Fig 2: CPT inhibits KMT1A enzymatic activity in an in vitro reconstituted system(A) HMTase assay with GST-KMT1A, [3H]-SAM cofactor, and either GST or GST-H3(N) substrate as indicated. Reactions contained either increasing concentrations of CPT (0.25 μM, 0.5 μM, 1 μM, 4 μM, and 5 μM) or maximum volume of DMSO, as indicated. H3(N) was visualized by coomassie staining, and 3H-[Me]-H3(N) by autoradiography. (B) Same as (A) except reactions were carried out using 2.5 μM or 5.0 μM of CPT, CPT-11 and SN38 as indicated. (C) HMTase assay with reactions carried out similarly as in (A), except samples were precipitated on filter paper, washed, dried, and subjected to scintillation counting. Counts per minute (CPM) were normalized by subtraction of background signal as measured by a control reaction lacking enzyme. Error bars represent ±SEM from reactions performed in duplicate.

Fig 3: Downregulation of KMT1A by CPT is independent of TOP1-DNA cleavage complex(A) Rh28 cells were treated with 63.0 nM LMP400, 17.0 nM LMP776, 30.0 nM CPT, or DMSO control as indicated for 24 hours. Rh30 cells were treated with 53.0 nM LMP400, 13.0 nM LMP776, 38.0 nM CPT, or DMSO control as indicated for 24 hours. KMT1A levels were then assessed by immunoblotting. (B) Rh28 and Rh30 cells were treated as in (A) and were subjected to immunoblot analysis to determine levels of γH2AX. Total H2A is used as additional loading control. (C) Rh30 cells were treated with LMP400, LMP776, or DMSO control as in (A), and MyoG levels were assessed via immunoblotting. (D) HCT116 and HCT116-G7 cells were treated with SN38 (2.5 nM and 5.0 nM) or DMSO control (-) as indicated for 48 hours. KMT1A levels were then assessed by immunoblotting. (E) HCT116-G7 cells were treated with increasing doses of CPT (5.0 nM, 10.0 nM, 25.0 nM, and 50.0 nM) or DMSO control (-) as indicated for 48 hours. KMT1A levels were then assessed by immunoblotting. For all immunoblot analysis, β-Actin is used for loading controls.

Fig 4: CPT-11 treatment permits differentiation of aRMS cells in vitro and in vivo(A) Quantified MyHC expression in Rh28 and Rh30 cell differentiation following treatment with 2.5 μM CPT-11 or DMSO for 7 days in DM, as indicated. Cells were fixed and subjected to immunofluorescence using MyHC antibodies, and counterstained with DAPI. Data is shown as percentage of MyHC+ cells per total cells showing elongated morphology. Error bars represent ±SEM from at least 3 randomly chosen fields. (B) Immunohistochemical staining of Ki67 and MyHC of tumor sections from PBS and CPT-11 treated mice bearing Rh30 xenografts. Mice were treated weekly with either PBS or CPT-11 for 3 weeks. Pictures were taken at 20X magnification. (C) Immunoblot analysis of lysates from tumor samples of PBS- or CPT-11-treated mice for KMT1A and MyoG. β-Actin is used for loading control.

Fig 5: Cell-based screening of a small molecule library identifies camptothecin (CPT) as a potent activator of MyoD in KMT1A-overexpressing myoblast cells(A) Diagram depicting the drug screen process which identified CPT as the strongest MyoD activator in C2-KMT1A-4RE reporter myoblast cells from a library of 2,000 compounds. Cells were treated for 36 hours in DM. (B) Luciferase activity of 37 initial hit compounds and DMSO control (-) as measured in the primary screen. The compound with the strongest activation of luciferase, camptothecin (14D7), is denoted CPT. (C) C2-KMT1A-4RE reporter cells were plated and treated with indicated concentrations of CPT, with DMSO (-) as vehicle control. After 36 hours of treatment in DM, luciferase activity was determined. Error bars represent ±SEM from biological replicates (n=3). ** indicates P<0.01; *** indicates P<0.001 relative to DMSO control.