Page 116 - 2020_09-Haematologica-web

J. Zhou et al.
were significantly decreased in ASLAN003-treated cells (Online Supplementary Table S2). The eIF proteins are essen- tial factors for protein synthesis, hence we performed an assay to determine the effects of ASLAN003 on protein synthesis. Indeed, ASLAN003 inhibited protein synthesis, as demonstrated by the reduced incorporation of OPP at protein translation sites in both MOLM-14 and KG-1 cells (Figure 5A). We also investigated proteins involved in the control of the mRNA translation process. In agreement with the RNA-sequencing data, western blot analysis con- firmed the downregulation of EIF4B, and RPL6 proteins (Figure 5B)
As revealed by RNA-sequencing and qRT-PCR analysis, FOS and JUN were upregulated by ASLAN003. We, there- fore, decided to delineate the role of activating protein 1 (AP-1) transcription factors in ASLAN003-mediated differ- entiation. Western blot analysis confirmed the dose- dependent increase of c-FOS protein levels in response to exposure to ASLAN003 (Figure 5B). T-5224 is a selective, small-molecule inhibitor of AP-1.23 Of note, T-5224 did not decrease cell viability when applied at doses of up to 125 μM in MOLM-14 and KG-1 cells (Online Supplementary Figure S4A). We then evaluated the effect of T-5224 on ASLAN003-mediated differentiation. Addition of 20 μM T-5224 completely abolished the differentiation effect of ASLAN003 in KG-1 cells and dampened the effect by half in MOLM-14 cells (both P<0.001) (Figure 5C, Online Supplementary Figure S4B). T-5224 alone had a minimal
AC
effect on differentiation (Figure 5C). These results there- fore suggest that ASLAN003-mediated differentiation is facilitated, at least partially, via activation of AP-1 tran- scription factors.
Robust in vivo efficacy of ASLAN003 in multiple mouse xenograft acute myeloid leukemia models
To determine in vivo efficacy of ASLAN003 in AML, we first used two mouse xenograft models of the human AML cell lines, MOLM-14, and THP-1. Treatment with ASLAN003 (50 mg/kg, once daily oral gavage) was well tolerated as evidenced by the fact that there were no sig- nificant differences in body weight, hemoglobin concen- tration or platelet counts between the vehicle control and treated groups in these two models (Online Supplementary Figure S5A). Survival was significantly prolonged in the ASLAN003-treated groups compared to the vehicle control groups in both xenograft models (P=0.03 and P<0.001) (Figure 6A). In the MOLM-14 xenograft model, ASLAN003 substantially reduced the number of disseminated tumors, but also the size of these tumors relative to those in con- trols (Online Supplementary Figure S5B). Interestingly, in the THP-1 xenograft model, we observed that the livers of control mice were swollen and the surfaces were covered by copious white dots, a manifestation of leukemic infiltra- tion. In sharp contrast, the appearance and size of the livers remained largely normal in ASLAN003-treated mice bear- ing THP-1 cells (Online Supplementary Figure S5B). Taken
B
Figure 5. Effects of ASLAN003 on protein synthesis and AP-1 transcription factors. (A) MOLM-14 and KG-1 cells were incubated with ASLAN003 at the doses of 1 μM and 2 μM for 1 h before addition of O-propargyl-puromycin (OPP) reagent for 1 h, followed by flow cytometry analyses. The graph represents fold decreases in OPP labeling [means ± standard deviation (SD)] (n=3) in MOLM-14 and KG-1 cells, with values for dimethylsulfoxide (DMSO)-treated cells set at 1.0. Statistical com- parisons between groups are shown (Student t-test). *P<0.05; **P<0.01. (B) Immunoblotting analysis of whole cell lysates extracted from KG-1 and MOLM-14 cells for markers as indicated. The treatment time was 48 h. GAPDH was used as a loading control. (C) MOLM-14 and KG-1 cells were treated with ASLAN003 alone, T- 5224 alone, the two drugs in combination, or DMSO as a control, for 96 h, and then subjected to FACS quantification of human CD11b antigen. The graphs show the percentage of the CD11b+ population (n=2, mean ± SD). Statistical comparisons between the effects of ASLAN003 as a single agent and in combination treat- ment are shown (**P<0.001). The percentage of CD11b+ cells were not statistically different between DMSO- and T-5224-treated samples (P>0.05).
2294
haematologica | 2020; 105(9)