Reduced MDM2 and MDM4 Protein, but Not mRNA, Expression Is Associated with More Severe Disease in Acute Myeloid Leukemia (AML), Myelodysplastic Syndromes (MDS), and Chronic Myelomoncytic Leukemia (CMML)

Background: Following studies suggesting overexpression of MDM2 and MDM4 in AML, clinical trials were conducted in AML with MDM2 inhibitors, generally combined with chemotherapy, with so far relatively limited results. On the other hand , in those studies, MDM2 and 4 expressionwas mainly assessed on mRNA and the indirect estimation of protein expression using immunohistochemistry on mononuclear cells (MNCs). In addition, results were often conflicting, based in particular on different cells studied and techniques used. We analyzed MDM2 and MDM4 expressioin AML, MDS and CMML, analyzing both mRNA and protein levels, the latter by flow cytometry.

Methods: Bone marrow samples from 113 (53 AML, 17 of them in complete remission (CR) after treatment, 50 MDS or 10 CMML) adult patients (classified according to WHO2016) seen at Hôpital Saint-Louis, Paris, were collected between 2018 and 2021 . Bone marrow samples collected from hip surgery of 17 elderly aged-matched donors without hematological disorder were used as controls. For the mRNA Human,CD34⁺ cells were purified from MNCs and total mRNA purified. RT-qPCR was performed and expression of each mRNA was analyzed in the CD34+ population. MDM2, MDM4 and p53 protein expression was determined in all samples with monoclonal antibody staining and flow cytometry analysis. In controls and in MDS and CMML patients without excess of marrow blasts (<5%), protein expression was assessed in the CD34+/CD45low population In patients with excess of marrow blasts (5% or greater,), because of the heterogeneity of CD34 expression on blast cells, protein expression was assessed on the CD45low/SSC blast population. Data were acquired using a BD FACS Canto II.

Results: For protein expression, In CD34+/CD45low cells from MDS/CMML patients without excess of blasts, MFI was 795.7 for MDM2, 2555 for MDM4 and 500.6 for p53, with no significant differences with controls for p53 (p=0.3620) but lower levels in patients for MDM2 (p=0.0123) and MDM4 (p=0.0337). In the blast population of patients with excess of blasts, MDM2, MDM4 and p53 protein expression were drastically decreased in MDS (MFI: MDM2=320.8, p<0.0001; MDM4=1017, p<0.0001; p53=137.5, p<0.0001), AML (MFI: MDM2=233.2, p<0.0001; MDM4=833.8, p<0.0001; p53=111.8, p<0.0001) and CMML (MFI: MDM2=262.2, p=0.0001; MDM4=851.7, p<0.0001; p53=113.3, p=0.0007) (Fiigure A) compared with control CD34+/CD45low cells. Comparison between CD34+/CD45low bone marrow cells from patients without excess of blasts and CD45low/SSC blasts of patients with excess of blasts (AML, MDS, CMML) showed in the latter group lower expression of each protein for MDS (MFI: MDM2 p<0.0001; MDM4 p=0.0005; p53 p=0.0007), AML (MFI: MDM2 p<0.0001; MDM4 p<0.0001; p53 p<0.0001) and CMML (MFI: MDM2 p=0.005; MDM4 p=0.0165; p53 p=0.0268). We found a negative correlation for MDM2 (r=-0.3191, p=0.0018), MDM4 (r=-0.3925, p<0.0001), p53 (r =0.3195, p=0.0018) between protein expression levels and marrow blast percentage. Expression of CD34+/CD45low cells from patients in CR (MFI: MDM2= 1797; MDM4 =2993; p53=971) showed no significant differences with the control CD34+/CD45low cells (MFI:MDM2 p=0,4533; MDM4 p=0.1420; p53 p=0.2891). For MDM2 and MDM4 proteins, patients whose expression value was lower than the median had significant poorer survival than those with a higher value (MDM2 p=0.0209; MDM4 p= 0.1025; p53 p=0.0304) (Figure B). In MDS, however, protein expression had no significant impact on the probability of progression to AML (MDM2 p=0.5636; MDM4 p= 0.1488; p53p=0.7816). No significant differences in mRNA expression were found between CD34+ cells from healthy donors and patients without excess blast or patients with excess blast. We observed no correlation between the mRNA and protein expression levels

Conclusions: We found in AML, MDS and CMML a discrepancy between mRNA levels and protein levels for MDM2, MDM4 and p53, and overall low MDM2 and MDM4 protein expression, which might explain the relatively low efficacy of MDM2 inhibitors observed so far in clinical trials . The relatively high MDM2 and p53 levels observed in control bone marrows possibly also explains, at least in part, the reported toxicity of these drugs on myeloid cells in clinical trials.

Fenaux:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Jazz: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Syros Pharmaceuticals: Honoraria.