Page 123 - Haematologica May 2020

MDM2 and BCR-ABL1 inhibition targets CML stem cell
increased levels of p53 and several of its targets in CML BM cells compared to those from normal controls, in both mouse models and human samples. These findings are consistent with a previous report that the BCR-ABL1 fusion protein in CML cells promotes p53 accumulation, but antagonizes its activity by modulating the p53-MDM2 regulatory loop.33 A recent study also showed that CD34+ progenitor cells from CML-CP patients expressed statisti- cally significantly higher phosphorylated p53 (Ser15) com- pared to CD34+ cells from health donors.34
We found that pro-apoptotic BAX and NOXA were increased in CML cells compared with normal controls. BCR-ABL1 is known to up-regulate anti-apoptotic pro- teins such as MCL-1 and BCL-XL to support CML cell survival. We previously reported higher anti-apoptotic BCL-2 levels in CML cells and LSK cells in the same mouse model.20 These anti-apoptotic BCL-2 proteins like- ly antagonize the pro-apoptotic BCL-2 proteins tilting the balance towards cell survival, which makes CML cells more dependent on anti-apoptotic BCL-2 proteins. This notion is consistent with our results showing that BM CD45+ cells and LSK cells from the CML mice were more sensitive to BH3 peptide-induced apoptosis than those cells from the control mice. Furthermore, it was previously reported that, although imatinib does not directly affect p53 levels, it abrogated nutlin-3-induced p21,35 which is known to block the cell cycle and sup- press apoptosis.36
We were able to detect increased p53, NOXA, and BAX in CML LSK cells from mouse BM treated with DS-5272, or the combination, 24 hours after treatments. The short in vivo half-life of DS-5272 may contribute to the dimin- ished induction of p53 and its target proteins. It is impor- tant to point out that although TKI are inactive against CML stem cells, they do inhibit BCR-ABL1 activity in these cells.6 The balance of pro- and anti-apoptotic pro- teins decides cell death or survival. Activation of p53- induced apoptotic signaling by MDM2 inhibition together with inhibition of BCR-ABL1-regulated survival pathway by TKI likely push CML cells/stem cells towards death. This is supported by our previous study in blast crisis CML demonstrating that nutlin3a induced p53 and pro- apoptotic proteins PUMA and BAX, while nilotinib sup- pressed BCR-ABL1 signaling and decreased anti-apoptotic proteins BCL-XL and MCL-1, and that their combination synergistically induced cell death even in blast crisis CML cells resistance to TKI.27 Wendel et al. reported that loss of p53 hampers the anti-leukemia response to BCR-ABL inhibition in a BCR-ABL1 transgenic mouse model,37 sug- gesting that activation of p53 signaling may enhance TKI activities in CML.
You et al. recently demonstrated that JNJ-26854165, another MDM2 inhibitor, is active in CML cells through promoting BCR-ABL proteosomal degradation, independ- ent of p53.38 This is not surprising since several reports have shown p53-independent anticancer activity of JNJ- 26854165.39-41 DS-5272, derived from a candidate MDM2 inhibitor by chemical modifications to improve its poten- cy and physicochemical property, is a highly selective and potent MDM2 inhibitor.42 Although we cannot state that DS-5272 works entirely in a p53-dependent manner, espe-
cially since MDM2 has functions other than antagonizing p53, its use alone or in combination with a TKI increased p53, NOXA, and BAX, suggesting that it functions, at least in part, through increasing the p53 signaling.
Tyrosine kinase inhibitors have been proven to be high- ly effective in controlling CML, but in most cases they do not cure the disease. Although imatinib significantly deceased CML LSK cells in BM at the end of the treatment in the transgenic mouse model, the combination was more effective. Imatinib had no effect on spleen leukemia LSK. The different effects of imatinib on BM and spleen CML LSK is not clear and it may involve microenviron- mental factors. However, the combination also signifi- cantly decreased spleen CML LSK cells. Importantly, ima- tinib by itself did not significantly reduce leukemia LT- HSC frequency in second transplantation, but rather did so only when combined with DS-5272. Similarly, nilotinib itself also did not decrease leukemia LT-HSC frequency in a second transplantation, as shown in our previous study.20
The mechanism of BCR-ABL1-driven p53 activation is not fully understood. However, BCR-ABL1-mediated hyper-proliferative signals likely contribute to the activa- tion. The combination of MDM2 inhibition and TKI pro- foundly prolonged overall survival in our mouse model. In addition to modulating apoptosis regulators, other path- ways may also be involved. For example, it was reported that TKI nilotinib inhibits MDM2 and induces a p53-inde- pendent apoptosis by down-regulating XIAP.43 Kojima et al.44 showed that inhibition of MDM2 with nutlin decreased CXCL12 in stromal cells, a critical component of the BM microenvironment that supports leukemia-BM microenvironment interactions and confers drug resist- ance. Furthermore, whether mouse immunity is regulated by the combination treatment is unknown, which war- rants future investigation.
Data from this study, together with our previous report in blast crisis CML,27 demonstrate that combined inhibi- tion of MDM2 and BCR-ABL1 tyrosine kinase can target CML cells and CML stem/progenitor cells, and it has the potential to overcome TKI resistance and significantly improve outcomes in CML. Furthermore, we have demonstrated that BCL-2 is a key survival factor of CML stem cells, and targeting BCL-2, combined with a TKI, had the potential to eradicate CML stem cells.20 Adding a MDM2 inhibitor, which activates p53 and induces pro- apoptotic BCL-2 proteins to the combination, will likely further improve the therapeutic potential for patients with CML, which certainly warrants future clinical investiga- tions.
Funding
The authors would like to thank the research funding from Daiichi Sankyo (to BZC) and by National Institutes of Health grants P01CA49639 and P30CA016672 and the Paul and Mary Haas Chair in Genetics (to MA) and by NCI-SBIR grant 2R44CA203610-02A1 (to Eutropics) for supporting this study.
Acknowledgments
Erica Goodoff from Scientific Publication at MD Anderson Cancer Center and Numsen Hail, Jr. for editing and assisting with the manuscript preparation.
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