Figure 2.
TFE3-dependent transcription does not contribute to leukemogenesis. (A) MLL-TFE3 mutant truncations used in this study: MLL-trunc, ΔHelix-LZip-Pro, ΔTAD, and ΔPro; as well as the full-length MLL-TFE3. (B) Kaplan-Meier curves of the syngeneic mouse model of the mutants and the full-length MLL-TFE3, all in the presence of NRasG12D. MLL-trunc (blue line; n = 6), ΔHelix-LZip-Pro mice (green line; n = 6), ΔPro (orange line; n = 9), ΔTAD (lavender line; n = 10), MLL-TFE3 (red line; n = 11), and NRasG12D only mutation as a control (brown line; n = 10). There is a significant difference in survival between ΔPro and MLL-TFE3 mice (P = .0002). There is no significant difference in survival between ΔTAD and MLL-TFE3 mice (P = .9). Tick on the line indicates nonleukemic deaths, while asterisk indicates the end of experimental cohort. Some cohorts were extended beyond 120 days, but there was no leukemic event observed. (C) Immunofluorescence showing 293T cells transfected with the cloned fusions. Cells transfected with NRasG12D was included as a staining control. Green, anti-MLL antibody + Alexa Fluor 488; red, DsRed expression; blue, 4′,6-diamidino-2-phenylindole (nucleus). Scale bars, 20 µm. (D) Representative hematoxylin and eosin staining of liver and spleen (original magnification ×10; scale bars, 200 µm) of the ΔTAD mouse, ΔPro mouse, and a nonleukemic ΔHelix-LZip-Pro mouse, all in the presence of NRasG12D cooperating mutation. Blasts can be seen in the spleen and liver of ΔTAD and ΔPro, but not in the nonleukemic ΔHelix-LZip-Pro. (E) Volcano plots showing differential expression analysis of MLL-TFE3 relative to MLL-AF9 (left panel) and ΔTAD relative to MLL-AF9 (right panel). E-box genes with significantly higher DE in MLL-TFE3 (log-fold change >2.0, P < .05) are shown with labels. This gene list is also shown on the volcano plot of differential expression analysis between ΔTAD and MLL-AF9. The majority of these genes also had significant higher expression in ΔTAD compared with MLL-AF9, with the exceptions labeled in red. Purple, higher or lower expressed genes in MLL-TFE3 cells, with high confidence (P < .05); aqua, higher or lower expressed genes in MLL-TFE3, with low confidence (P > .05); cream, similarly expressed genes, with high confidence; gray, similarly expressed genes, with low confidence; n.s., nonsignificant (genes with P < .05). The plot was generated using EnhancedVolcano package.

TFE3-dependent transcription does not contribute to leukemogenesis. (A) MLL-TFE3 mutant truncations used in this study: MLL-trunc, ΔHelix-LZip-Pro, ΔTAD, and ΔPro; as well as the full-length MLL-TFE3. (B) Kaplan-Meier curves of the syngeneic mouse model of the mutants and the full-length MLL-TFE3, all in the presence of NRasG12D. MLL-trunc (blue line; n = 6), ΔHelix-LZip-Pro mice (green line; n = 6), ΔPro (orange line; n = 9), ΔTAD (lavender line; n = 10), MLL-TFE3 (red line; n = 11), and NRasG12D only mutation as a control (brown line; n = 10). There is a significant difference in survival between ΔPro and MLL-TFE3 mice (P = .0002). There is no significant difference in survival between ΔTAD and MLL-TFE3 mice (P = .9). Tick on the line indicates nonleukemic deaths, while asterisk indicates the end of experimental cohort. Some cohorts were extended beyond 120 days, but there was no leukemic event observed. (C) Immunofluorescence showing 293T cells transfected with the cloned fusions. Cells transfected with NRasG12D was included as a staining control. Green, anti-MLL antibody + Alexa Fluor 488; red, DsRed expression; blue, 4′,6-diamidino-2-phenylindole (nucleus). Scale bars, 20 µm. (D) Representative hematoxylin and eosin staining of liver and spleen (original magnification ×10; scale bars, 200 µm) of the ΔTAD mouse, ΔPro mouse, and a nonleukemic ΔHelix-LZip-Pro mouse, all in the presence of NRasG12D cooperating mutation. Blasts can be seen in the spleen and liver of ΔTAD and ΔPro, but not in the nonleukemic ΔHelix-LZip-Pro. (E) Volcano plots showing differential expression analysis of MLL-TFE3 relative to MLL-AF9 (left panel) and ΔTAD relative to MLL-AF9 (right panel). E-box genes with significantly higher DE in MLL-TFE3 (log-fold change >2.0, P < .05) are shown with labels. This gene list is also shown on the volcano plot of differential expression analysis between ΔTAD and MLL-AF9. The majority of these genes also had significant higher expression in ΔTAD compared with MLL-AF9, with the exceptions labeled in red. Purple, higher or lower expressed genes in MLL-TFE3 cells, with high confidence (P < .05); aqua, higher or lower expressed genes in MLL-TFE3, with low confidence (P > .05); cream, similarly expressed genes, with high confidence; gray, similarly expressed genes, with low confidence; n.s., nonsignificant (genes with P < .05). The plot was generated using EnhancedVolcano package.

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