Fig. 2: Antileukemic activities of OST-01 in vivo.
A Effects of OST-01 on leukemic cell growth in vivo and AML mouse survival. Luc-Molm-13 cells (1.0 × 106) were intravenously injected in NSG mice. After 7 days of injection, mice were treated with ethanol control or OST-01 [1 µL/g/BID, oral gavage, continuously till the euthanized endpoint]. Leukemia growth was determined 14 days after the start of treatment using bioluminescence imaging. N =  10 mice per group. The survival curve of treated mice is shown on Fig. S4B. B Effects of OST-01 on leukemic cell growth in vivo using MllPTD/WT/Flt3ITD/ITD AML mouse model. MllPTD/WT/Flt3ITD/ITD AML cells (1 × 106 cells/mouse) were transplanted into WT mice to generate a cohort of AML-bearing mice, which were randomly divided into two groups and treated with ethanol control [1 µL/g/BID, oral gavage, (n =  8)] or OST-01 [1 µL/g/BID, oral gavage, (n =  10)] for 21 days. Leukemia burden as determined by number of LSKs and spleen size was shown on Fig. S4D. LSKs were isolated from the treated mice. Top, immunoblotting of USP36, c-Myc and NPM1 protein (left) and TEM images of nucleolus (right). Bottom, ribosome levels in LSKs (left) and Kaplan–Meier survival curve of primary transplanted leukemic mice (right). Ethanol control [blue line; n =  10; median survival (MS) 44 days] or OST-01 (red line; n =  10; MS 60 days). C and D Synergistic effects of OST-01 and VEN in vivo. C MllPTD/WT/Flt3ITD/ITD AML cells (0.5 × 106 cells/mouse) were transplanted into WT mice to generate a cohort of AML bearing mice, which were randomly divided into 4 groups (n =  15) and treated with ethanol control (1 µL/g/BID, oral gavage, continuously till the euthanized endpoint), OST-01 (1 µL/g/BID, oral gavage, continuously till the euthanized endpoint), VEN (50 mg/kg, oral gavage, 21 days), or combination of OST-01 and VEN at the same doses of single agents. (Left) Kaplan–Meier survival curve of primary transplanted leukemic mice treated with ethanol control (black line; MS 92 days), OST-01 (blue line; MS 123 days), VEN (green line; MS 92 days), or OST/VEN (red line; MS 156 days). (Right) Kaplan–Meier survival curve of secondary transplanted leukemic mice treated with ethanol control (black line; MS 26 days), OST-01 (blue line; MS 58 days), VEN (green line; MS 31 days), or OST/VEN (red line; MS 105 days). D Inv(16) AML mice. (Left) Kaplan–Meier survival curve of primary transplanted leukemic mice treated with ethanol control (black line; MS 58.5 days), OST-01 (blue line; MS 70 days), VEN (green line; MS 58 days), or OST/VEN (red line; MS 85 days). (Right) Kaplan–Meier survival curve of secondary transplanted leukemic mice treated with ethanol control (black line; MS 38 days), OST-01 (blueline; MS 62.5 days), VEN (green line; MS 48 days), or OST/VEN (red line; MS 95 days). E FLT3-WT PDX AML mice. (Left) Kaplan–Meier survival curve of primary PDX model treated with ethanol control (black line; MS 35.5 days), OST-01 (blue line; MS 45 days), VEN (green line; MS 37 days), or OST/VEN (red line; MS 54 days). (Right) Kaplan–Meier survival curve of secondary transplanted leukemic mice treated with ethanol control (black line; MS 34.5 days), OST-01 (blue line; MS 47 days), VEN (green line; MS 41 days), or OST/VEN (red line; MS 54 days). F Schematic model mechanism of action of OST-01 in AML cells. OST-01 inhibits deubiquitinase USP36 expression, leading to downregulation of nucleolar proteins including c-Myc, nucleophosmin (NPM1), and nucleostemin (NS). This consequently disrupts nucleolus structure and inhibits ribosome synthesis.
