Extended Data Fig. 7: Targeting NAT10 by small molecule inhibitors suppresses AML cells both in vitro and in vivo.
(a) MTT assays and the IC50 values of Remodelin, fludarabine, Fosaprepitant, Dantrolene and Folinic acid in MonoMac6 cells after 72 hours of treatment. (b) MTT assays showing the inhibitory effects of Remodelin in leukaemia cell lines with 72-hour treatment. IC50 in each cell line was shown. (c) MTT assays showing the inhibitory effects of Remodelin in C1498 cells. (d) Effect of fludarabine and Remodelin on the colony-forming capacity of blast cells from MLL-AF9-induced leukaemia mice. (e) Predicted binding modes of fludarabine (P-F-ara-A) and 3P-fludarabine (F-ara-ATP) to the enzymatic pocket of the NAT10 protein. (f) DARTS assays evaluating the efficacy of Remodelin, P-F-ara-A and F-ara-ATP on protecting METTL3 protein against pronase digestion in MOLM13 cell lysates. (g) Western blotting of NAT10 protein from CETSA assays in MonoMac6 pretreated with fludarabine (F-ara-A), Remodelin or DMSO. (h) Total cellular serine level in MOLM13 cells treated with or without Remodelin at 10 μM for 96 hours. (i) Histogram showing the cell proportions in different phases of MOLM13 cells treated with Remodelin (20 μM) or fludarabine (1 μM) for 72 hours. (j) An example showing the EdU labelling and PI staining followed by flow cytometry to assess cell cycle of MOLM13 cells. (k) Body weight of C1498 syngeneic AML mice after i.p. injection of fludarabine (200 mg/kg), Remodelin (30 mg/kg) or corresponding vehicle control. (l) Schematic structures depicting human wild-type MENIN protein and MENIN variants used in this study. The red lines indicated the mutation of M327 with methionine (M) to isoleucine (I) conversion in the M327I variant or the mutation of G331 with glycine (G) to arginine (R) conversion in the G331R variant, respectively. (m) Western blotting showing the overexpression of FLAG-tagged wild-type or mutant MENIN in stably transduced MOLM13 cells (n) The inhibition curves of MOLM13 cells transduced with wild-type or mutant MENIN treated with Revumenib at indicated concentration for 12 days. Values are mean of n = 2 biological replicates in d. Values are mean ± s.d. of n = 3 biological replicates in a, b, c, i and n or n = 4 biological replicates in h. Two-tailed Student’s t-tests were used. Images in f, g and m were representative of three independent experiments.