**Figure 4. Efficacy study of I-BET151 in xenograft mouse model of MLL-fusion leukaemia.** Female NOD-SCID mice were xenotransplanted with 1x10⁷ MV4;11 cells after conditioning with Busulfan. Following establishment of disease (detectable MV4;11 cells from retro-orbital bleeds), mice were randomly assigned to receive daily IP injections of 20 mg/kg I-BET151 or vehicle control. (**A**) Kaplan-Meier plot of survival during the course of the study. Green arrowhead indicates when treatment commenced on day 40 with treatment continuing for the pre-specified period of 21 days. Animals with no detectable disease at the time of sacrifice (less than 0.5% of MV4;11 cells) or that were unable to be evaluated at the time of death were censored (denoted by a cross). Number of mice monitored: n=11 for vehicle group and n=12 for I-BET151 group. Log-rank (Mantel-Cox) test of I-BET151 treatment compared to vehicle control (_p_ = .536). (**B**-**C**) At the time of sacrifice, disease burden was evaluated in peripheral blood (PB), bone marrow (BM), and spleen cells. Number of mice analyzed: n=8 for vehicle group and n=10 for I-BET151 group. (**B**) The percent of MV4;11 cells was determined by flow cytometric analysis as the percent of HLA-A,B,C⁺ cells in the total nucleated population (both mouse and human cells). Dot plot with medians reported as crossbars. (**C**) The absolute number of MV4;11 cells each sample was also determined using CountBright absolute counting beads. Dot plot with medians reported as crossbars. Access [full size image][1] and [R script][2] used to generate this figure. This script will call the [data][3] directly from the OSF. **Figure 4 - figure supplement 1. Mouse body weight and cell death analysis in I-BET151-treated xenograft mouse model of MLL-fusion leukaemia.** This is the same experiment as in Figure 4. Female NOD-SCID mice xenotransplanted with MV4;11 cells were randomly assigned to receive daily IP injections of 20 mg/kg I-BET151 or vehicle control after detection of disease. (**A**) During the course of treatment animals were monitored and their weight was measured twice a week over the 21 day treatment period. Number of mice monitored: n=11 for vehicle group and n=12 for I-BET151 group. (**B**-**C**) At the time of sacrifice, cell death (apoptotic and necrotic cells) was evaluated in peripheral blood (PB), bone marrow (BM), and spleen cells. Number of mice analyzed: PB: n=4 for vehicle group and n=7 for I-BET151 group; BM: n=6 for vehicle group and n=8 for I-BET151 group; Spleen: n=6 for vehicle group and n=8 for I-BET151 group. (**B**) The percent of MV4;11 cells that were apoptotic was determined by flow cytometric analysis as the percent of Annexin V⁺PI^- cells in the HLA-A,B,C⁺ population. Dot plot with medians reported as crossbars. (**C**) The percent of MV4;11 cells that were necrotic was determined by flow cytometric analysis as the percent of Annexin V⁺PI⁺ cells in the HLA-A,B,C⁺ population. Dot plot with medians reported as crossbars. Access [full size image][4] and [R script][5] used to generate this figure. This script will call the [data][6] directly from the OSF. **Figure 4 - figure supplement 2. Detection of HLA by immunohistochemistry in mouse tissues.** This is from the same experiment as in Figure 4. Female NOD-SCID mice xenotransplanted with MV4;11 cells were randomly assigned to receive daily IP injections of 20 mg/kg I-BET151 or vehicle control after detection of disease. Tissues containing observed tumors were fixed, sectioned, and stained with an antibody against human leukocyte antigen (HLA-A,B,C) and counterstained with hematoxylin. (**A**) Positive control staining of human tonsil tissue that was processed in parallel with other tissue sections (magnification: 400X left, 100X right). Representative images of various tissues from MV4;11 engrafted mice who received vehicle treatment, which include (**B**) stomach (magnification: 400X left, 100X right), (**C**) gastrointestinal tract (magnification: 400X left, 100X right), (**D**) mesentery (magnification: 400X left, 20X right), (**E**) ovary (magnification: 400X left, 20X right), and (**F**) peritoneal cavity (magnification: 400X). Mouse tissues (e.g. gastrointestinal mucosa) were negatively stained. Access the [full size image][7] and [svg file][8] made with [Inkscape][9], version 0.91. IHC images are from vehicle control treaded mice [5160, 5171] and can be found [here][10]. **Figure 4 - figure supplement 3. Flow cytometry gating strategies.** (**A**) Representative images of gating strategy to assess leukaemia burden in peripheral blood, spleen, and bone marrow cells. The FL-3 vs FSC plot was used to gate on CountBright absolute counting beads and total nucleated cells. From the total nucleated population, the HLA-A,B,C APC vs FSC plot was used to gate on HLA-A,B,C⁺ cells (human leukemia cells). (**B**) Representative images of gating strategy to assess cell death in peripheral blood, spleen, and bone marrow cells. The HLA-A,B,C APC vs FSC plot was used to gate on human leukemia cells. From the human leukemia cell population, the PI vs Annexin V FITC plot was used to gate on apoptotic cells (Annexin V⁺PI^- population) and necrotic cells (Annexin V⁺PI⁺ population). Access the [full size image][11] and [svg file][12] made with [Inkscape][9], version 0.91. ---------- [Statistical Analyses component][13] contains R scripts that were used for the analysis. [1]: https://osf.io/w27dt/ [2]: https://osf.io/6k72p/ [3]: https://osf.io/zty4c/ [4]: https://osf.io/xgkkv/ [5]: https://osf.io/myuzv/ [6]: https://osf.io/x6n9n/ [7]: https://osf.io/cfhve/ [8]: https://osf.io/re4md/ [9]: https://inkscape.org/en/ [10]: https://osf.io/xjekz/ [11]: https://osf.io/xrbex/ [12]: https://osf.io/xnhd7/ [13]: https://osf.io/73pwt/