nras g12d  (Jackson Laboratory)

Journal: Science Advances

Article Title: A germline ETV6 mutation disrupts hematopoiesis via de novo creation of a nuclear export signal

doi: 10.1126/sciadv.adu4058

Figure Lengend Snippet: ( A ) Schematic depicting the ETV6 activity in transformed Ba/F3 cells. ( B ) NRAS G12D -transformed Ba/F3 cell growth curves in the presence of ZsGreen or various ETV6 constructs. Error bars represent SD from n = 3 experimental replicates. ( C and D ) Hierarchical clustering analysis (C) and PCA analysis (D) of RNA-seq data collected from Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3). ( E ) Transcript abundance of known ETV6 target genes in Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3).

Article Snippet: To stably express the constitutively active NRAS G12D in Ba/F3 cells, a gene block corresponding to the NRAS G12D gene was ordered from Integrated DNA Technologies and assembled into the pLVX-IRES-TdTomato-FlagAkt1 plasmid using the Eco RI and Bam HI restriction sites.

Techniques: Activity Assay, Transformation Assay, Construct, RNA Sequencing

Journal: Science Advances

Article Title: A germline ETV6 mutation disrupts hematopoiesis via de novo creation of a nuclear export signal

doi: 10.1126/sciadv.adu4058

Figure Lengend Snippet: ( A ) Schematic depicting the ETV6 activity in transformed Ba/F3 cells. ( B ) NRAS G12D -transformed Ba/F3 cell growth curves in the presence of ZsGreen or various ETV6 constructs. Error bars represent SD from n = 3 experimental replicates. ( C and D ) Hierarchical clustering analysis (C) and PCA analysis (D) of RNA-seq data collected from Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3). ( E ) Transcript abundance of known ETV6 target genes in Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3).

Article Snippet: A population of positively transduced Ba/F3 NRAS G12D -IRES-TdTomato cells was sorted using a BD Biosciences FACSMelody Cell Sorter.

Techniques: Activity Assay, Transformation Assay, Construct, RNA Sequencing

Journal: Science Advances

Article Title: A germline ETV6 mutation disrupts hematopoiesis via de novo creation of a nuclear export signal

doi: 10.1126/sciadv.adu4058

Figure Lengend Snippet: ( A ) Schematic depicting the ETV6 activity in transformed Ba/F3 cells. ( B ) NRAS G12D -transformed Ba/F3 cell growth curves in the presence of ZsGreen or various ETV6 constructs. Error bars represent SD from n = 3 experimental replicates. ( C and D ) Hierarchical clustering analysis (C) and PCA analysis (D) of RNA-seq data collected from Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3). ( E ) Transcript abundance of known ETV6 target genes in Ba/F3 NRAS cell lines described in (B) at the 3-day time point ( n = 3).

Article Snippet: To stably express the constitutively active NRAS G12D in Ba/F3 cells, a gene block corresponding to the NRAS G12D gene was ordered from Integrated DNA Technologies and assembled into the pLVX-IRES-TdTomato-FlagAkt1 plasmid using the Eco RI and Bam HI restriction sites.

Techniques: Activity Assay, Transformation Assay, Construct, RNA Sequencing

Journal: bioRxiv

Article Title: TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

doi: 10.1101/2025.04.07.647605

Figure Lengend Snippet: (A) Normalized number of colony-forming units (CFU) formed by day 7 from 3 x10 c-Kit + cells harvested from Npm1 cA/+ mouse bone marrow after Ttll4 CRISPR editing. (B) Normalized number of colony-forming units (CFU) formed by day 7 from 3 x10 c-Kit + cells harvested from wildtype (C57/Bl6) mouse bone marrow after Ttll4 CRISPR editing. Experiments in (A) and (B) were performed three times with similar results. (C) Experimental design for primary transplant assays of Ttll4 CRISPR-Cas9 knockout in the Npm1 cA/+ ; Nras G12D/+ model of AML. Whole bone marrow cells were harvested from leukemic CD45.2 + Npm1 cA/+ ; Nras G12D/+ primary transplant-recipient mice, electroporated with sgNT or sgTTLL4 Cas9-ribonucleoproteins (RNPs), and transplanted into sub-lethally irradiated CD45.1 + B6.SJL recipient mice (n = 9). (D) Engraftment of donor cells was measured by flow cytometry for CD45.2 + cells in peripheral blood collected at 5 weeks post-transplantation. Representative flow plots for one mouse per group are shown. (E) Normalized median fluorescent intensity (MFI) of c-Kit expression in CD45.2 + peripheral blood cells at 5 weeks. Representative histograms from 3 mice per group are shown. (F) Wright-Giemsa staining of peripheral blood smears at 5 weeks. Representative images from one mouse per group are shown. Normalized data is presented as the ratio of individual replicates over the average of all sgNT replicates. Error bars indicate mean ± SEM; *p ≤0.05, ***p ≤0.001; unpaired t-test with Welch’s correction.

Article Snippet: Npm1 cA/+ or wildtype c-Kit + bone marrow or Npm1 cA/+ ; Nras G12D/+ whole bone marrow cells were pre-cultured for 1 hour at 37°C in PVA media, comprised of Ham’s F-12 media (Gibco) supplemented with 1% PVA (ThermoFisher), 10 mM HEPES (Life Technologies), 1X ITS-X (ThermoFisher), 2 mM L-glutamine (Gibco), 10 ng/mL mSCF (PeproTech), 10 ng/mL mTPO (Peprotech), and 1% penicillin/streptomycin (Corning).

Techniques: CRISPR, Knock-Out, Irradiation, Flow Cytometry, Transplantation Assay, Expressing, Staining

Journal: bioRxiv

Article Title: TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

doi: 10.1101/2025.04.07.647605

Figure Lengend Snippet: (A) Kaplan-Meier survival curves of mice transplanted with Npm1 cA/+ ; Nras G12D/+ leukemic bone marrow cells electroporated with sgNT or sgTTLL4 RNPs (n = 9 per group). (B) Quantification of flow cytometry analysis of Lin - Sca1 - c-Kit + (LK) myeloid progenitors in control (sgNT) or Ttll4 -deleted (sgTTLL4) CD45.2 + bone marrow collected at time of death. Representative flow plots of LK and LSK (Lin - Sca1 + c-Kit + ) populations for one mouse per group are shown. (C-E) Quantification of flow cytometry analysis of expression of the immature cell marker c-Kit against the myeloid differentiation markers (C) CD11b, (D) CD115, and (E) F4/80 in CD45.2 + bone marrow collected at time of death. Representative flow plots from one mouse per group are shown. Error bars indicate mean ± SEM; *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001; Log-rank Mantel-Cox test (A), unpaired t-test with Welch’s correction (B-E).

Article Snippet: Npm1 cA/+ or wildtype c-Kit + bone marrow or Npm1 cA/+ ; Nras G12D/+ whole bone marrow cells were pre-cultured for 1 hour at 37°C in PVA media, comprised of Ham’s F-12 media (Gibco) supplemented with 1% PVA (ThermoFisher), 10 mM HEPES (Life Technologies), 1X ITS-X (ThermoFisher), 2 mM L-glutamine (Gibco), 10 ng/mL mSCF (PeproTech), 10 ng/mL mTPO (Peprotech), and 1% penicillin/streptomycin (Corning).

Techniques: Flow Cytometry, Control, Expressing, Marker

Journal: bioRxiv

Article Title: TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

doi: 10.1101/2025.04.07.647605

Figure Lengend Snippet: (A) Normalized number of colony-forming units (CFU) formed by day 7 from 3 x10 c-Kit + cells harvested from Npm1 cA/+ mouse bone marrow after Ttll4 CRISPR editing. (B) Normalized number of colony-forming units (CFU) formed by day 7 from 3 x10 c-Kit + cells harvested from wildtype (C57/Bl6) mouse bone marrow after Ttll4 CRISPR editing. Experiments in (A) and (B) were performed three times with similar results. (C) Experimental design for primary transplant assays of Ttll4 CRISPR-Cas9 knockout in the Npm1 cA/+ ; Nras G12D/+ model of AML. Whole bone marrow cells were harvested from leukemic CD45.2 + Npm1 cA/+ ; Nras G12D/+ primary transplant-recipient mice, electroporated with sgNT or sgTTLL4 Cas9-ribonucleoproteins (RNPs), and transplanted into sub-lethally irradiated CD45.1 + B6.SJL recipient mice (n = 9). (D) Engraftment of donor cells was measured by flow cytometry for CD45.2 + cells in peripheral blood collected at 5 weeks post-transplantation. Representative flow plots for one mouse per group are shown. (E) Normalized median fluorescent intensity (MFI) of c-Kit expression in CD45.2 + peripheral blood cells at 5 weeks. Representative histograms from 3 mice per group are shown. (F) Wright-Giemsa staining of peripheral blood smears at 5 weeks. Representative images from one mouse per group are shown. Normalized data is presented as the ratio of individual replicates over the average of all sgNT replicates. Error bars indicate mean ± SEM; *p ≤0.05, ***p ≤0.001; unpaired t-test with Welch’s correction.

Article Snippet: Npm1 cA/+ , wildtype, or Npm1 cA/+ ; Nras G12D/+ mouse bone marrow was harvested from donor mice and subjected to red blood cell lysis (Qiagen).

Techniques: CRISPR, Knock-Out, Irradiation, Flow Cytometry, Transplantation Assay, Expressing, Staining

Journal: bioRxiv

Article Title: TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

doi: 10.1101/2025.04.07.647605

Figure Lengend Snippet: (A) Kaplan-Meier survival curves of mice transplanted with Npm1 cA/+ ; Nras G12D/+ leukemic bone marrow cells electroporated with sgNT or sgTTLL4 RNPs (n = 9 per group). (B) Quantification of flow cytometry analysis of Lin - Sca1 - c-Kit + (LK) myeloid progenitors in control (sgNT) or Ttll4 -deleted (sgTTLL4) CD45.2 + bone marrow collected at time of death. Representative flow plots of LK and LSK (Lin - Sca1 + c-Kit + ) populations for one mouse per group are shown. (C-E) Quantification of flow cytometry analysis of expression of the immature cell marker c-Kit against the myeloid differentiation markers (C) CD11b, (D) CD115, and (E) F4/80 in CD45.2 + bone marrow collected at time of death. Representative flow plots from one mouse per group are shown. Error bars indicate mean ± SEM; *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001; Log-rank Mantel-Cox test (A), unpaired t-test with Welch’s correction (B-E).

Article Snippet: Npm1 cA/+ , wildtype, or Npm1 cA/+ ; Nras G12D/+ mouse bone marrow was harvested from donor mice and subjected to red blood cell lysis (Qiagen).

Techniques: Flow Cytometry, Control, Expressing, Marker

Journal: bioRxiv

Article Title: Fetal context conveys heritable protection against MLL-rearranged leukemia that depends on MLL3

doi: 10.1101/2025.03.11.642680

Figure Lengend Snippet: (A, B) Kaplan-Meier survival curves after E10.5 or P0 MLL::ENL induction using Tet-On-ME and Tet-On-ME/Nras G12D models. (C) Summary of experimental set-up for MLL::ENL induction and transplantation experiments. (D) Kaplan-Meier survival curves for recipient mice transplanted with 300,000 P0 liver cells or 500 LSK cells after MLL::ENL induction at E10.5 or P14. (E) Donor (CD45.2) leukocyte engraftment in surviving recipient mice at 8 months after transplantation. (F) Summary of experimental set-up for panels G and H, and for CITE-seq experiments in . (G) Kaplan-Meier survival curves and 16-week donor chimerism for recipient mice after MLL::ENL induction at E10.5 or P0 and transplantation at P28. (H) MLL::ENL transgene expression (counts per million; CPM) based on RNA-seq. n= 4 biological replicates per group, ***p<0.001 by one-way ANOVA with Tukey’s posthoc test. For all survival curves, group sizes and p-values are shown in the panels. p-values were calculated by the log rank test.

Article Snippet: Vav1-Cre (RRID:IMSR_JAX:008610), Rosa26 LSL-rtTA-IRES-mKate2 (RRID:IMSR_JAX:008610), Rosa26 LSL-tTA (RRID:IMSR_JAX:011008), Col1a1 TetO-H2B-GFP (RRID:IMSR_JAX:016836) and Nras G12D (RRID:IMSR_JAX:008304), were obtained from Jackson Laboratory.

Techniques: Transplantation Assay, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Targeting CD38 effectively prevents myelofibrosis in myeloproliferative neoplasms

doi: 10.1101/2025.03.02.639410

Figure Lengend Snippet: A. Schematic of the transplanted Nras G12D/+ Jak2 V617F/+ -Mx1-Cre mouse model. B. White blood cell (WBC) counts, monocyte counts and hemoglobin level of wild type (WT, n = 10), Nras G12D/+ (N, n = 8), Jak2 V617F/+ (J, n = 8), and Nras G12D/+ Jak2 V617F/+ (NJ, n = 10) recipient mice following transplantation over a 28-week period. C-D. Representative flow cytometry dot plot and the percentage of CD11b + CD115 + monocyte and ckit + cells in bone marrow (BM). E. Kaplan-Meier survival curve for mice with NJ, J, N and WT. Statistics were assessed by Log-rank test. F. Spleen size and weight of four groups of mice at 20 weeks and 28weeks. G. Hematoxylin and eosin (H&E) staining of spleen of age-match (28 weeks) mice. Scale = 500 μm; Original magnification, 10×. H. Representative flow cytometry plots and the percentage of Lin − Sca1 + c-Kit + (LSK) and Lin − Sca-1 − c-kit + (myeloid progenitor, MP) cells in spleen from WT, N, J, and NJ mice 28 weeks post-transplantation. I. The percentage of CD11b + CD115 + monocyte in spleen 28 weeks post-transplantation. Red asterisks represent the statistical comparison of Ly6c high monocytes among the four groups, while blue asterisks represent the statistical comparison of Ly6c low monocytes among the four groups. All data represent mean ± standard error of the mean (SEM). Statistics were assessed by two-tailed Student’s T test. * P ≤ .05; ** P ≤ .01; *** P ≤ .001; **** P ≤ .0001.

Article Snippet: Nras G12D/+ mice were kindly provided by Dr. Xiaofan Zhu (State Key Laboratory of Experimental Hematology).

Techniques: Transplantation Assay, Flow Cytometry, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting CD38 effectively prevents myelofibrosis in myeloproliferative neoplasms

doi: 10.1101/2025.03.02.639410

Figure Lengend Snippet: A. Schematic of the Nras G12D/+ Jak2 V617F/+ mouse model treated with 78c. B. White blood cell (WBC) counts, monocyte counts and hemoglobin level of two groups. C. H&E and Reticulin staining of bone marrow (BM). Scale = 50 μm; Original magnification 40×. D. Comparison of BM fibrosis grade in each group. E. Quantification of mRNA levels of Cd38 in sorted BM CD11b + CD115 + Ly6c high monocyte by qRT-PCR. F. Schematic of LPS-induced Nras G12D/+ -Mx1-Cre transplanted mouse model induced treated with either solvent or 78c. G. H&E and Reticulin staining of BM. Scale = 50 μm; Original magnification 40×. H. Comparison of BM fibrosis grade in each group. I. Quantification of mRNA levels of Cd38 in sorted BM CD11b + CD115 + Ly6c high monocyte by qRT-PCR. J. The representative flow cytometry dot plot and the percentage of fibrocytes of two groups. All data represent mean ± standard error of the mean (SEM). Statistics were assessed by two-tailed Student’s T test. * P ≤ .05; ** P ≤ .01; *** P ≤ .001; **** P ≤ .0001.

Article Snippet: Nras G12D/+ mice were kindly provided by Dr. Xiaofan Zhu (State Key Laboratory of Experimental Hematology).

Techniques: Staining, Comparison, Quantitative RT-PCR, Solvent, Flow Cytometry, Two Tailed Test

Journal: Blood

Article Title: α-Ketoglutarate dehydrogenase is a therapeutic vulnerability in acute myeloid leukemia

doi: 10.1182/blood.2024025245

Figure Lengend Snippet: OGDH is a bona fide genetic dependency across AML systems. (A) Schematic of the TCA cycle highlighting the αKG dehydrogenase complex. (B) Differential gene dependency in AML vs all non-AML (other) cancer types, based on difference of mean depletion or enrichment of sgRNAs targeting individual genes across available CRISPR screens. An y-axis value <0 indicates preferential dependency in AML and a value >0 indicates preferential dependency in other. Each data point represents an individual gene. Selected genes are labeled. (C) OGDH , DLST , and DLD gene effect across CRISPR screens for AML vs other cell lines included in the DepMap database. A lower gene effect indicates an increased likelihood of gene dependency, with 0 indicating nondependency and –1 representing the median of all pan-essential genes. Each data point represents an individual cell line, and the mean ± standard error are shown. P values indicated (Wilcoxon rank-sum test). (D) OGDH gene dependency ranking across CRISPR screens for AML vs other cell lines included in the DepMap database. Each data point represents an individual cell line, and the mean ± standard error are shown. P value is indicated (Wilcoxon rank-sum test). (E) For each indicated gene, the violin plot shows change in sgRNA abundance (measured as log 2 fold change) in publicly available whole-genome CRISPR screens across multiple AML vs other cancer cell lines. P values are indicated (Wilcoxon rank-sum test). (F-G) Western blot analysis showing the expression of OGDH in AML cell lines transduced with the indicated shRNA after 3 days of doxycycline treatment. (F) Nras G12D ; MLL-AF9 cells. (G) p53 R172H cells. (H-I) Depletion of Ogdh shRNAs in the competition assay. (H) Nras G12D ; MLL-AF9 murine leukemia cells were infected with retroviruses encoding the indicated doxycycline-inducible shRNAs linked to a GFP reporter and admixed with 20% uninfected (GFP – ) cells. The relative percentages of GFP + cells (normalized to day 1 for each shRNA) were counted on the indicated days after doxycycline treatment. P values of shOgdh vs shControl groups indicated (2-way analysis of variance [ANOVA] with Sidak multiple comparisons test). (I) p53 R172H murine leukemia cells were infected with lentiviruses encoding reverse tetracycline-controlled transactivator and the indicated doxycycline-inducible shRNAs linked to a blue fluorescent protein (BFP) reporter on single backbone (LT3 BEPIR). Infected cells were admixed with 20% uninfected cells. The relative percentages of BFP + cells (normalized to day 2 for each shRNA) were counted on the indicated days after doxycycline treatment. (J) Western blot analysis showing expression of OGDH in PDX1 AML cells 4 days after transduction with the indicated shRNAs. (K) Cell proliferation of PDX1 AML cells transduced with the indicated shRNAs (normalized to day 6 after infection for each shRNA). (L) Depletion of OGDH shRNAs in a competition assay in PDX2 cells. PDX2 AML cells were infected at day –7 with a lentivirus encoding shControl or shOGDH linked to a GFP reporter and a second lentivirus encoding shControl linked to a red fluorescent protein (RFP) reporter. The GFP:RFP ratio (normalized to day 0 for each shRNA pair) was measured on the indicated days. P value of shOGDH vs shControl group is indicated (2-way ANOVA). αKGDC, α-ketoglutarate dehydrogenase complex; ns, not significant; OAA, oxaloacetate.

Article Snippet: Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras G12D ; MLL-AF9 AML cells (A) and p53 R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR).

Techniques: CRISPR, Labeling, Western Blot, Expressing, Transduction, shRNA, Competitive Binding Assay, Infection

Journal: Blood

Article Title: α-Ketoglutarate dehydrogenase is a therapeutic vulnerability in acute myeloid leukemia

doi: 10.1182/blood.2024025245

Figure Lengend Snippet: OGDH is required for AML progression in vivo. (A) Schematic of in vivo knockdown experiment using transplantation of shRNA-transduced Nras G12D ; MLL-AF9 AML. (B) Leukemia burden after shRNA knockdown of Ogdh in MLL-fusion AML. Nras G12D ; MLL-AF9 AML clones transduced with retroviruses encoding doxycycline-inducible sh Control or sh Ogdh were transplanted into sublethally irradiated recipient mice. Luminescence imaging was performed before (day 4) and 6 days after treatment with doxycycline vs vehicle (day 10). (C) Quantification of average radiance for the bioluminescence imaging shown in panel B. P values indicated (1-way ANOVA with Sidak multiple comparisons test; n = 5 per condition). (D) Survival curves are shown, and P values are indicated (log-rank test; n = 5-7 per condition). (E) Schematic of in vivo knockdown experiment using transplantation of shRNA-transduced p53 R172H AML. (F) Leukemia burden after shRNA knockdown of Ogdh in p53-mutant AML. p53 R172H AML clones transduced with retroviruses encoding doxycycline-inducible sh Control or sh Ogdh were transplanted into sublethally irradiated recipient mice. GFP + percentage in peripheral blood was measured at day 14. P values indicated (1-way ANOVA with Sidak multiple comparisons test; n = 7-8 per condition). Avg., average; max, maximum; min, minimum; Neo, neomycin resistance cassette; ns, not significant; p, photons; pre-tx, pre-treatment; puro, puromycin resistance cassette; PGK, phosphoglycerate kinase 1 promoter; rtTA, reverse tetracycline-controlled transactivator; s, seconds; sr, steradian; TRE, tetracycline response element.

Article Snippet: Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras G12D ; MLL-AF9 AML cells (A) and p53 R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR).

Techniques: In Vivo, Knockdown, Transplantation Assay, shRNA, Clone Assay, Transduction, Control, Irradiation, Imaging, Mutagenesis

Journal: Blood

Article Title: α-Ketoglutarate dehydrogenase is a therapeutic vulnerability in acute myeloid leukemia

doi: 10.1182/blood.2024025245

Figure Lengend Snippet: OGDH inhibition reduces TCA cycle flux and aspartate biosynthesis. (A-B) Analysis of mitochondrial bioenergetics. Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras G12D ; MLL-AF9 AML cells (A) and p53 R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR). Mitochondrial inhibitors are individually labeled and were added at the indicated time points. (C) TCA cycle and related metabolite levels in p53 R172H AML cells. LC-MS was performed at 48 hours after knockdown with the indicated shRNAs. Data are shown as a heat map of log 2 fold change relative to shControl-treated cells. Red color represents upregulated; blue color, downregulated. P values are indicated (2-way ANOVA with Sidak multiple comparisons test; n = 3-6 per condition). (D) Schematic of oxidative flux (carbons labeled in green) or reductive flux (carbons labeled in purple) through the TCA cycle by 13 C 5 -glutamine tracing. Red “X” indicates block observed in p53 R172H AML after OGDH knockdown. Labeled carbons are indicated in gray, green, or purple. (E) Fraction (%) of m+4 labeling from 13 C 5 -glutamine for the indicated metabolites in p53 R172H AML after induction of shRNAs targeting OGDH (or control) for 48 hours and labeling for 6 hours. P values indicated (1-way ANOVA with Dunnett multiple comparisons test; n = 3 per condition). (F) Fraction (%) of m+5 labeled citrate or m+3 labeled aspartate from 13 C 5 -glutamine in p53 R172H AML after induction of shRNAs targeting OGDH (or control) for 48 hours and labeling for 6 hours. P values indicated (1-way ANOVA with Dunnett multiple comparisons test or Kruskal-Wallis with Dunn multiple comparisons test as appropriate; n = 3 per condition). (G) After induction of shRNAs targeting OGDH (or control) in p53 R172H AML for 48 hours and labeling for 6 hours, LC-MS was used to determine the steady-state level and fraction of aspartate labeling from 13 C 6 -glucose (left) or 13 C 5 -glutamine (right). With 13 C 6 -glucose tracing, the m+2 and m+4 fractions represent 1 and 2 forward rotations through the TCA cycle, respectively, whereas with 13 C 5 -glutamine tracing m+2 represents 2 forward rotations and m+4 represents 1. Aspartate levels are shown as peak area in arbitrary units (a.u.) × fractional percentage of labeling with each tracer. P values are indicated for total labeled aspartate in shControl vs shOgdh groups (1-way ANOVA with Dunnett multiple comparisons test; n = 3 per condition). Ac-CoA, acetyl-coenzyme A; ctl, sh Control; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; ns, not significant.

Article Snippet: Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras G12D ; MLL-AF9 AML cells (A) and p53 R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR).

Techniques: Inhibition, Labeling, Liquid Chromatography with Mass Spectroscopy, Knockdown, Blocking Assay, Control

Journal: Blood

Article Title: α-Ketoglutarate dehydrogenase is a therapeutic vulnerability in acute myeloid leukemia

doi: 10.1182/blood.2024025245

Figure Lengend Snippet: Biosynthetic failure in AML impairs cell cycle progression and drives differentiation. (A) Representative Cytospin and Hema3 stains of Nras G12D ; MLL-AF9 clones transduced with the indicated shRNAs after 4 days of doxycycline treatment (n = 9 per condition). Scale bar, 10 μm. (B-C) Gene set enrichment analysis (GSEA) after OGDH depletion. Nras G12D ; MLL-AF9 AML clones (B) or p53 R172H AML cells (C) transduced with retroviruses encoding doxycycline-inducible sh Control or sh Ogdh (2 independent shRNAs sequenced separately and pooled during analysis) were subjected to RNA sequencing after 4 days of treatment with doxycycline. GSEA identified pathways of interest that were upregulated or downregulated. (D) Transcript levels of Gata2 (left) and Gypa (right) in p53 R172H AML cells treated with doxycycline for 4 days and sorted for BFP. BFP + indicates shRNA on; BFP– indicates shRNA off. P values for shOgdh off (BFP – ) vs on (BFP + ) indicated (unpaired t test; n = 4 per condition) (E-F) Cell cycle analysis by 5-ethynyl-2′-deoxyuridine (EdU) tracing. Nras G12D ; MLL-AF9 (E) or p53 R172H (F) AML cells treated with doxycycline for 4 days and sorted for GFP or BFP, respectively, were labeled with EdU for 1 hour. They were then fixed, permeabilized, stained with propidium iodide (PI), and subjected to cell cycle analysis by flow cytometry. Left lower gate represents G1 phase; upper gate, S phase; right lower gate, G2 phase/mitosis (M). Percentages of cells falling within each gate are indicated. (G) Extracellular aspartate levels in media were measured at 48 hours after knockdown of p53 R172H AML cells with the indicated shRNAs. (H) Baseline transcript levels of Slc1a3 and Slc1a5 in p53 R172H AML cells, MLL-fusion AML cells, and normal murine cKit-positive progenitor cells. (I) Relative number (% of control) of BFP + cells after 4 days of induction of the indicated shRNA in p53 R172H AML cells transduced with an empty vector or an exogenous Asp transporter (+SLC1A3). Media containing fetal bovine serum (FBS) was either unmodified (RPMI) or supplemented with 5 mM L-aspartate (L-Asp) or 10 mM L-glutamine (L-Gln) as indicated. P values are indicated (1-way ANOVA with either Dunn or Sidak multiple comparisons test as appropriate). (J) Median fluorescent intensity (MFI) of Cd11b after 4 days of induction of the indicated shRNAs in p53 R172H AML cells transduced with an empty vector or vector for expression of an exogenous aspartate transporter (+SLC1A3). Media containing FBS was supplemented with 5 mM L-Asp. P values are indicated (1-way ANOVA with Sidak multiple comparisons test). FDR, false discovery rate; LSC, leukemia stem cell; NES, normalized enrichment score; ns, not significant; TPM, transcripts per million.

Article Snippet: Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras G12D ; MLL-AF9 AML cells (A) and p53 R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR).

Techniques: Clone Assay, Transduction, Control, RNA Sequencing, shRNA, Cell Cycle Assay, Labeling, Staining, Flow Cytometry, Knockdown, Plasmid Preparation, Expressing