Clonal Expansion of Mutant p53 Clones By MDM2 Inhibition in Acute Myeloid Leukemias

MDM2 inhibition by small molecules as a means of restoring p53 function has shown clinical activity against acute myeloid leukemia (AML) (Andreeff, Clin Cancer Res 2015). However, we and others have found increased variant allele frequencies (VAFs) of TP53 mutations in AML cells after treatment with MDM2 inhibitors, either as monotherapy or in combination with other agents (Daver ASH 2019), which suggests that MDM2 inhibition selects preexisting clones or generates de novo clones with TP53 mutations. We performed a long-term culture of AML cells (MOLM-13, an AML cell line with wild-type p53 and FLT3-ITD) treated with increasing concentrations of an MDM2 inhibitor idasanutlin (up to 320 nM, less than 10% concentration of Cmax) (Selleck). We obtained MDM2 inhibitor-resistant (R) AML cells after 96 days of the drug exposure and found that the resistant cells harbor hotspot TP53 p.R248W (R248W) mutation. We next isolated single cell clones from MOLM-13 R cells by limiting dilution, and obtained twelve subclones (subclones #1-12 in order of development). All clones carried the same R248W mutation. To determine clonal patterns of these cells, we performed single cell DNA sequencing (scDNAseq) of MOLM-13 parental, R and subclone #1 and #2 (SC1 and SC2) cells using the MissionBio Tapestry system covering 125 amplicons of 19 genes frequently mutated in AML. scDNAseq identified FLT3-ITD mutations in all cells analyzed, as expected. In the parental cells we identified only 0.02% cells (1/ 5,240) with the identical R248W mutation found in MOLM-13 R cells. MOLM-13 R cells had only 0.6% of wild-type TP53 cells, 51% carrying R248W only, and 43% R248W/R213* mutation (R248W/R213*). SC1 and SC2 cells had 1% and 99% of R248W and R248W/R213* clones, respectively (Fig.1). Seven other mutations were detected by scDNAseq. Results suggest that MDM2 inhibition can accelerate the selection of TP53-mutant AML cells in vitro. Of note, the parental cells had remained mostly p53 wild-type, where the subclone with mutant R248W did not have a growth advantage over other cells. Next we analyzed patient samples enrolled in the phase 1 clinical trial (NCT02319369) for the MDM2 inhibitor milademetan (DS-3032b; Daiichi-Sankyo) in relapsed/refractory AML or high-risk MDS patients. Fifty seven patients were treated with single agent milademetan in the study. All but one patient had wild-type TP53 as determined by NGS at baseline. One patient (1.8%) had a TP53 p.R213* mutation at baseline with V