Fig. 6: AS-99 impairs transcriptional program of MLL fusion proteins and reduces leukemia burden. | Nature Communications

Fig. 6: AS-99 impairs transcriptional program of MLL fusion proteins and reduces leukemia burden.

From: Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity

Fig. 6: AS-99 impairs transcriptional program of MLL fusion proteins and reduces leukemia burden.The alternative text for this image may have been generated using AI.

a, b Quantitative RT-PCR performed in MOLM13 cells (a) or MV4;11 cells (b) after 7 days of treatment with AS-99. Gene expression was normalized to HPRT1 and referenced to the DMSO treated cells. Representative data from two independent experiments, each performed in triplicates (mean ± SD, n = 3 biological replicates) are shown. P values were calculated using unpaired 2-tailed t test. For gene expression in MOLM13 cells, the P values are as follows: MEF2C: 6 µM: 0.0017, 4 µM: 0.0035, 2 µM: 0.036, DLX2: 6 µM 0.0017, 4 µM: 0.0003, 2 µM: 0.0032, MEIS1: 6 µM: < 0.0001, 4 µM: 0.011, 2 µM: 0.12; FLT3: 6 µM: 0.0007, 4 µM: 0.0018, 2 µM: 0.086; HOXA9: 6 µM: 0.026, 4 µM: 0.038, 2 µM: 0.064; MNDA: 6 µM: 0.0021, 4 µM: 0.0068, 2 µM: <0.0001. For gene expression in MV4;11 cells, the P values are as follows: MEF2C: 3 µM: 0.0007, 1.5 µM: 0.009, DLX2: 3 µM: <0.0001, 1.5 µM: 0.0017, MEIS1: 3 µM: 0.16, 1.5 µM: 0.074, FLT3: 3 µM: 0.0010, 1.5 µM: 0.57, HOXA9: 3 µM: 0.010, 1.5 µM: 0.044, MNDA: 3 µM: 0.0031, 1.5 µM: 0.0004. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. c Results from RNA-seq studies for AS-99 (3 µM) versus DMSO-treated MV4;11 cells. Volcano plot showing q-values for differential expression analysis of each gene versus fold-change (FC) for AS-99 over DMSO-treated cells. Blue dots indicate significantly downregulated genes, red dots indicate significantly upregulated genes (q < 0.05, fold-change > 2). d, e Plots showing enriched gene sets upon treatment of MV4;11 cells with AS-99 or DMSO determined by fgsea for defined targets of MLL-AF927 (d) or targets of NUP98-HOXA928 (e). The top 20 differentially expressed genes from the Leading Edge of the indicated fgsea analysis are shown below each of the enrichment plots. padj = adjusted p value; ES enrichment score, NES normalized enrichment score. f–h Effect of AS-99 (30 mg/kg, q.d., i.p.) on leukemia burden in the MV4;11 xenotransplantation murine model. Quantification of bioluminescence signal in mice treated with AS-99 (n = 7 mice) or vehicle (n = 6 mice) at the indicated days. Mean ± SEM (f). Quantification of bioluminescence signal shown for individual mice at the last day of treatment (day 19 post-transplantation). Mean ± SD (g). Flow cytometry quantification of human CD45+ cells in spleen, and blood samples harvested from the mice after last day of treatment with AS-99 (n = 7 mice) or vehicle (n = 6 mice) (h). Mean ± SD. P values were calculated using the unpaired 2-tailed t test. Gating strategy is presented in Supplementary Fig. 11e.

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