Identification of a novel MLL-TFE3 in infant leukemia patient. (A) The reported fusion partners of TFE3-rearrangements in renal cell carcinoma and alveolar soft part sarcoma. The figure is modified from St Jude PeCan protein viewer (https://pecan.stjude.cloud/proteinpaint/).¹⁹  The breakpoint is shown relative to the TFE3 domains and the location of the amino acid, with the number of reported cases in the St Jude database shown in circles. (B) RNA-seq analysis using JAFFA²⁰  identified a novel MLL-TFE3. Image shown is modified from a fusion visualization tool, Clinker.²¹  RNA read coverage is shown as reads per million (RPM), across the genes involved in the rearrangement. Protein domains involved in the fusion arrangement are also shown. (C) RT-PCR of the patient with MLL-TFE3 (1) and a leukemia patient with different rearrangement (2) using a primer set flanking the predicted breakpoint sequence of MLL-TFE3 (MLL-TFE3 BP) and GAPDH as an internal control for RT-PCR. (D) Sanger sequencing of patient cDNA showing the breakpoint sequence of MLL-TFE3. (E) Kaplan-Meier curves of the syngeneic mouse model. MLL-r with the NRas^G12D cooperating mutation (solid lines), MLL-AF9 (blue line; n = 4), and MLL-TFE3 (red line; n = 5) are shown. MLL-r without the NRas^G12D cooperating mutation (dashed lines), MLL-AF9 (blue dashes; n = 2), and MLL-TFE3 (red dashes; n = 3) are shown. Mice expressing only the NRas^G12D mutation were included as control (brown line; n = 4). P value between MLL-AF9 and MLL-TFE3 mice, both in the presence of the NRas^G12D mutation, is shown. A tick on the line indicates nonleukemic deaths, while the asterisk indicates the end of experimental cohort. (F) Representative hematoxylin and eosin staining of liver and spleen (original magnification ×10; scale bars, 200 µm) of MLL-AF9+NRas^G12D, MLL-TFE3+NRas^G12D, and normal mice. Blasts can be seen in the spleen and liver of MLL-AF9 and MLL-TFE mice, but not in the normal control. (G) Summary of the immunophenotyping result of the ex vivo bone marrow cells in the absence (−) or the presence (+) of doxycycline (Dox), analyzing the expression of myeloid markers. The data are plotted as scatter dot plot with mean value. (H) Summary of drug assay treatments using birinapant (±emricasan) (n = 5) and venetoclax (n = 5). The data are shown as percent viability of cells (by 4′,6-diamidino-2-phenylindole exclusion), normalized to untreated cells, and plotted against log concentration of drugs tested, with nonlinear regression analysis (variable slope). AT, AT hooks; bHLH, basic Helix-loop-Helix domain; bromo, bromodomain; CxxC, cysteine-rich region; D.P., double-positive stained cells; EFS, event-free survival; FYRC, FY-rich domain (C-terminal); FYRN, FY-rich domain (N terminal); LMI, LEDGF and menin interaction domain; LZip, leucine zipper; PHD, plant homeodomain; Pro, proline-rich domain; SET, Su(var)3-9, enhancer-of-zeste and trithorax domain; SNL, speckled nuclear localization signals.