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CUDC-907 combined with venetoclax in AML
by treatment with CUDC-907 and venetoclax, alone or in combination. Further, combination treatment resulted in increased cleavage of caspase 3 and PARP in both AML cell lines and primary patient samples, confirming the flow cytometry results shown in Figure 2A; Figure 3A; Online Supplementary Figure S1; Online Supplementary Figure S3A. In order to determine if combined CUDC-907 and venetoclax treatment disrupts the binding of Bim to Mcl- 1 and Bcl-2, co-immunoprecipitation of Bcl-2 was per- formed using both U937 and MOLM-13 cells. CUDC-907 treatment caused increased binding of Bim to Bcl-2, while venetoclax treatment reduced Bim bound to Bcl-2 (Figure 3D-E). Co-immunoprecipitation of Bim revealed that venetoclax treatment increased binding of Bim to Mcl-1. CUDC-907 treatment reduced Bim bound to Mcl-1 in MOLM-13 cells, though not in U937 cells. However, com- bined treatment reduced both Bim bound to Bcl-2 and pre- vented increased Bim bound to Mcl-1 induced by veneto- clax treatment. Knockdown of Bim (50%) significantly reduced apoptosis induced by CUDC-907 alone and in combination with venetoclax (Figure 3F). Mcl-1 overex- pression also partially rescued cells from CUDC-907- induced apoptosis when treated alone and in combination with venetoclax (Figure 3G). Taken together, these results demonstrate that Bim and Mcl-1 play important roles in combined CUDC-907 and venetoclax treatment in AML cells. Further, CUDC-907 and venetoclax reciprocally overcome mechanisms of resistance to single drug treat- ment.
Venetoclax enhances CUDC-907-induced DNA damage in acute myeloid leukemia cells
Based on our previous study in which we show down- regulation of key DNA repair proteins by CUDC-907 treatment,11 we sought to determine if these changes also occurred in the presence of venetoclax. Consistent with our published work, CUDC-907 downregulated RRM1, CHK1, and Wee1, which was maintained by the com- bined treatment in both AML cell lines and primary patient samples (Figure 4A-B). CUDC-907 treatment increased chromatin-bound RPA32 and γH2AX, reflecting increased DNA replication stress and DNA damage (Figure 4C). Venetoclax treatment increased chromatin- bound RPA32 and γH2AX in MOLM-13 cells, though not in U937 cells. Combined CUDC-907 and venetoclax treat- ment resulted in more chromatin-bound RPA32 and γH2AX compared to single drug treatments. Alkaline comet assay results revealed that individual treatment with CUDC-907 induced DNA damage, while combined treatment significantly increased DNA damage compared to individual drug treatment for both AML cell lines and a primary AML patient sample (Figure 4D; Online Supplementary Figure S4). We previously reported that CHK1 plays a role in the mechanism of action of veneto- clax in AML cells.6 In order to confirm the roles of Wee1 and RRM1 in venetoclax activity, U937 cells were treated with the selective Wee1 inhibitor MK-1775 or the ribonu- cleotide reductase inhibitor hydroxyurea (HU) alone or combined with venetoclax, and then subjected to comet assays. As shown in Figure 4E and Online Supplementary Figure S5, Wee1 inhibition and HU treatment both induced DNA damage, which was significantly increased by the addition of venetoclax. HU and MK-1775 treat- ment increased the percent of Annexin V positive cells, which was significantly enhanced by venetoclax. In order
to further confirm the role of Wee1, Wee1 was overex- pressed in U937 cells (Online Supplementary Figure S6A), which partially prevented apoptosis induced by CUDC- 907 alone and in combination with venetoclax (Online Supplementary Figure S6B). Further, overexpression of Wee1 decreased DNA damage induced by CUDC-907 treatment alone and in combination with venetoclax (Online Supplementary Figure S6C). These results show that Wee1 plays a role in apoptosis induced by CUDC-907 or combined CUDC-907 and venetoclax treatment in AML cells. In order to determine the effect of venetoclax on DNA damage, MOLM13 cells were treated with CUDC- 907 for 16 hours, washed, given fresh media with or with- out venetoclax for up to 12 hours. Following CUDC-907 treatment, DNA damage decreased, suggesting that repair progressed after removal of CUDC-907. However, addi- tion of venetoclax significantly slowed down the decrease of the percent DNA in the comet tails, indicating inhibi- tion of DNA repair (Figure 4F; Online Supplementary Figure S7). Taken together, these results suggest that venetoclax impairs repair of DNA damage induced by CUDC-907 in AML cells.
CUDC-907 decreases Mcl-1 protein stability and transcriptionally regulates Bim, CHK1, Wee1, and RRM1, enhancing venetoclax activity in acute myeloid leukemia cells
In order to begin to determine if CUDC-907 modulates Mcl-1, Bim, CHK1, Wee1, and RRM1 by altering tran- scription, transcripts were measured via real-time RT-PCR after drug treatment. In AML cell line U937 and primary patient sample AML#23, CUDC-907 treatment, both alone and in combination with venetoclax, significantly increased Mcl-1 transcripts, while in MOLM13 and AML#22 no significant change was detected (Figure 5A). CUDC-907 treatment alone and in combination with venetoclax significantly increased Bim and reduced CHK1, Wee1, and RRM1 transcripts in all samples tested. These results suggest that the upregulation of Bim and downreg- ulation of CHK1, Wee1, and RRM1 by CUDC-907 treat- ment is likely through transcriptional mechanisms, while Mcl-1 is not. In order to determine if CUDC-907 and venetoclax have an impact on Mcl-1 protein stability, MOLM-13 and U937 cells were treated with CUDC-907 and venetoclax, alone or in combination, for 16 hours, washed, and then treated with cycloheximide (10 mg/mL) for up to 120 minutes. Western blots revealed that Mcl-1 levels decreased significantly faster in cells treated with CUDC-907 compared to control cells (MOLM13: 68 vs. 82 minutes, P=0.0189; U937: 82 vs. 94 minutes, P=0.0423). Venetoclax treatment significantly increased Mcl-1 half- life (MOLM13: 115 vs. 82 minutes, P=0.0005; U937: 115 vs. 94 minutes, P=0.0061), while combination treatment prevented venetoclax-induced Mcl-1 half-life increase (Figure 5B-C). Proteasome inhibition prevented downreg- ulation of Mcl-1 by CUDC-907 both alone and in combi- nation with venetoclax in AML cell lines and a primary AML patient sample (Figure 5D-F). Since phosphorylation of Mcl-1 at T163 has been shown to stabilize Mcl-1 by prolonging its half-life,25 we looked at phosphorylation of Mcl-1 post-drug treatment. CUDC-907 treatment alone and in combination with venetoclax reduced phosphory- lation of Mcl-1 at T163 (Figure 5D-F). Taken together, these results suggest that CUDC-907 downregulates Mcl- 1 by decreasing its protein stability in AML cells.
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