Decitabine, Etoposide-Based Regimen Greatly Improved T53 Mutation Elderly AML/MDS Survival By Activating Notch1 Signaling Pathway

The number of elderly myelodysplastic syndrome/acute leukemia(MDS/AML) patients is increasing every year while the treatment is limited and outcome is poor, which is also faced with the lack of basic research and breakthrough. The expectation of better life quality and longer survival in elderly patients is increasing with the improvement of living standards.

We compared two different regimens decitabine with CEG as well as decitabine with CAG in random clinical trials(ChiCTR-INR-16009337) enrolled 72 elderly MDS/AML patients and conducted multivariate factor regression to determine the independent prognostic factor of decitabine with CEG regimen. We also compared the sensitivity of different cell lines to etoposide, decitabine and aclarubicin in vitro and explored the possible molecular mechanism as well as signaling pathway by RNA-seq.

We found TP53 mutation is an independent prognostic factor(HR for OS: 3.4(1.2-9.9)) in decitabine with CEG group, with a significant prolonged survival of 31 months(p=0.019) and progression free survival of 24 months(p=0.034) among these patients. While TP53 wild type patients could get benefit from decitabine with CAG regimen, with a prolonged overall survival(p=0.034) and progression free survival(p=0.009). We also found decitabine with etoposide could greatly reduce tumor burden and prolong T53 mutation leukemic mice survival in NOD/SCID mice.TP53 mutation cells were sensitive to etoposide-induced apoptosis, while TP53 wide type cells were relatively sensitive to Aclarubicin-induced apoptosis in vitro. Etoposide and decitabine could induce differentiation in TP53 mutation clones, while aclarubicin didn't have such effect. RNA-sequence data gave us a hint that etoposide might activate Notch1 signaling pathway. We then use CRISPR to knockout Notch1 and find drug induced apoptosis and differentiation were reduced to a half of before(p<0.01), which demonstrate that etoposide induced apoptosis and differentiation as well as decitabine induced differentiation were all dependent on Notch1 signaling pathway. We employed CRISPR again followed by gene re-expression in TP53 wide type cells as well as gene restoration in TP53 mutation cells, we found that after TP53 knockout, etoposide induced apoptosis was increased by approximately two times(p<0.01), CD11b expression(differentiation) was from 2% to 19%(p<0.01) accompanied with Notch1 pathway activation. Aclarubicin induced apoptosis reduced to about a half of before(p<0.01). While after TP53 re-expression, all apoptosis and differentiation as well as Notch1 pathway activation could be rescued, which identify p53 negatively regulate Notch1 signaling pathway.

Decitabine with CEG regimen significantly prolonged TP53 mutation MDS/AML survival. TP53 mutational clone might be sensitive to etoposide, which was dependent on Notch1 signaling pathway. p53 function presence would affect Notch1 activation. We will consider how to make decision on elderly MDS/AML patients treatment by TP53 status in larger clinical trials.

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