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X. Li et al.
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Figure 7. (continued from the previous page) (D) MV4-11, U937 and MOLM-13 AML cell lines and two primary AML patient samples were treated with 0-100 nM CUDC-907 for 24 h. Total RNA was isolated and c-Myc transcripts were determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). **P<0.01, ***P<0.001. (E) Cells obtained from the MV4-11 xenografts, which were treated with a single dose of CUDC-907, were enriched for human cells. Then total RNA was isolated and real-time RT-PCR performed to determine c-Myc tran- scripts. **P<0.01. (F) U937 cells were infected with Precision LentiORF c-Myc and red fluorescent protein (RFP) control lentivirus particles overnight, then washed and incubated for 24 h. The whole cell lysate from one aliquot of the cells was subjected to western blotting. The fold changes for the c-Myc densitometry measurements, normalized to β- actin and then compared to non-treated control (NTC), are indicated (left panel). The other aliquot of the cells was treated with CUDC-907 for 24 h and then subjected to annexin V/propidium iodide (PI) staining and flow cytometry analysis. ***P<0.001 (right panel). (G) Proposed mechanism of action of CUDC-907 treatment: (i) CUDC-907 inactivates ERK and inhibits PI3K resulting in reduced Mcl-1 expression; (ii) CUDC- 907 inhibits histone deacetylases (HDAC) which downregulates CHK1, Wee1, and/or RRM1, reducing DNA repair; (iii) CUDC-907 inhibits HDAC decreasing c-Myc; and (iv) CUDC-907 inhibits HDAC which upregulates Bim. The foregoing molecular changes lead to apoptosis.
Mcl-1 and shRNA knockdown of Bim demonstrated that both proteins play important roles in CUDC-907-induced apoptosis in AML cells (Figure 5A, B). Our results are con- sistent with the known effects of PI3K and HDAC inhibi- tion, which have been shown to decrease the anti-apop- totic protein Mcl-1 and upregulate the pro-apoptotic pro- tein Bim.22-25 In addition, they are in agreement with the findings of Rahmani et al. who demonstrated that Bim and Mcl-1 play a role in HDAC and PI3K inhibitor lethality in non-Hodgkin lymphoma.12 Our data show that CUDC- 907 treatment decreases the stability of Mcl-1, at least par- tially through its ability to inactivate ERK (Figure 5D-H). Based on the reported transcriptional regulation of Bim following HDAC inhibitor treatment31,32 and the increase in Bim transcripts following CUDC-907 treatment (Figure
7F), demonstrating that c-Myc plays a role in CUDC-907- induced apoptosis in AML cells.
Discussion
A major hurdle in the successful treatment of AML is resistance to standard therapies, which warrants the development of novel strategies. Here, we showed that CUDC-907 has promising antileukemic activity against AML cell lines, both in vitro and in vivo, and against leukemia progenitor cells from primary AML patient sam- ples. CUDC-907 treatment decreased expression of the anti-apoptotic protein Mcl-1 and increased expression of the pro-apoptotic protein Bim. Ectopic overexpression of
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haematologica | 2019; 104(11)