Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Isogenic single, double and triple-mutant iPS cell lines generated through sequential CRISPRCas9-mediated gene editing of a normal iPS cell line (Parental). b , Overview of in vitro and in vivo phenotypic assessment of iPS-HSPCs. c , Fraction of CD34 − /CD45 + cells, i.e. hematopoietic cells that have lost CD34 expression upon maturation, on day 14 of hematopoietic differentiation. Mean and SEM from n = 8(P), 10(A, SA), 12(S), 7(R), 3(AR, SR), and 19(SAR) independent differentiation experiments with 2 (A, S, SA, AR, SR, SAR) or 3(R) iPS cell lines per genotype are shown. *P < 0.05, ****P < 0.001, ns: not significant (two-tailed unpaired t test). d , Number of methylcellulose colonies obtained from iPS-HSPCs on day 14 of hematopoietic differentiation. Mean and SEM from n = 6(P, A, S, SA, SAR), 3 (R, AR) and 4(SR) independent differentiation experiments with 2 iPS cell lines per genotype are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (two-tailed unpaired t test). e , Cell counts of iPS-HSPCs in liquid hematopoietic differentiation culture. Mean and SEM of n = 2(P, R, AR, SR), 4(A, SAR), 6(S) and 11(SA) independent differentiation experiments with 1 (P, R, SAR) or 2 (A, S, AR, SR, SA) iPS cell lines per genotype are shown. f , Competitive growth assay. The cells were mixed 1:1 at the onset of hematopoietic differentiation with an isogenic normal iPS cell line stably expressing GFP. The relative population size was estimated as the percentage of GFP- cells (calculated by flow cytometry) at each time point relative to the population size on day 2. Mean and SEM from n = 3(P), 4(A), 6(S, R), 5(SA), 2(AR,SR) and 8(SAR) independent differentiation experiments with 2 (P, A, R, SA, AR, SR, SAR) or 3 (S) iPS cell lines per genotype are shown. P values were calculated with a two-tailed unpaired t test. g , Cell cycle analyses of iPS-HSPCs. Mean and SEM from 3 (P, A, S, SA, SAR) and 4 (R) independent differentiation experiments with one line per genotype are shown. *P < 0.05 (R vs P: P = 0.0336; SAR vs P: P = 0.0279), ns: not significant (two-tailed unpaired t test). h , Human engraftment in the BM of NSG mice 13-15 weeks after transplantation with HSPCs derived from the indicated gene-edited mutant iPS cell lines (1 or 2 lines per genotype). Error bars show mean and SEM of values from individual mice. n = 2 (P); 2(A); 2(S); 8(R); 2(SA); 6(AR); 8(SR); 27(SAR). P values were calculated with a two-tailed unpaired t test. i , Representative flow cytometry for evaluation of human engraftment in mouse BM. j , Wright-Giemsa-stained BM cells retrieved from a mouse transplanted with SAR iPS-HSPCs. Scale bar, 25 μm. P: Parental; A: ASXL1 -mutant; S: SRSF2 -mutant; R: NRAS -mutant; SA: SRSF2 - ASXL1 double mutant; AR: ASXL1 - NRAS - double mutant; SR: SRSF2 - NRAS double mutant; SAR: SRSF2 - ASXL1 - NRAS triple mutant.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Mutagenesis, Generated, In Vitro, In Vivo, Expressing, Two Tailed Test, Growth Assay, Stable Transfection, Flow Cytometry, Transplantation Assay, Derivative Assay, Staining

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Gene editing strategy to generate a heterozygous DNMT3A R882H mutation in the same normal parental iPS cell line used to generate the lines shown in Extended Data Fig. , through homology-directed repair with simultaneous delivery of one mutant and one WT donor templates. Schematic representation of the DNMT3A locus with the position of the gRNA target sequence and the PCR primers used for RFLP analysis shown. Silent mutations introduced in the donor to create the DdeI restriction site (underlined) and inactivate the PAM motif are indicated in green font. The G→A mutation giving rise to the R882H amino acid substitution is shown in red font. b , Sanger sequencing confirming the G→A heterozygous point mutation giving rise to the R882H amino acid substitution in one edited DNMT3A R882H iPS cell line selected after screening. c , d , Schematic of gene editing steps to generate the iPS cell lines with single, double and triple driver mutations starting from the parental WT (c) or an iPS cell line derived from a RUNX1- familial platelet disorder (FPD) patient harboring a germline RUNX1 V118Gfs*11 mutation (d). e , Human engraftment in the BM of NSG mice 13-15 weeks after transplantation with gene-edited iPS-HSPCs. Mean and SEM is shown. RAR: RUNX1 - ASXL1 - NRAS triple mutant (n = 5 mice); DFR: DNMT3A - FLT3-NRAS triple mutant (n = 2 mice). f , Gene targeting strategy used to introduce a tetracycline response element (TRE)-driven Cas9 and the reverse tetracycline transactivator (rtTA), respectively, into the two alleles of the AAVS1 locus using TALEN-mediating targeting. g , Karyotype of iPS cell line NRAS G12D -iCas9-10 confirming a normal diploid karyotype. h , Confirmation of induction of iCas9 expression by DOX in the NRAS G12D -iCas9-10 iPS cells by qRT-PCR. i , Representative flow cytometric evaluation of engraftment in mice transplanted with the iPS-HSPCs shown in Fig. . j , Representative flow cytometric evaluation of transduction efficiency of iPS-HSPCs with the lentiviral constructs shown in Fig. , co-expressing the indicated fluorescent protein genes.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Mutagenesis, Sequencing, Derivative Assay, Transplantation Assay, Introduce, Expressing, Quantitative RT-PCR, Transduction, Construct

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , iPS cells with heterozygous SRSF2 and ASXL1 (top) or NRAS (bottom) mutations were differentiated into HSPCs and transduced with lentiviral vectors encoding NRAS G12D (top) or SRSF2 P95L and either a truncated dominant-negative ASXL1 transgene (ASXL1 del1900–1922 ) or a gRNA targeting exon 12 of ASXL1 (bottom) and transplanted intravenously into NSGS mice. b , Human engraftment in the bone marrow of mice 13–15 weeks post-transplantation. Each data point represents one mouse: n = 3 (R + mCherry), 4 (R + SA del ), 8 (R + SA gRNA ) and 6 (SA + R) from two experiments. Mean and s.e.m. are shown. P values were calculated with a two-tailed unpaired t -test. c , CB CD34 + cells were transduced with the lentiviral vectors shown at the indicated time intervals of in vitro culture, with Dox added to the culture at the indicated time point to induce NRAS G12D expression. The cells were prestimulated for 4 days before and were injected into NSGS mice 7 days after the first transduction. d , Human engraftment in the bone marrow of NSGS mice transplanted with CB CD34 + cells shown in c . P values were calculated with one-way ANOVA; n = 2 (mCherry/GFP), 3 (SA), 6 (R + SA) and 8 (SA + R) mice. Mean and s.d. are shown. e , Survival of mice from c ; n = 5 (SA + R) and 3 (R + SA) mice. f , Bone marrow and spleen images from a mouse transplanted with CB SA + R cells representative of at least three experiments. Left, haematoxylin and eosin (H&E) staining. Middle and right, immunohistochemistry for hCD45 (pan-haematopoietic) and hCD33 (myeloid) markers. g , Wright–Giemsa-stained human cells retrieved from the bone marrow of a mouse transplanted with SA + R CB cells. Image representative of at least three independent experiments. h , Representative images of spleens from mice transplanted as shown in c . BM, bone marrow; UT, untransplanted. Scale bars, 500 μm ( f , lower magnification panels), 100 μm ( f , higher magnification panels), 50 μm ( g ).

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Transduction, Dominant Negative Mutation, Transplantation Assay, Two Tailed Test, In Vitro, Expressing, Injection, Staining, Immunohistochemistry

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Experimental scheme. CB CD34 + cells were transduced with lentiviral vectors as indicated and analysed by flow cytometry (right panels) on the day of transplantation, that is, 7 days after the first transduction (day 8). b , Uniform manifold approximation and projection (UMAP) representation of integrated single-cell transcriptome data from the six groups of cells shown in a , on day 8. c , Stacked barplots showing fraction of cells in each cluster. With the exception of clusters marked as not significant (NS), all other cluster sizes were significantly different from the respective cluster size in the Ctrl (mCherry/GFP) group (logistic regression). d , iPS-HSPCs with NRAS (R) or SRSF2 and ASXL1 mutations (SA) were transduced with lentiviral vectors as indicated. Right panels, principal component analysis of RNA-seq and ATAC-seq data from sorted CD34 + CD45 + cells; n = 3 independent experiments for all groups. e , Normalized enrichment scores (NES) and adjusted P values derived from gene set enrichment analysis (GSEA) for gene sets corresponding to the human AML developmental hierarchy from ref. using gene lists ranked by the −log 10 ( P adjusted (padj)) × log 2 FC from the indicated differential expression comparisons. f , Heatmap showing Pearson correlation values for the ATAC-seq peak normalized read counts in the iPS-HSPC dataset from d and overlapping peaks in primary normal haematopoietic cell subpopulations from ref. . g , Experimental scheme (left) and survival (right) of animals transplanted with CMPs and GMPs transduced with SA and sorted prior to induction of R with Dox. n = 1 for each CMP group (±Dox); n = 3 for each GMP group (±Dox). h , Experimental scheme (left) and survival (right) of mice transplanted with CMPs and GMPs sorted prior to SAR transduction. n = 4 for all groups. i , FACS-sorted CMPs and GMPs from g . CLP, common lymphoid progenitor; DC P, dendritic cell progenitor; EBM, eosinophil/basophil/mast cell; Ery/Meg P, erythrocyte/megakaryocyte progenitor; Ery P, erythrocyte progenitor; LMPP, lymphoid-primed MPP; Meg P, megakaryocyte progenitor; Mono, monocyte; Mono P, monocyte progenitor; ProMono, promonocyte.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Transduction, Flow Cytometry, Transplantation Assay, RNA Sequencing, Derivative Assay, Quantitative Proteomics

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Two iPS cell lines derived from a patient with AML with a clonal t(1;7;14) translocation and a subclonal KRAS G12D mutation, one capturing the RASWT major clone (AML-4.24) and one the KRAS G12D subclone (AML-4.10) of the patient AML were differentiated to HSPCs, transplanted into NSGS mice, allowed to generate lethal leukaemias, collected, sorted and subjected to scRNA-seq analysis. b , UMAP representation of single-cell transcriptome data. The dashed line delineates the monocytic metacluster. c , Cell density across the UMAP coordinates from b . Cells coloured by sample (top panel) or phase of the cell cycle (bottom panel). d , Percentage of cells expressing the indicated genes (normalized counts > 0.5) or contained in the monocytic metacluster (shown in b). e , Schematic of the GoT experiment. f , Number of cells that could be genotyped as NRAS -WT (423 cells) or MT (576 cells) by GoT. g , h , Fraction ( g ) and absolute number ( h ) of cells belonging to each cell type assigned from transcriptome data in the NRAS -WT and NRAS -MT cells (NA, not assigned to a NRAS genotype). Cells belonging to the NRAS -MT clone contain a higher fraction of monocytic cells (Fisher’s exact test P value = 0.00028, odds ratio = 3.255) and lower fraction of immature HSC/MPP-like cells (Fisher’s exact test P value = 3.044 × 10 −11 , odds ratio = 0.3345) than NRAS -WT cells. i , Expression of a monocytic priming gene module (IRF7/IRF8) from ref. in NRAS -WT and MT cells. The whiskers denote the 1.5× interquartile range (IQR). The lower and upper hinges of the boxes represent the first and third quartiles, respectively. The middle line represents the median. Points represent values outside the 1.5× IQR. The P value was calculated with a two-sided Wilcoxon test.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Derivative Assay, Translocation Assay, Mutagenesis, Expressing

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , UMAP of integrated single-cell transcriptome data from Fig. , at resolution 0.4. b , Expression of selected marker genes in each annotated cluster from the GoT data. c , Fraction of CD14+ monocytic blasts in AML patients with mutations in RAS pathway genes ( NRAS , KRAS or PTPN11 ) or without any RAS pathway mutation (RAS WT). “Any RAS MT” denotes cases with mutations in either of the 3 genes NRAS , KRAS or PTPN11 . The whiskers denote the 1.5* IQR (interquartile range). The lower and upper hinges of the boxes represent the first and third quartiles, respectively. The middle line represents the median. Points represent values outside of the 1.5* IQR. The P value was calculated with a two-sided Wilcoxon test. d , FAB subtype of AML patients with mutations (MT) in RAS pathway genes ( NRAS , KRAS or PTPN11 ) or without any RAS pathway mutation (RAS WT). “Any RAS MT” denotes cases with mutations in either of the 3 genes NRAS , KRAS or PTPN11 . Two-tailed Fisher test, ns: not significant. e , Flow cytometry for myelomonocytic markers CD68 and CD11b in CD34+ cells from 4 patient-derived AML-iPS cell lines with or without (Control) lentiviral expression of NRAS G12D or KRAS G12D . MLLr: MLL-rearranged; SF: splicing factor; CBF: core binding factor. f , Fraction of CD14+ monocytic blasts in AML patients with or without mutations (MT) in SRSF2 and ASXL1 genes. SA denotes cases with double SRSF2 and ASXL1 mutations; S/A WT denotes cases without SRSF2 or ASXL1 mutations. The whiskers denote the 1.5* IQR (interquartile range). The lower and upper hinges of the boxes represent the first and third quartiles, respectively. The middle line represents the median. Points represent values outside of the 1.5* IQR. ns: not significant (two-sided Wilcoxon test). g , FAB subtype of AML patients with or without mutations (MT) in SRSF2 and ASXL1 genes. SA denotes cases with double SRSF2 and ASXL1 mutations; S/A WT denotes cases without SRSF2 or ASXL1 mutations. Two-tailed Fisher test, ns: not significant. h , Proportion of cases with or without NRAS or KRAS mutations (RASMT and RAS–WT, respectively) with or without combined SRSF2 and ASXL1 mutations (SA and not SA, respectively) among 399 CMML patients from the MDS International Working Group cohort (Bernard et al. 2022). (P value: one tail Fisher test). i , j , Experimental scheme. Schematic of lentiviral vectors used (i). Vectors N, K and F are DOX-inducible. k , Myelomonocytic markers CD11b and CD14 in GMPs with various transgene combinations, shown in i,j, cultured for 5 days after sorting. N: NRAS G12D , K: KRAS G12D , F: FLT3-ITD, IDH: IDH1 R132H , S: SRSF2 P95L ; A: ASXL1 Del . l , Heatmap showing differential expression of the indicated granulocytic (MPO, AZU1, ELANE) and monocytic (CD14, CD52, S100A6, S100A8, S100A9, CCL2, CCL3, CCL4) lineage genes in the GMP cluster, in the indicated comparisons, from the single-cell transcriptome data from Fig. . *P < 0.05, ***P < 0.001 (two-sided Wilcoxon test).

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Expressing, Marker, Mutagenesis, Two Tailed Test, Flow Cytometry, Derivative Assay, Control, Binding Assay, Cell Culture, Quantitative Proteomics

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a – d , Outcome data from 118 older or unfit patients with newly diagnosed AML treated in a prospective trial with 10-day DEC and VEN (DEC10-VEN). DOR ( a ) and OS ( b ) in patients with monocytic versus non-monocytic AML. DOR ( c ) and OS ( d ) in patients with AML with TP53 -WT with versus without N/KRAS mutations. log-rank test, two-tailed unadjusted P values. e , Two iPS cell lines derived from a patient with AML, one capturing the RAS-WT major clone (AML-4.24) and one the KRAS G12D subclone (AML-4.10), were differentiated in vitro to LSCs and to monocytic blasts. f , HSPCs and monocytes derived from normal iPS cells and from the indicated AML-iPS cell lines were treated with VEN and viability was measured by CellTiter-Glo. Viability compared with dimethylsulfoxide (DMSO)-treated is shown. HSPCs, n = 3 normal, 5 AML-4.24, 7 AML-4.10 and 2 AML-9.9; monocytes, n = 3 normal, 4 AML-4.24, 2 AML-4.10 and 1 AML-9.9 independent experiments; mean and s.d. are shown. P values were calculated with a two-tailed unpaired t -test. g , CB CD34 + cells transduced with SA + R, as shown in Fig. were transplanted into NSGS mice. The mice were treated with VEN (100 mg kg −1 day −1 by oral gavage) or vehicle, starting 1 week post-transplant, daily, for 3 weeks. h , Survival of mice from the experiment shown in g ; n = 3 (VEN) and 4 (Vehicle). P value was calculated with a log-rank (Mantel–Cox) test. i , NRAS G12D expression in hCD45 + cells from the bone marrow of a moribund mouse treated with VEN. CI, confidence interval; CR, complete remission; CRi, CR with incomplete haematologic recovery; HR, hazard ratio; NR, not reached.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Two Tailed Test, Derivative Assay, In Vitro, Transduction, Expressing

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Violin plots showing expression of anti- and pro-apoptotic genes of the BCL2 family in monocytic blasts (monocytic metacluster generated by merging all monocytic and dendritic cell clusters shown in Fig. and Extended Data Fig. ) or LSCs (cluster 28 shown in b-d) within the AML-4.10 and AML-4.24 leukemia cells from xenografts. P values were calculated with a two-sided Wilcoxon test. b , Expression of anti- and pro-apoptotic genes in the LSC cluster shown in Fig. without subclustering in the iPS cell-derived leukemia cells from xenografts. P values were calculated with a two-sided Wilcoxon test. c , Expression of HSC markers SPINK2 and HOPX projected onto the integrated UMAP. The red squares indicate the LSC subcluster (cluster 28 shown in c,d). d , UMAP representation of single-cell transcriptome data in resolution 3.2, yielding 46 clusters. e , Left panel: UMAP representation of the LSC cluster (from resolution 0.4 clustering shown in Fig. ) subdivided into 5 clusters (from resolution 3.2 clustering shown in c). Middle and right panels: Expression of HSC markers CD34, HOPX and SPINK2 projected onto the LSC cluster UMAP. f , Split-violin plots showing expression of anti- and pro-apoptotic genes in monocytic blasts or immature MPP-like and GMP-like cells of the NRAS MT and NRAS WT genetic clones from GoT data. **P < 0.01, ****P < 0.0001, ns: not significant (two-tailed Wilcoxon test). g , Normalized expression of the indicated pro- and anti- apoptotic genes in the genetically engineered iPS-HSPCs shown in Fig. (n = 3 independent experiments for all groups). *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant (two-tailed unpaired t-test).

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Expressing, Generated, Derivative Assay, Clone Assay, Two Tailed Test

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Experimental design. b , UMAP representation of integrated single-cell transcriptome data from FACS-sorted SA + R CMPs and GMPs. c , Viability of FACS-sorted CMPs, GMPs and HSC/MPPs, untransduced (UT) or transduced with SA + R, treated with VEN. ** P < 0.01, **** P < 0.0001, NS (one-way ANOVA). Mean and s.d. from n = 3 independent experiments are shown. d , Cells expressing ΔLNGFR-NRAS G12D (NRAS G12D+ , red) or none of the transgenes (WT, green) projected in the UMAP from b . e , Expression of MCL1 and BCL2 in NRAS G12D+ versus WT cells belonging to the GMP cluster. P values were calculated with a two-sided Wilcoxon test. f , Viability of CD34 + LSCs from the indicated patient-derived AML-iPS cell lines with or without (Control) ectopic lentiviral expression of NRAS G12D or KRAS G12D , as indicated, treated with VEN and/or RASi. Viability compared with DMSO-treated group is shown. Mean and s.d. from n = 3 or 4 independent experiments is shown. P values were calculated with a two-tailed unpaired t -test. g , Detection of the indicated proteins by western blotting in CD34 + LSCs from the indicated patient-derived AML-iPS cell lines with or without (Control) ectopic lentiviral expression of NRAS G12D or KRAS G12D , with or without treatment with RASi. Samples were derived from the same experiment and processed in parallel. β-Actin controls were run on different gels as sample processing controls. For source data, see Supplementary Fig. . CBF, core binding factor; SF-mutated, splicing factor-mutated; MLLr, MLL-rearranged.

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Transduction, Expressing, Derivative Assay, Control, Two Tailed Test, Western Blot, Binding Assay

Journal: Nature

Article Title: RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax

doi: 10.1038/s41586-024-08137-x

Figure Lengend Snippet: a , Expression of the indicated pro- and anti- apoptotic genes in FACS-sorted SA + R CMPs and GMPs measured by bulk RNA-Seq. Mean and SD from n = 3 independent experiments are shown. ns: not significant (two-tailed unpaired t-test). b , UMAP representation of integrated single-cell transcriptome data from FACS-sorted SA + R CMPs (left) and GMPs (right) from Fig. . c , Expression of the indicated pro- and anti- apoptotic genes in the different clusters. d , Flow cytometry analysis of SA + R CB cells from the experiment shown in Fig. on day 6, showing that the vast majority of ΔLNGFR-NRAS G12D + cells also express the other two transgenes (GFP-ASXL1 del1900-1922 and mCherry-SRSF2 P95L ). e , Volcano plot showing differentially expressed genes between NRAS G12D+ and WT cells of the GMP cluster from Fig. . Significantly upregulated and downregulated genes (Wilcoxon test) are shown in red and blue, respectively. Granulocytic (MPO, AZU1, ELANE) and monocytic (S100A8, S100A9, S100A12, CD52, CCL2) lineage genes, downregulated and upregulated, respectively, are highlighted. Downregulated genes encoding ribosomal proteins are shown in green. f , Top 20 most enriched HALLMARK pathways in NRAS G12D+ vs WT cells belonging to the GMP cluster from Fig. . NES: normalized enrichment score. g , Viability of CD34+ LSCs from the indicated patient-derived AML-iPS cell lines with or without ectopic lentiviral expression of NRAS G12D , treated with VEN at the indicated concentrations. %Viability compared to DMSO-treated group is shown. n = 3 for AML-4.24 treated with 12 μM VEN and n = 4 for all other groups. Mean and SD are shown. P values were calculated with a two-tailed unpaired t test. h , Summary schematic of the effects of RAS mutation acquisition in different HSPC types. RAS mutations acquired by more primitive HSPCs (HSC/MPPs or CMPs) result in reduction of GMP formation and reciprocal increase in megakaryocyte and erythroid progenitors (MEP) (left panel). Acquisition of RAS mutations in GMPs drives their differentiation towards the monocytic and away from the granulocytic lineage (right panel).

Article Snippet: Amplicon libraries for NRAS G12D were generated and sequenced on an Illumina MiniSeq 300 cycle mid-output kit in 147:8:16:147 configuration.

Techniques: Expressing, RNA Sequencing, Two Tailed Test, Flow Cytometry, Derivative Assay, Mutagenesis

Journal: Nature Communications

Article Title: Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

doi: 10.1038/s41467-022-35700-9

Figure Lengend Snippet: a Cytokine levels in bone marrow (BM) extracellular fluid (BMEF) from control ( Mx1-Cre − NRAS G12D ) and NRAS-G12D + ( Mx1-Cre + NRAS G12D ) mice. IL-1β (control, n = 6; NRAS-G12D + , n = 5), IL-6 (control, n = 4; NRAS-G12D + , n = 3), IL-10 ( n = 5 per group), IFN-γ (control, n = 6; NRAS-G12D + , n = 5) and TNF-α (control, n = 6; NRAS-G12D + , n = 5) levels are shown. b IL-1rn levels in BMEF (control, n = 17; NRAS-G12D + , n = 16). c Numbers of cells in total BM (TBM), and TBM number of CD11b + Ly6G + neutrophils, CD11b + Gr-1 + f4/80 + monocytes and Lin − Sca-1 + c-Kit + (LSK) cells ( n = 3 per group). d, e qRT-PCR mRNA expression in ( d ) CD11b + Gr-1 hi f4/80 − granulocytes of Il1b (control, n = 11; NRAS-G12D + , n = 9), Il1rn (control, n = 16; NRAS-G12D + , n = 9) and Il1rap (control, n = 3; NRAS-G12D + , n = 5) and in CD11b + Gr-1 + f4/80 + monocytes of Il1b (control, n = 13; NRAS-G12D + , n = 11), Il1rn (control, n = 17; NRAS-G12D + , n = 16) and Il1rap (control, n = 3; NRAS-G12D + , n = 5). e LSK subsets: LSK CD34 − Flt3 − , long-term hematopoietic stem cells (LT-HSC); LSK CD34 + Flt3 − , short-term HSC (ST-HSC); LSK CD34 + Flt3 + , multipotent progenitors (MPP) of Il1b (LT-HSC control, n = 7; NRAS-G12D + , n = 8; ST-HSC control, n = 8; NRAS-G12D + , n = 7; MPP, n = 8 per group), Il1rn (LT-HSC control, n = 4; NRAS-G12D + , n = 6; ST-HSC, MPP control, n = 4; NRAS-G12D + , n = 8) and Ilrap ( n = 4 per group). f Frequency of TBM CD34 − Flt3 − LT-HSC, CD34 + Flt3 − ST-HSC, CD34 + Flt3 + MPP, CD11b + Gr-1 hi f4/80 − granulocytes and CD11b + Gr-1 + f4/80 + monocytes expressing IL-1 receptor accessory protein (IL-1RAP, n = 4 per group). g NFκB transcription factor activity calculated based on NFκB target gene expression levels, identified from Synapse ID syn4956655 (Supplementary Data ), from RNA sequencing data in LT-HSC, ST-HSC and MPP ( n = 3 per group). h Number of monocytes in peripheral blood (PB) from NRAS-G12D + mice treated with vehicle ( n = 3) or bortezomib ( n = 4). Data are biologically independent animals, and means ± S.E.M for bar plots or medians for violin plots. Statistical analyses were performed with two-tailed Student’s t -test ( a – d, f LT-HSC, g, h ) or two-tailed Mann–Whitney U test ( e, f except LT-HSC). p values ≤ 0.05 are reported. Source data are provided as a Source Data file.

Article Snippet: Age and gender matched Il1rn −/− , B6.SJL (CD45.1 + ), C57BL/6J, immunodeficient NOD Scid Gamma (NSG) mice expressing human IL3 , GM-CSF and SCF (NSG-SGM3) , (The Jackson Laboratory), Nes-gfp (pure C57BL/6J background; gift from S. Mendez-Ferrer, own colony maintained at Charles River Laboratories CRL, Germany) , Mx1-Cre NRAS G12D , (pure C57BL/6J background; gift from P. Garcia, own colony maintained at CRL) and Vav-Cre NRAS G12D (pure C57BL/6J background; J. Zhang) were used in experiments.

Techniques: Control, Quantitative RT-PCR, Expressing, Activity Assay, Targeted Gene Expression, RNA Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

doi: 10.1038/s41467-022-35700-9

Figure Lengend Snippet: a Representative fluorescence-activated cell sorting (FACS) analysis (cells in total bone marrow; TBM, %) and TBM number of CD45 − CD31 − Ter119 − CD63 + stromal cells from control ( Mx1-Cre − NRAS G12D ) and NRAS-G12D + ( Mx1-Cre + NRAS G12D ) mice ( n = 3 per group). b Representative FACS analysis (cells in BM CD63 + stromal cells, %) and frequencies of live, early apoptotic (E.A.) and late apoptotic (L.A.) cells within the CD45 − CD31 − Ter119 − CD63 + stromal compartment in TBM from control ( n = 8) and NRAS-G12D + ( n = 5) mice. c qRT-PCR mRNA expression in CD45 − CD31 − Ter119 − CD63 + stromal cells of Il1b (control, n = 15; NRAS-G12D + , n = 18), Ilrn (control, n = 16; NRAS-G12D + , n = 18) and Il1rap (control, n = 4; NRAS-G12D + , n = 5). Data are biologically independent animals, and means ± SEM for bar plots or medians for violin plots. Statistical analyses were performed with two-tailed Student’s t -test ( a, b, c except Il1rn ) or two-tailed Mann-Whitney U test ( c Il1rn ). p values ≤ 0.05 are reported. Source data are provided as a Source Data file.

Article Snippet: Age and gender matched Il1rn −/− , B6.SJL (CD45.1 + ), C57BL/6J, immunodeficient NOD Scid Gamma (NSG) mice expressing human IL3 , GM-CSF and SCF (NSG-SGM3) , (The Jackson Laboratory), Nes-gfp (pure C57BL/6J background; gift from S. Mendez-Ferrer, own colony maintained at Charles River Laboratories CRL, Germany) , Mx1-Cre NRAS G12D , (pure C57BL/6J background; gift from P. Garcia, own colony maintained at CRL) and Vav-Cre NRAS G12D (pure C57BL/6J background; J. Zhang) were used in experiments.

Techniques: Fluorescence, FACS, Control, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

doi: 10.1038/s41467-022-35700-9

Figure Lengend Snippet: a – c C57BL/6J wild-type (WT) mice were used as recipients in competitive transplants (1:1) of bone marrow (BM) nucleated cells from WT and IL-1rn knockout (IL-1rn-KO) mice ( n = 5), WT and NRAS-G12D + mice ( Mx1-Cre + NRAS G12D , n = 5) or IL-1rn-KO and NRAS-G12D + mice ( n = 6). a Illustration of the experimental design. R: recipient. D: donor cells. b Number of CD11b + myeloid cells, CD11b + Gr-1 + f4/80 + monocytes and CD11b + Gr-1 hi f4/80 − granulocytes in peripheral blood (PB). c Number of B220 + lymphocytes in PB. ( d–f ) C57BL/6J WT mice were used as recipients of BM nucleated cells from NRAS-G12D + mice ( n = 20; monocytes n = 21), and IL-1rn-KO mice were used as recipients of BM nucleated cells from control ( Mx1-Cre − NRAS G12D , n = 19) and NRAS-G12D + mice ( n = 19), over 4 independent experiments. d Illustration of the experimental design. e Number of CD11b + myeloid cells, CD11b + Gr-1 + f4/80 + monocytes and CD11b + Gr-1 hi f4/80 − granulocytes in PB. f Number of B220 + lymphocytes in PB. g – i NRAS-G12D + mice were treated with vehicle ( n = 9) or IL-1RN ( n = 8). g Illustration of the experimental design. pIpC, poly-inosine:poly-cytosine. h Number of CD11b + myeloid cells, CD11b + Gr-1 + f4/80 + monocytes and CD11b + Gr-1 hi f4/80 − granulocytes in PB. i Number of B220 + lymphocytes in PB. Data are biologically independent animals, and means ± SEM for bar plots or medians for violin plots. Statistical analyses were performed with two-tailed Student’s t -test ( b, c, e myeloid D NRAS-G12D + :R IL-1rn-KO vs R WT, monocytes D NRAS-G12D + :R WT vs D control:R IL-1rn-KO, D NRAS-G12D + :R IL-1rn-KO vs R WT, h ) or two-tailed Mann–Whitney U test ( e myeloid D NRAS-G12D + :R WT vs D control:R IL-1rn-KO, D NRAS-G12D + :R IL-1rn-KO vs D control, monocytes D NRAS-G12D + :R IL-1rn-KO vs D control, granulocytes, f ). p values ≤ 0.05 are reported. Source data are provided as a Source Data file.

Article Snippet: Age and gender matched Il1rn −/− , B6.SJL (CD45.1 + ), C57BL/6J, immunodeficient NOD Scid Gamma (NSG) mice expressing human IL3 , GM-CSF and SCF (NSG-SGM3) , (The Jackson Laboratory), Nes-gfp (pure C57BL/6J background; gift from S. Mendez-Ferrer, own colony maintained at Charles River Laboratories CRL, Germany) , Mx1-Cre NRAS G12D , (pure C57BL/6J background; gift from P. Garcia, own colony maintained at CRL) and Vav-Cre NRAS G12D (pure C57BL/6J background; J. Zhang) were used in experiments.

Techniques: Knock-Out, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a Workflow for CRISPR screens in MM cell lines. b Scatter plot of the average CRISPR screen scores (CSS) for RAS-dependent MM lines (x-axis) vs. RAS-independent MM lines ( y -axis). Outliers were determined by an extra sum-of-squares F test ( P < 0.05) and are labeled in purple and blue. c Workflow of BioID2-based mutant KRAS and NRAS proximity labeling SILAC mass spectrometry (MS) studies. d Essential interactome of G12V RAS isoforms in RAS-dependent MM. Average CSS for KRAS/NRAS-dependent MM cell lines ( x -axis) plotted by average combined enrichment of BioID2-KRAS G12V / BioID2-NRAS G12V relative to empty vector. Essential interactome (≤−1.0 CSS and ≥1.0 log2fc BioID2-RAS) is labeled in pink. e Venn diagram of protein interaction partners substantially enriched (≥2.0 log2fc) in BioID2-KRAS G12V and BioID2-NRAS G12V with RAS-dependent outlier genes identified in Fig. 1b. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: CRISPR, Labeling, Mutagenesis, Multiplex sample analysis, Mass Spectrometry, Plasmid Preparation

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a Co-immunoprecipitation of SLC3A2 with mutant isoforms of mNeonGreen-tagged KRAS and NRAS. mNeonGreen-tagged KRAS G12D was used in RPMI 8226 and XG2, NRAS G12D in SKMM1 and NRAS Q61L in L363. Representative blots; n = 4. b Essential interactome of SLC3A2 in RPMI 8226. CRISPR screen score (CSS; x-axis) plotted by the BioID2-SLC3A2/empty vector enrichment (y-axis). Essential interactome is labeled in pink (≤−1.0 CSS and ≥1.0 log2fc BioID2-SLC3A2). c Proximity ligation assay (PLA) (red) of SLC3A2 and RAS in RPMI 8226 and SKMM1 cells. Cells were counterstained with DAPI (blue) and wheat germ agglutinin (WGA; green). Scale bar is 10 μm, representative images; n = 3. d PLA score of cells transduced with indicated shRNAs following puromycin selection for two days. Data from three independent experiments for KRAS, NRAS and SLC3A2 knockdown, and 2 independent experiments for SLC7A5 knockdown. Data pooled from independent experiments; the number of cells quantified per condition is listed in the source data file. *** denotes P value < 0.0001 by one-way ANOVA with Dunnett’s post test; box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, and outliers are displayed as dots. e Global changes in phosphorylation measured by quantitative MS following knockdown of SLC3A2 in SKMM1. Change in phosphorylation (log2fc) in cells expressing shSLC3A2 vs. shCTRL ( x -axis) plotted by the measured intensity ( y -axis). f Identification of genes that regulate phosphorylation of RPS6 at S240/244 by CRISPR screening. Averaged CRISPR screen results from sorted RPMI 8226 and SKMM1 cells comparing cells with low p-RPS6 vs. high p-RPS signal. All values are averaged for both cell lines except for KRAS and NRAS, which are shown for RPMI 8226 and SKMM1, respectively. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: Immunoprecipitation, Mutagenesis, CRISPR, Plasmid Preparation, Labeling, Proximity Ligation Assay, Transduction, Selection, Knockdown, Phospho-proteomics, Expressing

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a BioID2-SLC3A2 interactomes in XG2 cells transduced with control shRNA (x-axis) or shKRAS (y-axis), proteins are colored that decreased by at least 0.5 log2fc (purple) or increased by at least 0.5 log2fc (blue). b Proximity ligation assay (PLA) (red) of SLC3A2 with MTOR in RPMI 8226 and SKMM1 cells. Cells counterstained with DAPI (blue) and wheat germ agglutinin (WGA; green); Scale bar is 10 μm. Representative images; n = 3. c SLC3A2-MTOR PLA score of cells transduced with indicated shRNAs. Details about box plots below. d Gene Ontology pathway enrichment of SLC3A2 interactors that decreased by at least 0.5 log2fc following RAS knockdown. Bonferroni corrected P value plotted on the x-axis. e PLA of SLC3A2 with LAMP1 (red) in RPMI 8226 and SKMM1 cells. Cells counterstained with DAPI (blue) and WGA (green); Scale bar is 10 μm. Representative images; n = 3. f SLC3A2-LAMP1 PLA score of cells transduced with control shRNA or shRNAs specific for KRAS, NRAS, SLC3A2 and MTOR. Details about box plots below. g Immunofluorescence images of MTOR (green), LAMP1 (red) and DAPI (blue) in RPMI 8226 and SKMM1 cells expressing shCTRL, shKRAS.2 or shNRAS.1. Scale bar is 10 μm. Representative images; n = 3. h PLA of MTOR with LAMP1 (red) in RPMI 8226 and SKMM1 cells. Cells counterstained with DAPI (blue) and WGA (green); Scale bar is 10 μm. Representative images; n = 3. i MTOR-LAMP1 PLA score of cells transduced with control shRNA or shRNAs specific for MTOR, KRAS, NRAS, SLC3A2 and RPTOR. Details about box plots below. For all box plots: Data are pooled independent experiments; the number of cells quantified per condition indicated in the source data file. *** denotes P value <0.0001, ** denotes P value = 0.0023, n.s. denotes not significant, as determined by one-way ANOVA with Dunnett’s post test. The number of independent experiments and the number of cells quantified for each condition can be found in the source data. Box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, outliers are displayed as dots. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: Transduction, Control, shRNA, Proximity Ligation Assay, Knockdown, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a Pathway analysis of proteins with ±0.8 log2 fold changes in phosphorylation in SKMM1 cells transduced with shNRAS compared to control shRNA as determined by quantitative mass spectrometry. b Scatter plot of changes in phosphorylation following NRAS knockdown (shNRAS/shCTRL; x-axis) vs. intensity (y-axis). Proteins in the MTOR and MAPK signaling pathways are labeled. c Western blot analysis of mTORC1 and MEK signaling following KRAS or NRAS knockdown in the indicated MM lines. Representative blots, n = 5. d Co-immunoprecipitation of MTOR with mutant isoforms of mNeonGreen-tagged KRAS and NRAS. KRAS G12D was used in RPMI 8226 and XG2, NRAS G12D in SKMM1 and NRAS Q61L in L363. Representative blots; n = 3. e Proximity ligation assay (PLA) of MTOR-RAS (red) in RPMI 8226 and SKMM1 cells. Cells counterstained with DAPI (blue) and wheat germ agglutinin (WGA; green); Scale bar is 10 μm. Representative images; n = 3. f MTOR-RAS PLA score of cells transduced with control shRNA or shRNAs specific for KRAS ( n = 3), NRAS ( n = 3), SLC3A2 ( n = 3), SLC7A5 ( n = 2) and MTOR ( n = 3). Data pooled from independent experiments; the number of cells quantified per condition listed in the source data file. *** denotes P value <0.0001 by one-way ANOVA with Dunnett’s post test. Box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, outliers are displayed as dots. g MTOR-RAS PLA in RPMI 8226 and SKMM1 cells expressing shCTRL ( n = 3), shRPTOR ( n = 3) or shRICTOR ( n = 3) shRNAs. Data pooled from independent experiments; the number of cells quantified per condition listed in the source data file. *** denotes P value <0.0001, n.s. denotes not significant, by one-way ANOVA with Dunnett’s post test. Box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, outliers are displayed as dots. h Immunofluorescence of MTOR (red), ectopically expressed mNeonGreen-KRAS G12D in RPMI 8226 or mNeonGreen-NRAS G12D SKMM1 cells (green), SLC3A2 (magenta) and LAMP1 (cyan). Yellow arrows highlight areas of overlap. Scale bar is 10 μm. Representative images; n = 3. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: Phospho-proteomics, Transduction, Control, shRNA, Mass Spectrometry, Knockdown, Protein-Protein interactions, Labeling, Western Blot, Immunoprecipitation, Mutagenesis, Proximity Ligation Assay, Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a Pathway diagram of MTOR signaling. Symbols are colored by the average CRISPR screen score (CSS) in RAS-dependent (pink) and RAS-independent (purple) MM cell lines and marked with a cyan dot containing an “R” if they were found to interact with mutant KRAS and NRAS in BioID2 experiments (≥2.0 log2fc). b Indicated proximity ligation assays (PLA) in RPMI 8226 and SKMM1 cells with PLA (red), wheat germ agglutinin (WGA; green) and DAPI (blue). Scale bar is 10 μm. Representative images; n = 2. c Western blot analysis of mTORC1 signaling in RPMI 8226 and SKMM1 cells following KRAS or NRAS knockdown and expression of either control (sgCTRL) or TSC2 sgRNAs. Representative blots; n = 3. d CRISPR modifier screen results identify genes with differential essentialities under glutamine restriction vs. normal glutamine conditions in SKMM1. CSS of genes from cells grown under glutamine restriction vs. normal glutamine conditions on y -axis. e Change in CSS for LZTR1, SLC3A2 and NRAS from Day 21 vs. Day 0 for normal glutamine conditions (gray) and under glutamine restriction (pink). f MTOR-RAS Proximity ligation assay (PLA) scores for cells under normal glutamine conditions (gray; n = 3) and under acute (12 h) glutamine restriction (pink; n = 3). Data pooled from independent experiments; the number of cells quantified per condition indicated in the source data file. *** denotes P value <0.0001 determined by Mann–Whitney unpaired two-tailed t -test. Box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, outliers are displayed as dots. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: CRISPR, Mutagenesis, Ligation, Western Blot, Knockdown, Expressing, Control, Proximity Ligation Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet: a Proximity ligation assay (PLA) between MTOR and RAS in formalin-fixed paraffin-embedded (FFPE) bone marrow aspirates from MM patients with PLA (red), CD138 (white) and DAPI (blue). Scale bar is 10 μm. b Changes in expression for the mTORC1 signature genes following treatment with 100 nM everolimus for the indicated cell lines and times. c Kaplan–Meier survival plots of MM patients from the MMRF CoMMpass trial divided into tertials by gene expression of the mTORC1 down signature in panel ( b ). The P value was determined using a two-sided likelihood-ratio test based on a Cox proportional hazard model with the mTORC1 signature treated as a continuous variable. d Gene Set Enrichment Analysis (GSEA) of mTORC1 signature for KRAS, NRAS and FGFR3 mutations in the MMRF CoMMpass patient cohort. Source data are provided as a Source Data file.

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques: Proximity Ligation Assay, Formalin-fixed Paraffin-Embedded, Expressing, Gene Expression

Journal: Nature Communications

Article Title: Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma

doi: 10.1038/s41467-022-33142-x

Figure Lengend Snippet:

Article Snippet: For co-immunoprecipitation and imaging studies, mutant RAS isoforms of KRAS G12D , KRAS S17N , NRAS G12D , NRAS Q61L and NRAS S17N were linked to mNeonGreen as gene fragments (Twist Biosciences).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Embryonic Expression of Nras G 12 D Leads to Embryonic Lethality and Cardiac Defects

doi: 10.3389/fcell.2021.633661

Figure Lengend Snippet: Nras G12D/ + ; Mox2 Cre/ + (G12D/ +; Mox2) embryos exhibit whole body edema and the small liver at E15.5. (A) Schematic illustration of floxed and activated oncogenic Nras alleles. (B) Representative images of E13.5 and E15.5 WT control and G12D/ +; Mox2 mutant. The G12D/ +; Mox2 embryo looks grossly normal at E13.5 but at E15.5 looks pale and shows whole body edema as indicated by an arrow. (C) Quantitative analysis of cell numbers in E13.5 fetal livers from G12D/ +; Mox2 vs. control embryos. (D) Flow cytometric analysis and quantification of HSCs (defined as Lin – CD41 – CD48 – Mac1 + CD150 + Sca1 + cKit + cells) in total live fetal liver cells. HSC percentages in control fetal liver cells were arbitrarily set as 1. (E) Flow cytometric analysis and quantification of LSKs (defined as Lin – Sca1 + cKit + cells) in total fetal liver live cells. LSK percentage in control fetal liver cells were arbitrarily set as 1. (F) Flow cytometric analysis and quantification of erythroid differentiation based on CD71 and Ter119 expression. Unpaired 2-tailed Student’s t tests were used to determine the significance. Results are presented as mean + SD. * p < 0.05; *** p < 0.001.

Article Snippet: Mice bearing the conditional oncogenic Nras mutation ( Nras loxp stop cassett loxp (LSL) G12D/ + ) ( ) were crossed to Mox2 Cre/+ mice ( ) (Jackson Laboratory #003755) to generate Nras LSL G12D/ + ; Mox2 Cre/ + ( Nras G12D/ + ; Mox2 Cre/ + ) embryos.

Techniques: Control, Mutagenesis, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Embryonic Expression of Nras G 12 D Leads to Embryonic Lethality and Cardiac Defects

doi: 10.3389/fcell.2021.633661

Figure Lengend Snippet: Embryonic lethality of Nras G12D/ + ; Mox2-Cre mutants.

Article Snippet: Mice bearing the conditional oncogenic Nras mutation ( Nras loxp stop cassett loxp (LSL) G12D/ + ) ( ) were crossed to Mox2 Cre/+ mice ( ) (Jackson Laboratory #003755) to generate Nras LSL G12D/ + ; Mox2 Cre/ + ( Nras G12D/ + ; Mox2 Cre/ + ) embryos.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Embryonic Expression of Nras G 12 D Leads to Embryonic Lethality and Cardiac Defects

doi: 10.3389/fcell.2021.633661

Figure Lengend Snippet: Nras G12D/ + ; Mox2 Cre/ + (G12D/ +; Mox2) embryos exhibit cardiac developmental defects. Representative images of H&E stained transverse sections of control (A) vs. two G12D/ +; Mox2 mutant (B,C) embryos at E13.5. The mutant hearts show VSD (indicated by arrow in right panels B,C ), DORV (indicated by arrowhead in middle panels B,C ) and the thin ventricular wall (indicated by * in right panels B,C ). The pulmonary valves were not remodeled properly (indicated by # in left panels B,C ). Ao, Aorta; P, Pulmonary valve; R, Right ventricle; L, Left ventricle; Vs, Ventricular septum.

Article Snippet: Mice bearing the conditional oncogenic Nras mutation ( Nras loxp stop cassett loxp (LSL) G12D/ + ) ( ) were crossed to Mox2 Cre/+ mice ( ) (Jackson Laboratory #003755) to generate Nras LSL G12D/ + ; Mox2 Cre/ + ( Nras G12D/ + ; Mox2 Cre/ + ) embryos.

Techniques: Staining, Control, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Embryonic Expression of Nras G 12 D Leads to Embryonic Lethality and Cardiac Defects

doi: 10.3389/fcell.2021.633661

Figure Lengend Snippet: Global gene expression analyses of E13.5 Nras G12D/ + ; Mox2 Cre/ + (G12D/ + ; Mox2) heart. Agilent microarray analysis was performed on E13.5 hearts from control and G12D/ +; Mox2 embryos. (A) Heat map depicting differentially expressed genes ( p < 0.01 and fold change >2) between control and mutant hearts. (B) Gene Set Enrichment Analysis identified altered gene expression profiles in mutant hearts. Representative examples in non-canonical Wnt signaling pathway, BMP signaling pathway, and MAPK cascade are shown here. NES, normalized enrichment score. (C) Analysis of MEK/ERK and AKT signaling in E13.5 mutant hearts by Western blotting. Actin antibody was used for a loading control.

Article Snippet: Mice bearing the conditional oncogenic Nras mutation ( Nras loxp stop cassett loxp (LSL) G12D/ + ) ( ) were crossed to Mox2 Cre/+ mice ( ) (Jackson Laboratory #003755) to generate Nras LSL G12D/ + ; Mox2 Cre/ + ( Nras G12D/ + ; Mox2 Cre/ + ) embryos.

Techniques: Gene Expression, Microarray, Control, Mutagenesis, Western Blot

Journal: Cancers

Article Title: Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia

doi: 10.3390/cancers12102766

Figure Lengend Snippet: NUP98-NSD1 immortalizes murine hematopoietic cells and cooperates with mutated NRASG12D in vitro. ( A ) Percentage of 5-fluorouracil (5-FU)-treated bone marrow cells that were transduced with either NRASG12D-BFP, NUP98-NSD1-GFP, or both NUP98-NSD1-GFP and NRASG12D-BFP from day 4 to day 12 after transduction (mean ± SEM). ( B ) Cumulative Colony-forming cell unit (CFU) yield is shown for an initial plating of 1000 NRASG12D, NUP98-NSD1, or NRASG12D+NUP98-NSD1 transduced cells (mean ± SEM). ( C ) Percentage of apoptotic cells transduced with NRASG12D, NUP98-NSD1, or NRASG12D+NUP98-NSD1 until 12 days after transduction (mean ± SEM). ( D ) Representative immunophenotype of cells transduced with NRASG12D, NUP98-NSD1, or NRASG12D+NUP98-NSD1. ( E ) Representative Wright–Giemsa–stained cytospin preparations of NRASG12D, NUP98-NSD1, and NUP98-NSD1+NRASG12D transduced mouse bone marrow cells (1000×).

Article Snippet: The NRASG12D plasmid was obtained from ADDGENE (Plasmid #83176) and cloned in the retroviral expression vector MSCV-IRES-BFP.

Techniques: In Vitro, Transduction, Staining

Journal: Cancers

Article Title: Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia

doi: 10.3390/cancers12102766

Figure Lengend Snippet: NUP98-NSD1 induces a long-latency AML and cooperates with NRASG12D to induce an aggressive acute myeloid leukemia in vivo. ( A ) Development of syngeneic mouse models using constitutively expressed NRASG12D, NUP98-NSD1, or both. ( B ) Engraftment of transduced cells in peripheral blood of the indicated groups 4, 8, and 12 weeks after transplantation (mean ± SEM). ( C ) Survival of mice that received transplants of cells transduced with the indicated constructs. ( D ) White blood cell count in peripheral blood at time of sacrifice from mice, which received transplants of the indicated cells (mean ± SEM). ( E ) Hemoglobin levels in peripheral blood at time of sacrifice of mice, which received transplants of the indicated cells (mean ± SEM). ( F ) Platelet count in peripheral blood at time of sacrifice of mice, which received transplants of the indicated cells (mean ± SEM). ( G ) Average spleen weight in mice at time of sacrifice of mice, which received transplants of the indicated cells (mean ± SEM). ( H ) Representative image of a spleen at time of sacrifice.

Article Snippet: The NRASG12D plasmid was obtained from ADDGENE (Plasmid #83176) and cloned in the retroviral expression vector MSCV-IRES-BFP.

Techniques: In Vivo, Transplantation Assay, Transduction, Construct, Cell Counting

Journal: Cancers

Article Title: Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia

doi: 10.3390/cancers12102766

Figure Lengend Snippet: Common and distinct pathways are regulated by NUP98-NSD1 and NRASG12D. ( A ) Heat map from hierarchical clustering showing differential expression of HOX genes in NRASG12D, NUP98-NSD1, and NUP98-NSD1 + NRASG12D transformed cells. ( B ) Enrichment plot for the gene sets HOXA9_DN.V1_DN of Gene Set Enrichment Analysis (GSEA) comparing NUP98-NSD1 and NRASG12D transformed cells. ( C ) Quantitative RT-PCR of Hoxa7 in NRASG12D, NUP98-NSD1, and NUP98-NSD1+NRASG12D transformed mouse bone marrow cells (mean ± SEM). ( D ) Quantitative RT-PCR of Hoxa9 in NRASG12D, NUP98-NSD1, and NUP98-NSD1+NRASG12D transformed mouse bone marrow cells (mean ± SEM). ( E ) Quantitative RT-PCR of Hoxa10 in NRASG12D, NUP98-NSD1, and NUP98-NSD1+NRASG12D transformed mouse bone marrow cells (mean ± SEM). ( F ) Pie chart showing the top 39 biological categories regulated by NRASG12D from the GO biological process pathway gene set analysis comparing NUP98-NSD1+NRASG12D with NUP98-NSD1.

Article Snippet: The NRASG12D plasmid was obtained from ADDGENE (Plasmid #83176) and cloned in the retroviral expression vector MSCV-IRES-BFP.

Techniques: Quantitative Proteomics, Transformation Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Hematopoietic p53 loss cell-extrinsically defines an immune infiltrated microenvironment in leukemia and pre-leukemia

doi: 10.1101/2020.03.22.002774

Figure Lengend Snippet: (A) Immune landscape across several hematopoietic tissues in normal tissue and MLL-AF9 or Trp53 -/- ; Nras G12D leukemia. (B) Detailed immunophenotyping of Trp53 -/- ; Nras G12D and MLL-AF9 leukemia.

Article Snippet: The pBabe-MSCV-Nras G12D -Ires-RFP vector was cloned by excision from a donor Nras 12D open reading frame plasmid (Addgene # 14725) using BamHI.

Techniques:

Journal: bioRxiv

Article Title: Hematopoietic p53 loss cell-extrinsically defines an immune infiltrated microenvironment in leukemia and pre-leukemia

doi: 10.1101/2020.03.22.002774

Figure Lengend Snippet: (A) Immunophenotype of Trp53 -/- ; Nras G12D leukemia indicating minimal but present erythroid maturation. (B) Representative flow plots of various hematopoietic tissues and (C) corresponding H&E staining of spleen and liver

Article Snippet: The pBabe-MSCV-Nras G12D -Ires-RFP vector was cloned by excision from a donor Nras 12D open reading frame plasmid (Addgene # 14725) using BamHI.

Techniques: Staining