primary antibodies were used: PTEN (Cell Signaling, 9552), E-cadherin (Thermo Fisher Scientific, MA5-11496), claudin (Thermo Fisher Scientific, 37-4900), vimentin (Cell Signaling, 3390), p21 (Cell Signaling, 2946), p27 (Cell Signaling, 2552), KLF4 (cell signaling 4038), nanog (cell signaling 4903) and ß-actin (Cell Signaling, 3700).

Techniques: Quantitative RT-PCR, Western Blot, Control, Over Expression, Immunostaining, Transfection, Expressing, Marker