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X. Li et al.
CUDC-907 downregulates c-Myc expression enhancing the antileukemic activity of venetoclax in acute myeloid leukemia cells
Our previous work shows that CUDC-907 inhibits the expression of c-Myc,11 which is an oncoprotein that is fre- quently activated in AML cells and plays an important role in leukemogenesis.26,27 Western blot analyses revealed that CUDC-907 downregulation of c-Myc occurs in the pres- ence or absence of venetoclax in AML cell lines and pri- mary AML patient sample AML#24 (Figure 6A). c-Myc transcript levels were significantly reduced by CUDC-907 treatment alone and in combination with venetoclax (Figure 6B). In order to confirm the functional role of c- Myc, we transiently overexpressed c-Myc in U937 cells. Overexpression of c-Myc significantly reduced the effects of CUDC-907 alone and in combination with venetoclax (Figure 6C). Treatment with the c-Myc inhibitor 10058-F4 significantly enhanced venetoclax-induced apoptosis (Figure 6D). Further, c-Myc inhibition induced DNA dam- age that was further enhanced by combination with vene- toclax (Figure 6E). These results indicate that c-Myc downregulation by CUDC-907 plays an important role in the enhancement of venetoclax-induced death of AML cells.
Discussion
We previously reported that Mcl-1 and Bim are impor- tant for the antileukemic activity of venetoclax1 and they are also important for CUDC-907 activity in AML cells.11 In this study, we found that CUDC-907 treatment decreases Mcl-1 protein, overcoming a mechanism of resistance to venetoclax. Our previous study showing that inhibition of Mcl-1 enhances the antileukemic activity of venetoclax,3 further supports that downregulation of Mcl- 1 by CUDC-907 plays an important role in the combined antileukemic activity of CUDC-907 and venetoclax. Venetoclax prevents Bcl-2 from sequestering Bim, over- coming a mechanism of resistance to CUDC-907. In agreement with our previous study,11 Bim transcripts were increased after treatment with CUDC-907 alone and in combination with venetoclax (Figure 5A). Further, CUDC- 907 treatment decreased Mcl-1 protein stability, which does not appear to be effected by venetoclax treatment. While Mcl-1 and Bim play important roles in CUDC-907 and venetoclax activity, knockdown of Bim and overex- pression of Mcl-1 only partially prevented apoptosis induced by CUDC-907 alone and in combination with venetoclax, suggesting that other mechanisms exist.
In our previous study, we found that CHK1, Wee1, and RRM1 play important roles in the antileukemic activity of CUDC-907 against AML.11 We have also shown enhanced DNA damage in AML cells treated with combined vene- toclax and DNA damaging agents1,6 and that CHK1 inhibi- tion enhances the antileukemic activity of venetoclax.6 Pham and colleagues recently reported that venetoclax treatment activates the DNA damage response in lym- phoma cells, though the mechanism is unknown.28 In agreement, we show that inhibition of Wee1 or RRM1 in combination with venetoclax co-operatively induce DNA damage in AML cells. Further, we found that venetoclax significantly prolonged persistence of CUDC-907-induced DNA damage, suggesting that Bcl-2 plays a role in the DNA repair.
There is preclinical evidence that CUDC-907 downreg- ulates c-Myc in diffuse large B-cell lymphoma.9 Additionally, we previously reported that CUDC-907 downregulates c-Myc in vitro, in as little as 4 hours, and in vivo.11 This downregulation is maintained in the presence of venetoclax in vitro (Figure 6A). c-Myc overexpression partially rescues AML cells from both CUDC-907 monotherapy and combination therapy. Further, combina- tion of venetoclax plus the c-Myc inhibitor 10058-F4 results in significant induction of apoptosis, supporting the assertion that c-Myc inhibition is a key mechanism underlying the combination’s synergy. Based on the roles PI3K/mTOR and c-Myc play in proliferation,29,30 this com- bination may inhibit cell proliferation in addition to induc- ing apoptosis. Similar to our results, Cinar et al. reported that the c-Myc inhibitor 10058-F4 enhances venetoclax activity in double-hit and triple-hit lymphoma.31 Though the mechanism remains to be fully understood, c-Myc inhibition and venetoclax cooperatively induce DNA damage in AML cells (Figure 6E), suggesting that Bcl-2 is involved in the DNA damage response.
Our data demonstrates that CUDC-907 can enhance venetoclax activity in vivo. The mice were given a 4-day break out of an abundance of caution since a higher dosed group in a concurrent trial (150 mg/kg CUDC-907, pub- lished in 11) experienced moderate body weight loss (3%). Additionally, treatment was stopped after 14 doses of CUDC-907, again due to the moderate body weight loss in the concurrent trial (150 mg/kg CUDC-907 group, which was completely reversible within 4 days). In hind- sight, the body weight loss associated with combination treatment was minimal, suggesting that the 4-day drug holiday may not have been necessary or that we could have potentially given more than 14 doses. Nonetheless, our results show proof-of-concept; the combination of CUDC-907 and venetoclax increased the lifespan of the mice by 60%, while CUDC-907 and venetoclax alone increased the lifespan by 33% and 27%, respectively. Additionally, our unpublished data using the MV4-11- derived xenograft mouse model, shows that cytarabine in combination with venetoclax increased the lifespan of these mice by only 7.5% (unpublished data).
In summary, combined CUDC-907 and venetoclax shows great synergistic antileukemic activity against AML cells, at least partially mediated by Bim, Mcl-1, CHK1, Wee1, RRM1, and c-Myc (Figure 6F). Both CUDC-907 and venetoclax are orally available agents, which would sim- plify their administration in the clinical setting.32 More importantly, both agents are already in regular clinical use in experimental and non-experimental contexts. Venetoclax has been approved by the FDA for use in the context of AML, and CUDC-907 carries an FDA Fast Track designation for use in relapsed or refractory diffuse large B-cell lymphoma and a phase II trial is presently underway examining its efficacy in patients with MYC- altered relapsed/refractory diffuse large B-cell lymphoma. The results of this study support further development of this promising combination for the treatment of AML.
Contributions
XL, YS, KH, GM, RM, and GW performed the in vitro stud- ies; LP, JK, SHD, and KW performed the in vivo mouse studies; LP, JY, GW, LZ, YW, HL, JWT and YG participated in the design and coordination of the study; XL, YS, KH, GM, HE, TK, LP, JK, SHD, KW, JY, RM, LZ, YW, HL, JWT and YG
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haematologica | 2021; 106(5)