aml cell line sig m5  (DSMZ)

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a Domain architecture of DNMT1 and DNMT3A proteins. Domain abbreviations: DMAP1, DNMT1-associated protein motif; PCNA, proliferating cell nuclear antigen motif; RFTS, replication focus targeting sequence; CXXC, zinc-finger binding domain; BAH, bromo-adjacent homology domain; PWWP, Pro-Trp-Trp-Pro domain; ADD, ATRX-DNMT3-DNMT3L domain; MTase, methyltransferase domain. sgRNAs were designed to tile the coding regions of both genes. b Screening procedure. sgRNAs targeting DNMT1 and DNMT3B were synthesized and constructed as a pooled virus. Non-targeting control sgRNAs and sgRNAs targeting common essential genes were included as negative and positive controls, respectively. Cells were transduced at an MOI between 0.3 and 0.5 and grown on puromycin selection for 3 weeks. Cells were collected 3 days post-puromycin selection and on the last day of the screen. Isolation of genomic DNA and NGS were performed to calculate sgRNA enrichment and depletion over time. sgRNA enrichment and depletion was mapped along the coding regions of DNMT1 and DNMT3B to determine any functional domains essential for viability. c AML cell lines chosen for pooled screens. THP-1, OCI-M1, and NOMO-1 cells are wild-type for DNMT3A , whereas OCI-AML2, SIG-M5, and OCI-AML3 contain mutations within the MTase domain of DNMT3A .

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Sequencing, Binding Assay, Synthesized, Construct, Selection, Isolation, Functional Assay

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a – c sgRNAs tiling DNMT3B in AML cell lines wild-type for DNMT3A , NOMO-1 ( a ), OCI-M1 ( b ), and THP-1 ( c ) are shown with respect to their cDNA position along the coding region of DNMT3B. d – f sgRNAs tiling DNMT3B in AML cell lines mutant for DNMT3A , OCI-AML2 ( d ), SIG-M5 ( e ), and OCI-AML3 ( f ) are shown with respect to their cDNA position along the coding region of DNMT3B. Each circle represents an individual sgRNA targeting DNMT3B . The standardized LFC value for each sgRNA is plotted on each graph. The dashed black bar denotes the mean standardized LFC of the non-targeting controls for each cell line. The mean standardized LFC of the common essential sgRNAs is denoted by the red dashed line. The color of each circle represents the functional domain it targets: PWWP (light gray); ADD (black); MTase domain (blue). n = 3 replicates for each cell line screened.

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Mutagenesis, Functional Assay

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a – c sgRNAs tiling DNMT1 in AML cell lines wild-type for DNMT3A , NOMO-1 ( a ), OCI-M1 ( b ), and THP-1 ( c ) are shown with respect to their cDNA position along the coding region of DNMT1. d – f sgRNAs tiling DNMT1 in AML cell lines mutant for DNMT3A , OCI-AML2 ( d ), SIG-M5 ( e ), and OCI-AML3 ( f ), are shown with respect to their cDNA position along the coding region of DNMT1. Each circle represents an individual sgRNA targeting DNMT1 . The standardized LFC value for each sgRNA is plotted on each graph. The dashed black bar denotes the mean standardized LFC of the non-targeting controls for each cell line. The mean standardized LFC of the common essential sgRNAs is denoted by the red dashed line. The color of each circle represents the functional domain it targets: DMAP1 (yellow); PCNA (purple); RFTS (orange); CXXC (red); BAH1 (dark gray); BAH2 (green); MTase domain (blue). n = 3 replicates for each cell line screened.

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Mutagenesis, Functional Assay

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a , b The CKHS profile (from ProTiler) of DNMT1 ( a ) and DNMT3B ( b ) in DNMT3A wild-type cell lines NOMO-1, OCI-M1, and THP-1. c , d The CKHS profile of DNMT1 ( c ) and DNMT3B ( d ) in DNMT3A mutant cell lines OCI-AML2, SIG-M5, and OCI-AML3. e STARS analyses on bins of 11 sgRNAs along the span of the DNMT1 protein in SIG-M5, OCI-AML3, OCI-AML2, THP-1, OCI-M1, and NOMO-1 cells. A bin with an FDR of less than 0.25 was considered to have a significant impact on cell proliferation. The PCNA (purple) and CXXC (red) functional domains are not labeled on the DNMT1 protein structure. f 3D structure of DNMT1 (amino acids 291-1620; PDB ID: 4WXX) with functional domains RFTS (orange), CXXC (red), BAH1 (gray), BAH2 (green), and MTase (blue) domains labeled. The amino acids spanning the sgRNA bins in the MTase domain that gave a STARS FDR < 0.25 are colored in magenta and labeled on the structure with the respective amino acids position(s) that the sgRNAs targeted. The 2 bins that were the most consistent hits across the cell lines screened screens (aa 1514–1525 and aa 1561–1575) are highlighted in yellow.

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Mutagenesis, Functional Assay, Labeling

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a Domain architecture of the DNMT3A protein. The R882C/+ and R635W mutations in the MTase domain of DNMT3A are labeled. b The R882C/+ clinically relevant DNMT3A mutation was incorporated into OCI-M1 and NOMO-1 cells, which are normally wild-type for DNMT3A . OCI-AML2 cells, which harbor a homozygous R635W mutation in DNMT3A , were engineered to express wild-type DNMT3A. SIG-M5 cells, which harbor a heterozygous R882C mutation in DNMT3A , were engineered to express wild-type DNMT3A. c – f Sanger sequencing of the DNMT3A gene verifying the engineered mutation in OCI-M1 ( c ), NOMO-1 ( d ), OCI-AML2 ( e ), and SIG-M5 ( f ) cells. Sanger sequencing of the control parental pool of cells (bottom row), as well as the engineered clone (top row), is shown for each cell line. sgRNAs tiling DNMT1 are shown with respect to their cDNA position in OCI-M1 parental ( DNMT3A WT, g and OCI-M1 DNMT3A [R882C/+] ( DNMT3A mutant, h ) cells. i , j sgRNAs tiling DNMT1 are shown with respect to their cDNA position in NOMO-1 parental ( DNMT3A WT, i ) and NOMO-1 DNMT3A [R882C/+] ( DNMT3A mutant, j ) cells. k , l sgRNAs tiling DNMT1 are shown with respect to their cDNA position in OCI-AML2 parental ( DNMT3A mutant, k ) and OCI-AML2 DNMT3A [W635R] ( DNMT3A WT, l ) cells. m , n sgRNAs tiling DNMT1 are shown with respect to their cDNA position in SIG-M5 parental ( DNMT3A mutant, m ) and SIG-M5 DNMT3A [C882R/+] ( DNMT3A WT, n ) cells. Black dashed line denotes the mean standardized LFC of the non-targeting controls. The mean standardized LFC of the common essential sgRNAs is denoted by the red dashed line. The color of each circle represents the functional domain it targets: DMAP1 (yellow); PCNA (purple); RFTS (orange); CXXC (red); BAH1 (dark gray); BAH2 (green); MTase domain (blue). n = 3 replicates for each screen.

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Labeling, Mutagenesis, Sequencing, Functional Assay

Journal: Communications Biology

Article Title: The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation

doi: 10.1038/s42003-022-04139-5

Figure Lengend Snippet: a – d The differential LFC was calculated for each sgRNA targeting DNMT1 across the OCI-M1 ( a ), NOMO-1 ( b ), OCI-AML2 ( c ), and SIG-M5 ( d ) isogenic cell line pairs, using the parental line as a reference. Each circle represents an individual sgRNA spanning the coding region of DNMT1 e STARS analyses was performed using bins of 11 sgRNAS along the span of the DNMT1 protein in the OCI-M1, NOMO-1, OCI-AML2, and SIG-M5 isogenic cell line pairs. The differential LFC values for each isogenic cell line pair was used for these analyses. A bin with an FDR of less than 0.25 was considered to have a significant impact on cell proliferation. The PCNA (purple) and CXXC (red) functional domains are not labeled on the DNMT1 protein structure. f – i Residual analyses on the OCI-M1 ( f ), NOMO-1 ( g ), OCI-AML2 ( h ), and SIG-M5 ( i ) isogenic cell line pairs. A positive residual (red) indicates more depletion in the parental line compared to the clone, whereas a more negative residual value (blue) indicates more depletion in the clone compared to the parental line. The FDR values across the span of the DNMT1 bins are shown, with an FDR < 0.25 cutoff for significance. The PCNA (purple) and CXXC (red) functional domains are not labeled on the DNMT1 protein structure.

Article Snippet: Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates.

Techniques: Functional Assay, Labeling