Combination of Decitabine and Etoposide Is Highly Effective in Treating p53-Mutated MDS/AML Via Activating Notch Signaling

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy.

A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients’ records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059).

Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment.

Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients’ cells in vitro.

We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state.

Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.