Figure 2.
Certain PDX models depend on WT1 and DNMT3A for in vivo growth. (A) Competitive in vivo assays were performed, analyzed, and depicted as in Figure 1C,D, except that WT1 and DNMT3A were studied (see supplemental Figure 6 for quality controls). (B) Comparing gene dependency in PDX models vs cell lines. Raw data from Figure 2A and supplemental Figures 8 and 9 are summarized using a single dot for each single KO of each PDX model or cell line. For each PDX model or cell line, 3 single-guide RNAs (sgRNAs) per gene were studied. Results of an unpaired t-test are shown if they were significant (∗P < .05, ∗∗P < .01, and ∗∗∗P < .001). (C) Comparing behavior of PDX cells with KO in vitro vs in vivo. Experiment with AML-346 cells was performed, analyzed, and depicted as in Figure 2A, except that the incubation time was 26 days and an aliquot of cells was kept in vitro (∗P < .05, ∗∗P < .01, and ∗∗∗P < .001). (D) Transcriptomes of AML-356, AML-388, AML-661, and AML-346 cells with DNMT3A knockout were compared with nontargeting (NT) control (raw and complementary data in supplemental Figure 10). Gene enrichment map shows gene overlap (lines) in gene sets of hallmarks (orange nodes) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (blue nodes) pathways. Node size is proportional to the number of genes in each set; the proportion of shared genes between gene sets is depicted by the thickness of the line between nodes. Enrichment plot shows the genes differentially regulated in the hallmark oxidative phosphorylation on KO of DNMT3A (normalized enrichment score [NES] = 2.1537; P < .001; adjusted P [false discovery rate q-value] < 0.001). (E) Limiting dilution transplantation assay. PDX AML-346 cells were transduced with sgRNAs against WT1 or DNMT3A or control (CTRL), enriched, mixed in a 1:1 ratio for WT1:CTRL or DNMT3A:CTRL, and injected into 4 mice each at 400 000, 128 000, or 32 000 cells per mouse (WT1, n = 12; and DNMT3A, n = 11 mice). After 14 weeks, bone marrow was analyzed by flow cytometry, and data were analyzed using the ELDA software. Mean (solid lines) and 95% confidence interval (CI; dashed line) are depicted.

Certain PDX models depend on WT1 and DNMT3A for in vivo growth. (A) Competitive in vivo assays were performed, analyzed, and depicted as in Figure 1C,D, except that WT1 and DNMT3A were studied (see supplemental Figure 6 for quality controls). (B) Comparing gene dependency in PDX models vs cell lines. Raw data from Figure 2A and supplemental Figures 8 and 9 are summarized using a single dot for each single KO of each PDX model or cell line. For each PDX model or cell line, 3 single-guide RNAs (sgRNAs) per gene were studied. Results of an unpaired t-test are shown if they were significant (∗P < .05, ∗∗P < .01, and ∗∗∗P < .001). (C) Comparing behavior of PDX cells with KO in vitro vs in vivo. Experiment with AML-346 cells was performed, analyzed, and depicted as in Figure 2A, except that the incubation time was 26 days and an aliquot of cells was kept in vitro (∗P < .05, ∗∗P < .01, and ∗∗∗P < .001). (D) Transcriptomes of AML-356, AML-388, AML-661, and AML-346 cells with DNMT3A knockout were compared with nontargeting (NT) control (raw and complementary data in supplemental Figure 10). Gene enrichment map shows gene overlap (lines) in gene sets of hallmarks (orange nodes) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (blue nodes) pathways. Node size is proportional to the number of genes in each set; the proportion of shared genes between gene sets is depicted by the thickness of the line between nodes. Enrichment plot shows the genes differentially regulated in the hallmark oxidative phosphorylation on KO of DNMT3A (normalized enrichment score [NES] = 2.1537; P < .001; adjusted P [false discovery rate q-value] < 0.001). (E) Limiting dilution transplantation assay. PDX AML-346 cells were transduced with sgRNAs against WT1 or DNMT3A or control (CTRL), enriched, mixed in a 1:1 ratio for WT1:CTRL or DNMT3A:CTRL, and injected into 4 mice each at 400 000, 128 000, or 32 000 cells per mouse (WT1, n = 12; and DNMT3A, n = 11 mice). After 14 weeks, bone marrow was analyzed by flow cytometry, and data were analyzed using the ELDA software. Mean (solid lines) and 95% confidence interval (CI; dashed line) are depicted.

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