Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , The Bdnf -TMKI model. CaRE, calcium regulatory element binding site; CRE, cAMP response element; WT, wild type. b , Optogenetic paradigm. M2, mouse premotor frontal cortex; P, postnatal day. c , Representative images of glioma (SU-DIPG-VI) xenografted into wild-type and Bdnf -TMKI cortex following blue-light stimulation of ChR2 + cortical neurons. HNA (grey) marks glioma cells, Ki67 (red) marks proliferating cells. Scale bar, 50 µm. d , Proliferation index (Ki67 + cells/HNA + glioma cells) of xenografted SU-DIPG-VI glioma in wild-type or Bdnf -TMKI mice stimulated optogenetically (ChR2 + cortical neurons) or mock-stimulated (ChR2 − neurons). n = 6 (wild-type ChR2 − ), 4 ( Bdnf -TMKI ChR2 − ), 7 (wild-type ChR2 + ) and 4 ( Bdnf -TMKI ChR2 + ) mice. e , Survival curves of wild-type and Bdnf -TMKI mice bearing SU-DIPG-XIII-P* xenografts. n = 7 (wild type) and 8 ( Bdnf -TMKI mice). f , Survival curves of mice bearing wild-type and NTRK2 -KO orthotopic xenografts (SU-DIPG-VI and SU-pcGBM; n = 7 mice per group). g , Survival curves of SU-DIPG-XIII-P* xenografted mice treated with entrectinib versus vehicle-treated controls. Grey shading indicates drug treatment. h , Representative images (left) and proliferation index (right; EdU + cells/DAPI cells) of wild-type and NTRK2 -KO glioma cultures (SU-DIPG-VI) with or without BDNF treatment ( n = 5 coverslips per group). Scale bar, 100 µm. i , Representative images (left) and proliferation index (right; EdU + cells/Nestin + glioma cells) of wild-type and NTRK2 -KO glioma (SU-DIPG-VI) cultured alone or with neurons ( n = 3 coverslips per group). Scale bar, 50 µm. j , Proliferation index of SU-DIPG-VI wild-type and NTRK2- KO glioma co-culture with neurons (as in representative image in i ), with or without NBQX ( n = 3 coverslips per group; repeated in Extended Data Fig. ). k – m , Experimental scheme ( k ), Representative images ( l ) and quantification of proliferation rate (Ki67 + cells/HNA + glioma cells) of wild-type and NTRK2 -KO glioma xenografts (SU-DIPG-VI) treated with perampanel or vehicle control ( m ). n = 6 (wild type + vehicle), 7 (wild type + perampanel), 5 ( NTRK2 -KO + vehicle) and 6 ( NTRK2- KO + perampanel) mice. Scale bar, 50 µm. Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis ( d , h – j , m ); two-tailed log rank analysis ( e – g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant.

Article Snippet: Four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2 -KO glioma cells, mice were treated with oral administration of the AMPAR blocker perampanel (5 mg kg −1 ; Adooq Biosciences; formulated in 10% DMSO, 60% PEG300, 30% water) via oral gavage for three weeks (5 days per week) and controls treated with equivalent volume of vehicle.

Techniques: Binding Assay, Cell Culture, Co-Culture Assay, Control, Two Tailed Test

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Proliferation index of SU-DIPG-VI WT and NTRK2 KO glioma monoculture (left), or glioma co-culture with neurons (right, as in Fig. ), in the presence and absence of the AMPAR blocker NBQX (10 μM) (quantified as fraction of EdU + /HNA+ co-positive tumor cells assessed by confocal microscopy, n = 3 coverslips/group for glioma monoculture experiments and 6 coverslips/group for neuron-glioma co-culture; experiment replicated in Fig. , WT vehicle vs WT + neurons vehicle P < 0.0001, WT + neurons vehicle vs WT + neurons NBQX P < 0.0001, WT + neurons vs NTRK2 KO + neurons P < 0.0001). b , Representative images of data quantified in a ; wild-type and NTRK2 KO glioma cells (SU-DIPG-VI) co-cultured with neurons in the presence and absence of NBQX (10 μM). Blue denotes HNA positive glioma cells; red denotes EdU (proliferative marker); green denotes MAP2 (neurons). Scale bar = 30 µm. c , Proliferation index of SU-DIPG-VI (red data points) and SU-DIPG-XIII-FL (blue data points) as a monoculture or cocultured with neurons in the presence of a CAMKII inhibitor, KN-93 (10 μM) or vehicle control (quantified as fraction of EdU + /HNA + glioma cells; n = 7 coverslips/group, vehicle vs vehicle + neurons P < 0.0001, vehicle + neurons vs KN-93 + neurons P = 0.0017, vehicle vs KN93 + neurons P = 0.0212). d , Representative images of data quantified in c ; glioma cells (SU-DIPG-VI) in monoculture, or co-cultured with neurons, in the presence and absence of KN-93 (10 μM). Blue denotes HNA positive glioma cells; red denotes EdU (proliferative marker); green denotes MAP2 (neurons). Scale bar = 100 µm. Data are mean ± s.e.m., *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant, one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis.

Article Snippet: Four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2 -KO glioma cells, mice were treated with oral administration of the AMPAR blocker perampanel (5 mg kg −1 ; Adooq Biosciences; formulated in 10% DMSO, 60% PEG300, 30% water) via oral gavage for three weeks (5 days per week) and controls treated with equivalent volume of vehicle.

Techniques: Co-Culture Assay, Confocal Microscopy, Cell Culture, Marker, Control

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Representative image of Alexa 568 (red)- filled GFP+ glioma cell following whole-cell patch clamp recording. Co-labelled with GFP (green) and human nuclear antigen (HNA, grey). Scale bars = 10 µm. b , Representative voltage-clamp traces of whole cell patch-clamp electrophysiological recordings in glioma cells. Hippocampal slices were perfused with ACSF containing tetrodotoxin (TTX, 0.5 µM), and response to a local puff (250 msec) application of 1 mM glutamate (black square) was recorded from xenografted glioma cells with sequential application of NMDAR blocker (AP-5, 100 µM), TBOA (200 µM), AMPAR blocker (NBQX, 10 µM). c , Quantification of data in b ( n = 7 glioma cells, 4 mice, P = 0.0165). d , Whole cell patch-clamp electrophysiological recording of glioma cell with ACSF puff, representative voltage clamp trace. e , Representative traces of glutamate-evoked inward currents (black square) in patient-derived glioma xenografted cells before (grey) and after 30-minute perfusion with NLGN3 recombinant protein (100 ng/ml) in ACSF (containing TTX, 0.5 µM) (purple). f , Quantification of data in e ( n = 5 glioma cells, 3 mice). g , Model of calcium imaging of tdTomato nuclear tagged (red nuclei), GCaMP6s-expressing (green calcium transients) glioma cells xenografted into the mouse hippocampal region. h , Quantification of number of xenografted SU-DIPG-XIII-FL or SU-DIPG-VI cells glioma cells demonstrating a calcium transient (as depicted in Fig. and Extended Data Fig. 6j) in response to a glutamate puff (responders, grey, non-responders, white). i , Baseline GCaMP6s intensity in SU-DIPG-VI glioma cells before and 30-min after BDNF exposure, in the absence of glutamate puff ( n = 7 cells, 3 mice). j , GCaMP6s intensity trace of SU-DIPG-VI glioma cells response to glutamate puff before (3 cells, 3 mice: light grey, average: dark grey) and after BDNF perfusion (three cells: light blue, average intensity: dark blue). k , SU-DIPG-VI GCaMP6s cell response to glutamate puff at baseline and after BDNF perfusion (100 ng/ml, 30 min, n = 7 cells, 4 mice, P = 0.0174). l , Duration of calcium transient response to glutamate puff in SU-DIPG-VI hippocampal xenografted cells, before and after perfusion with BDNF (100 ng/ml, 30 min, n = 6 cells, 4 mice, P = 0.0302). m , Representative traces of SU-DIPG-XIII glioma GCaMP6s intensity in the presence of BDNF (100 ng/ml, 30 min). Response to glutamate application (black) recorded with BDNF perfusion (3 cells, 2 mice: light blue, average: dark blue) or with BDNF and NBQX (10 µM, 3 cells: light red, average: red). n , Response of GCaMP6s cells to glutamate puff with BDNF application, in the presence and absence of NBQX ( n = 6 cells, 3 mice, P = 0.0002). Data are mean ± s.e.m., *P < 0.05, ***P < 0.001, ns = not significant, two-tailed paired Student’s t -test for k , l , n , and two-tailed Wilcoxon signed pairs matched rank test for d , f and i .

Article Snippet: Four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2 -KO glioma cells, mice were treated with oral administration of the AMPAR blocker perampanel (5 mg kg −1 ; Adooq Biosciences; formulated in 10% DMSO, 60% PEG300, 30% water) via oral gavage for three weeks (5 days per week) and controls treated with equivalent volume of vehicle.

Techniques: Patch Clamp, Derivative Assay, Recombinant, Imaging, Expressing, Two Tailed Test

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Schematic depicting AMPAR trafficking downstream of BDNF–TrkB–CAMKII signalling . b , Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c , Quantification of cell surface GluA4 in b ( n = 3 independent biological replicates). d , Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e , Quantification of cell surface GluA3 in d ( n = 3 independent biological replicates). f , Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g , Quantification of cell surface GluA4 data in f ( n = 3 independent biological replicates). h , Schematic showing GluA2–SEP experiments. i , j , Validation of pHluorin approach. i , Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture with neurons. Right, representative GluA2(Q)–SEP puncta. Scale bars, 5 µm (left) and 1 µm (right). Cells were exposed to pH 7.4 followed by pH 5.5 and then pH 7.4. j , Quantification of fluorescence intensity of GluA2(Q)–SEP puncta before, during and after acidic exposure ( n = 4 puncta from a representative cell). k , Top, representative images of two processes from glioma cells expressing GluA2(Q)–SEP, PSD95–RFP and TAG-BFP2 in co-culture with neurons (scale bar, 5 µm). Middle and bottom, representative images of GluA2(Q)–SEP puncta at 0, 5, 15 and 20 min of BDNF incubation (scale bar=1 µm). l , Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta over time with BDNF treatment ( n = 8 puncta, 6 cells). m , Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta after 15 min versus basal fluorescence in control (vehicle, n = 4 puncta, 2 cells) or BDNF-treated cells ( n = 8 puncta, 6 cells). Data are mean ± s.e.m. Two-tailed unpaired Student’s t -test ( c , e , g , m ); two-tailed paired Student’s t -test ( j ); two-tailed one-sample t -test ( l ).

Article Snippet: Four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2 -KO glioma cells, mice were treated with oral administration of the AMPAR blocker perampanel (5 mg kg −1 ; Adooq Biosciences; formulated in 10% DMSO, 60% PEG300, 30% water) via oral gavage for three weeks (5 days per week) and controls treated with equivalent volume of vehicle.

Techniques: Western Blot, Biomarker Discovery, Expressing, Co-Culture Assay, Fluorescence, Incubation, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance

doi: 10.3390/ijms20174300

Figure Lengend Snippet: PKA inhibitor treatment associated PKA activity changes and cell resistance to permethrin in Sf9 cell lines. ( A ) The effector expressions of Sf9 cell lines (CAT, GPCR020021, Gαs006458, AC007240, and PKA018257) treated with 60 µM PKA activity inhibitor (H89 2HCl). PKA activity was measured in different effector expression cell lines with or without H89 2HCl treatment. The x-axis represents different effector expressions of Sf9 cell lines following H89 2HCl or DMSO treatments; the y-axis represents the PKA activity (U/mL/mg protein). Blue columns represent cell lines treated with DMSO alone; the orange columns represent H89 2HCl-treated cell lines. ( B ) Percentage of cell survival was measured in different effector expression of Sf9 cell lines treated with 60 µM H89 2HCl and a serial of concentrations of permethrin (50, 100, 200, 400 µM). The x-axis represents the different effector expression cell lines following H89 2HCl and/or permethrin treatments; the y-axis represents the percentage of cell survival ratio. The blue, orange, grey, yellow, and light blue columns represent H89 2HCl plus acetonitrile, 50, 100, 200, and 400 μM permethrin treatments, respectively. Results are shown as the mean ± S.E ( n ≥ 3). Statistical significance ( p ≤ 0.05) for the changes in PKA activity in H89 2HCl-treated or control cell lines are represented by *** p < 0.001; for the percentage of cell survival ratio in the effector expression cell lines treated by permethrin and/or H89 2HCl, statistical significance ( p ≤ 0.05) is represented by different alphabetic letters (i.e., a, b, or c), which mean significances among samples.

Article Snippet: The GPCR, Gαs, AC, PKA expression (MOI = 0.5) Sf9 cells were pretreated with 60 μM H89 2HCl (in DMSO) at 27 °C for 1 h avoiding light, then 10 μM forskolin (in DMSO) (CAS no. 66575-29-9, Selleck Chemicals, Houston, TX, USA) [ ] and 30 μM H89 2HCl were added to the fresh medium 5 min prior to the cell lysis preparation.

Techniques: Activity Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance

doi: 10.3390/ijms20174300

Figure Lengend Snippet: Synergistic roles of Bupivacaine HCl and H89 2HCl to the toxicity of permethrin in insecticide resistant mosquitoes. The x-axis represents LC 50 (ppm) of permethrin to mosquito larvae; the y-axis represents the permethrin with or without inhibitor treatments to mosquito larvae. ( A ) Early 3rd instar larvae of HAmCq G8 treated with serial concentrations of Bupivacaine HCl (Bup.HCl) (25, 50, 100, and 200 µM) and different concentrations of permethrin (0.1, 0.3, 1, and 3 ppm); permethrin alone and permethrin plus DMSO-treated larvae served as controls. ( B ) Early 3rd instar larvae of MAmCq G6 treated with serial concentrations of Bupivacaine HCl (Bup.HCl) (25, 50, 100, and 200 µM) and different concentrations of permethrin (0.03, 0.1, 0.3, and 1 ppm); permethrin alone and permethrin plus DMSO-treated larvae served as controls. ( C ) Early 3rd instar larvae of HAmCq G8 treated with serial concentrations of H89 2HCl (6.25, 12.5, 25, and 50 µM) and different concentrations of permethrin (0.03, 0.1, 0.3, 1, and 3 ppm); permethrin alone and permethrin plus DMSO-treated larvae served as controls. ( D ) Early 3rd instar larvae of MAmCq G6 treated with serial concentrations of H89 2HCl (6.25, 12.5, 25, and 50 µM) and different concentrations of permethrin (0.01, 0.03, 0.1, 0.3 and 1 ppm); permethrin alone and permethrin plus DMSO-treated larvae served as controls. Results are shown as the mean ± S.E ( n ≥ 3). Statistical significance ( p ≤ 0.05) for LC 50 in insecticide resistance mosquito larvae treated by permethrin with or without inhibitor treatments are represented by different alphabetic letters (i.e., a, b, or c), which mean significances among samples.

Article Snippet: The GPCR, Gαs, AC, PKA expression (MOI = 0.5) Sf9 cells were pretreated with 60 μM H89 2HCl (in DMSO) at 27 °C for 1 h avoiding light, then 10 μM forskolin (in DMSO) (CAS no. 66575-29-9, Selleck Chemicals, Houston, TX, USA) [ ] and 30 μM H89 2HCl were added to the fresh medium 5 min prior to the cell lysis preparation.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A -Rearranged Acute Lymphoblastic and Myeloid Leukemia

doi: 10.3390/ijms25116020

Figure Lengend Snippet: Responses to revumenib in KMT2A -rearranged AML and ALL cells. ( A ) Cell viability in response to increasing concentrations of revumenib as assessed by 4-day MTT assays in KMT2A -rearranged (n = 6) and wildtype KMT2A (n = 3) AML cell line models. The dashed line shows the 50% viability threshold. Experiments were performed in technical triplicates and data consisted of three biological replicates. ( B ) IC 50 -values (i.e., the inhibitory concentration to 50% of the leukemic cells) for revumenib as determined by nonlinear regression in KMT2A -rearranged and wildtype KMT2A AML cell lines, statistically evaluated by an unpaired two-tailed t -test, with ns showing no significant differences. ( C ) Cell viability in response to increasing concentrations of revumenib using 4-day MTT assays in ex vivo pediatric KMT2A -rearranged AML patient samples obtained from patient-derived xenograft mouse models (n = 3). The dashed line shows the 50% viability threshold. Experiments were performed in technical triplicates. ( D ) Cell viability in response to increasing concentrations of revumenib as assessed by 4-day MTT assays in KMT2A -rearranged ALL cell line models (n = 5) and wildtype KMT2A ALL cell lines (n = 2). The dashed line shows the 50% viability threshold. Experiments were performed in technical triplicates and data consisted of three biological replicates. ( E ) IC 50 -values for revumenib as determined by nonlinear regression in KMT2A -rearranged and wildtype KMT2A ALL cell lines, statistically evaluated by an unpaired two-tailed t -test; * p < 0.05 ( F ) Cell viability in response to increasing concentrations of revumenib using 4-day MTT assays in ex vivo pediatric KMT2A -rearranged ALL patient samples (n = 5). The dashed line shows the 50% viability threshold Experiments were performed in technical triplicates.

Article Snippet: The KMT2A -rearranged AML cell lines include MV4-11 ( KMT2A::AFF1 + ; DMSZ: ACC 102), ML2 ( KMT2A::AFDN + ; DMSZ: ACC 15), SHI-1 ( KMT2A::AFDN + ; DSMZ: ACC 645), NOMO-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 542), THP-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 16), and MONO-MAC-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 252), and the wildtype KMT2A AML cell lines include HL-60 (DMSZ: ACC 3), Kasumi-1 (AML1-ETO); DSMZ: ACC 220), and OCI-AML3 (NPM1 mutated; DSMZ: ACC 582).

Techniques: Concentration Assay, Two Tailed Test, Ex Vivo, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A -Rearranged Acute Lymphoblastic and Myeloid Leukemia

doi: 10.3390/ijms25116020

Figure Lengend Snippet: Prolonged revumenib exposure induces myeloid differentiation in KMT2A -rearranged AML cells. ( A ) Percentage of viable cells after 7 and 14-day exposures to indicated concentrations of revumenib as determined by trypan blue exclusion in KMT2A -rearranged AML cell line models (n = 5). Responsive cell lines are in blue and unresponsive cell lines are in purple. The dashed line shows the 50% viability threshold. Experiments were performed in technical duplicates and data consisted of two biological replicates. ( B ) Percentages of live (grey), apoptotic (orange), and dead (red) cells after 7 and 14-day exposures to indicated concentrations of revumenib as determined by an Annexin V/7AAD staining and flowcytometry in KMT2A -rearranged AML cell line models. Differences in live, apoptotic, and death cells induced by revumenib as compared to untreated controls were statistically verified by two-way ANOVA Tukey’s multiple comparisons tests. Data consisted of two biological replicates. ( C ) Flow cytometric assessment of the expression of the myeloid differentiation marker CD14 and of stem cell marker CD117 (c-Kit) after 7 and 14 days of revumenib exposure in the responsive KMT2A -rearranged AML cell lines SHI-1, MONO-MAC-1, and NOMO-1, from duplicate experiments. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005 and ns for no significant p .

Article Snippet: The KMT2A -rearranged AML cell lines include MV4-11 ( KMT2A::AFF1 + ; DMSZ: ACC 102), ML2 ( KMT2A::AFDN + ; DMSZ: ACC 15), SHI-1 ( KMT2A::AFDN + ; DSMZ: ACC 645), NOMO-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 542), THP-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 16), and MONO-MAC-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 252), and the wildtype KMT2A AML cell lines include HL-60 (DMSZ: ACC 3), Kasumi-1 (AML1-ETO); DSMZ: ACC 220), and OCI-AML3 (NPM1 mutated; DSMZ: ACC 582).

Techniques: Staining, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A -Rearranged Acute Lymphoblastic and Myeloid Leukemia

doi: 10.3390/ijms25116020

Figure Lengend Snippet: Revumenib readily induces apoptosis in KMT2A -rearranged ALL cells. ( A ) Percentage of viable cells after 4-day exposures to indicated concentrations of revumenib as determined by trypan blue exclusion in KMT2A -rearranged ALL cell line models (n = 5; in orange) and the highly sensitive KMT2A -rearranged AML cell line MV4-11 (in blue). The dashed line shows the 50% viability threshold. Experiments were performed in technical duplicates and data consisted of two biological replicates. ( B ) Percentages of live (grey), apoptotic (orange), and dead (red) cells after 4-day exposures to indicated concentrations of revumenib as determined by flow cytometry and Annexin V/7AAD staining in the KMT2A -rearranged AML cell line MV4-11 and ( C ) in the KMT2A -rearranged ALL cell lines. Differences in live, apoptotic, and death cells induced by revumenib as compared to untreated controls were statistically verified by two-way ANOVA Tukey’s multiple comparisons tests. Data consisted of two biological replicates. ( D ) Cell cycle analysis showing the percentages of cells residing in the G1-phase, S-phase, and GM2-phase as determined by Hoechst 33342/7AAD staining and flow cytometry after 4-day exposures to indicated concentrations of revumenib. Differences in cell cycle phases induced by revumenib as compared to untreated controls were statistically verified by two-way ANOVA Tukey’s multiple comparisons tests. Data consisted of two biological replicates. ( E ) Cell viability (as determined by trypan blue exclusion) and cell cycle analysis (as determined by Hoechst 33342/7AAD staining and flow cytometry) after 4-day exposures to indicated concentrations of revumenib in a representative KMT2A -rearranged infant ALL patient sample obtained from a patient-derived xenograft mouse model. The dashed line shows the 50% viability threshold. * p < 0.05, ** p < 0.005, **** p < 0.00005 and ns for no significant p .

Article Snippet: The KMT2A -rearranged AML cell lines include MV4-11 ( KMT2A::AFF1 + ; DMSZ: ACC 102), ML2 ( KMT2A::AFDN + ; DMSZ: ACC 15), SHI-1 ( KMT2A::AFDN + ; DSMZ: ACC 645), NOMO-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 542), THP-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 16), and MONO-MAC-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 252), and the wildtype KMT2A AML cell lines include HL-60 (DMSZ: ACC 3), Kasumi-1 (AML1-ETO); DSMZ: ACC 220), and OCI-AML3 (NPM1 mutated; DSMZ: ACC 582).

Techniques: Flow Cytometry, Staining, Cell Cycle Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A -Rearranged Acute Lymphoblastic and Myeloid Leukemia

doi: 10.3390/ijms25116020

Figure Lengend Snippet: Induction of acquired resistance to revumenib in KMT2A -rearranged ALL cells. Induction of acquired resistance to revumenib was accomplished by exposing the KMT2A::AFF1 + ALL cell lines SEM and RS4;11 to increasing concentrations of revumenib of up to 10 µM for 10 weeks. Revumenib-resistant daughter cell lines are indicated as SEM REV_RES#1–4 and RS4;11 REV_RES#1–4 , respectively. ( A ) Cell viability in response to increasing concentrations of revumenib as assessed by 4-day MTT assays in SEM (orange) and revumenib-resistant daughter cell lines SEM REV_RES#1–4 (purple). The dashed line shows the 50% viability threshold. Experiments were performed in technical triplicates and data consisted of three biological replicates. ( B ) IC 50 -values for revumenib as determined by nonlinear regression in SEM (orange) and revumenib-resistant daughter cell lines SEM REV_RES#1–4 (purple) evaluated by an unpaired two-tailed t -test. ( C ) MEN1 mutation analysis (i.e., Sanger sequencing results) showing an M322T MEN1 mutation in all revumenib-resistant SEM daughter lines SEM REV_RES#1–4 (blue). ( D ) Cell viability in response to increasing concentrations of revumenib as assessed by 4-day MTT assays in RS4;11 (orange) and revumenib-resistant daughter cell lines RS4;11 REV_RES#1–4 (purple). The dashed line shows the 50% viability threshold. Experiments were performed in technical triplicates and data consisted of three biological replicates. ( E ) IC 50 -values for revumenib as determined by nonlinear regression in RS4;11 (orange) and revumenib-resistant daughter cell lines RS4;11 REV_RES#1–4 (purple) evaluated by an unpaired two-tailed t -test. ( F ) MEN1 mutation analysis reveals an M322T MEN1 mutation only in revumenib-resistant RS4;11 REV_RES#2 cells (blue).

Article Snippet: The KMT2A -rearranged AML cell lines include MV4-11 ( KMT2A::AFF1 + ; DMSZ: ACC 102), ML2 ( KMT2A::AFDN + ; DMSZ: ACC 15), SHI-1 ( KMT2A::AFDN + ; DSMZ: ACC 645), NOMO-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 542), THP-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 16), and MONO-MAC-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 252), and the wildtype KMT2A AML cell lines include HL-60 (DMSZ: ACC 3), Kasumi-1 (AML1-ETO); DSMZ: ACC 220), and OCI-AML3 (NPM1 mutated; DSMZ: ACC 582).

Techniques: Two Tailed Test, Mutagenesis, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A -Rearranged Acute Lymphoblastic and Myeloid Leukemia

doi: 10.3390/ijms25116020

Figure Lengend Snippet: Synergy between revumenib and pinometostat in KMT2A -rearranged ALL. Three-dimensional synergy plots showing drug synergy (red) or antagonism (green) between indicated concentrations of revumenib (x-axis) and pinometostat (y-axis) as determined by 6-day pinometostat pre-treated cells followed by 4-day MTT assays of revumenib exposures in ( A ) KMT2A -rearranged ALL cell lines, ( B ) a PDX derived KMT2A -rearranged infant ALL patient sample, and ( C ) KMT2A -rearranged AML cell lines. Drug synergy/antagonism is expressed as Zero Interaction Potency (ZIP) scores (z-axis), with scores of >5 being considered as synergistic effects (red areas), and ZIP scores below −5 are deemed as antagonistic effects (green areas). On top of each 3D synergy plot, the average ZIP score over the entire range of pinometostat and revumenib concentrations is listed.

Article Snippet: The KMT2A -rearranged AML cell lines include MV4-11 ( KMT2A::AFF1 + ; DMSZ: ACC 102), ML2 ( KMT2A::AFDN + ; DMSZ: ACC 15), SHI-1 ( KMT2A::AFDN + ; DSMZ: ACC 645), NOMO-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 542), THP-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 16), and MONO-MAC-1 ( KMT2A::MLLT3 + ; DSMZ: ACC 252), and the wildtype KMT2A AML cell lines include HL-60 (DMSZ: ACC 3), Kasumi-1 (AML1-ETO); DSMZ: ACC 220), and OCI-AML3 (NPM1 mutated; DSMZ: ACC 582).

Techniques: Derivative Assay

Journal: Cell Death & Disease

Article Title: The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

doi: 10.1038/cddis.2013.387

Figure Lengend Snippet: Amounts of endocannabinoids (panel a ), number of Nissl-stained cells (panels b , d and e ) and GFAP immunostaining (panel c ), measured in the striatum of malonate-lesioned rats after MAGL inhibition with OMDM169 (500 ng administered locally) or JZL184 (4 mg/kg weight administered i.p.), or after the administration of PGE 2 -G (10 μ g administered locally) and/or AGN220675 (300 ng administered locally), and of their sham-operated controls. See details in the text. Values are means±S.E.M. of 4–6 animals per group. Data were assessed by one-way analysis of variance followed by the Student–Newman–Keuls test (* P <0.05, ** P <0.001, *** P <0.005 versus controls; # P <0.05 versus the group treated with malonate and PGE 2 -G)

Article Snippet: In another experiment, JZL184 (purchased from Tocris Bioscience, Bristol, UK, and dissolved in DMSO-Tween 80-saline, 1 : 1 : 18) was administered to malonate-lesioned rats at the dose of 4 mg/kg weight in two injections, 30 min before and 2 h after the intrastriatal injection of malonate, following our previously published procedure.

Techniques: Staining, Immunostaining, Inhibition

Journal: Cell Death & Disease

Article Title: The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

doi: 10.1038/cddis.2013.387

Figure Lengend Snippet: Amounts of 2-AG (panel a ) and PGE 2 -G (panel b ), and LDH activity, measured in cultured M-213 cells treated for 6 h with 40 mM malonate and/or the inhibitors of MAGL, OMDM169, at 1 μ M (panel c ) or JZL184 at 10 nM (panel d ), or with 40 nM malonate and/or 2-AG at 25 μ M (panel e ). See details in the text. Values are presented as means±S.E.M. of 4–6 cases per group. Data were assessed by the one-way analysis of variance followed by the Student–Newman–Keuls test (* P <0.05, ** P <0.005 versus controls; # P <0.05 versus malonate)

Article Snippet: In another experiment, JZL184 (purchased from Tocris Bioscience, Bristol, UK, and dissolved in DMSO-Tween 80-saline, 1 : 1 : 18) was administered to malonate-lesioned rats at the dose of 4 mg/kg weight in two injections, 30 min before and 2 h after the intrastriatal injection of malonate, following our previously published procedure.

Techniques: Activity Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

doi: 10.1038/cddis.2013.387

Figure Lengend Snippet: LDH activity measured in cultured M-213 cells treated for 6 h with 40 mM malonate and/or the inhibitor of MAGL JZL184 at 10 nM in the presence or absence of PGE 2 -G antagonist AGN220675 at 25 μ M (panel a ) or the COX-2 inhibitor celecoxib at 12.5 μ M (panel b ), or with 40 nM malonate and/or 2-AG at 25 μ M combined with celecoxib at 12.5 μ M or AGN220675 at 25 μ M (panel c ), or treated directly with 2-AG at 25 or 50 μ M and/or celecoxib at 12.5 μ M (panel d ). See details in the text. Values are presented as means±S.E.M. of 6-8 cases per group. Data were assessed by the one-way analysis of variance followed by the Student–Newman–Keuls test (* P <0.05, ** P <0.005, *** P <0.0005 versus controls; # P <0.05 versus malonate+JZL184; ## P <0.005 versus malonate)

Article Snippet: In another experiment, JZL184 (purchased from Tocris Bioscience, Bristol, UK, and dissolved in DMSO-Tween 80-saline, 1 : 1 : 18) was administered to malonate-lesioned rats at the dose of 4 mg/kg weight in two injections, 30 min before and 2 h after the intrastriatal injection of malonate, following our previously published procedure.

Techniques: Activity Assay, Cell Culture