PPM1D mutations confer resistance to venetoclax in pre-leukemic and AML populations

Abstract

Abstract BACKGROUNDVenetoclax (VEN) combined with lower-intensity therapies are routinely used as first-line therapy for older AML patients (pts) ineligible for intensive chemotherapy (IC). Several mechanisms of primary or acquired resistance have been described, involving mutations in TP53, BAX, FLT3, N/KRAS or PTPN11 (DiNardo and Tiong, Blood 2020; Garciaz, Cancers 2024). Protein phosphatase Mg2+/Mn2+ 1D (PPM1D) frameshift or nonsense variants in exon 6 cause protein truncation and loss of a C-terminal degradation domain, enhancing capacity to stabilize MDM2 or mediate TP53 dephosphorylation, leading to impaired TP53 function and chemoresistance (Husby, BJH 2021). Although PPM1D variants are frequently observed in patients with therapy-related myeloid neoplasms, the fate of PPM1D variants after VEN exposure remains unexplored. Here, we report PPM1D variants (PPM1Dmut) emerging after VEN therapy and shed light on its role in mediating VEN resistance. METHODSWe analyzed clinical and genomic data from pts with AML treated in France and Australia. Multiomic single-cell DNA+protein studies (Mission Bio) were performed on selected bone marrow (BM) samples. MOLM13 and MV4-11 cell lines were CRISPR/Cas9 modified to truncate PPM1D at exon 6 or delete TP53. Competitive co-culture assays were used to assess clonal selection under VEN or chemotherapy exposure. RESULTSWe first analyzed 72 AML pts treated with low-dose cytarabine + VEN, who developed either primary (refractory) or secondary resistance (relapse). As previously reported, we identified mutations in TP53, RAS or BAX. Interestingly, we also identified 7 pts (10%) with persistence or emergence of PPM1Dmut, including 5 that co-occurred with TP53mut. To characterize the trajectory of PPM1D variants after AML therapy, we examined a cohort of 1,046 newly diagnosed pts and identified 32 cases with PPM1D variants at relapse and/or during follow-up. 21 pts were treated with VEN-based therapy and 11 received intensive or other targeted therapy. PPM1Dmut were frameshift or nonsense in nature and all were found in exon 6, resulting in C-terminal truncation and loss of the degradation motif. Among patients with PPM1Dmut treated with VEN, 29% had TP53 co-mutations, 43% had secondary or therapy-related AML (s/t-AML). Single-cell analysis from 3 AML BM samples harboring both TP53mut and PPM1Dmut showed mutual exclusivity of these two variants at the single-cell level. These studies also showed that PPM1Dmut was present in both progenitor and mature lymphoid populations, suggesting pre-leukemic biology in some cases.In patients with paired samples taken at diagnosis and after treatment, longitudinal NGS analyses showed an absolute rise in VAF ≥ 5% in 50% of the VEN cohort (n=8/16), compared to 0% in the IC cohort (0/8). These findings suggested potential for PPM1Dmut clones to undergo therapeutic selection when exposed to VEN-based therapy.To further examine the impact of PPM1Dmut on VEN sensitivity, we generated CRISPR-Cas9-edited MOLM13 and MV4-11 cell lines carrying truncating PPM1Dmut in exon 6. In long-term co-culture assays, PPM1Dmut cells were competitively enriched, compared to WT cells upon exposure to either chemotherapy (Ara-C or 5-FU) or VEN. Interestingly, co-culture of TP53KO with dual TP53KO/ PPM1Dmut cells showed no competitive advantage for compound TP53KO/PPM1Dmut cells, suggesting the mode of resistance mediated by PPM1Dmut and TP53KO was convergent.To explore therapeutic options, we assessed several VEN-based combinations in cells that harbored PPM1Dmut or TP53KO. VEN combined with either small molecule PPM1D (GSK-2830371) or MDM2 inhibitors (RG-7388) showed promising efficacy against PPM1Dmut, but were ineffective against TP53KO cells. In contrast, VEN + MCL1 inhibitor (S63845) was highly cytotoxic and efficacious across all genotypes tested, including both PPM1Dmut and TP53KOcells. CONCLUSIONWe describe emergent PPM1Dmut clones as a novel mechanism of VEN resistance in AML. These mutations are enriched in s/t-AML and often co-occurred with TP53 mutations, although they appeared mutually exclusive at the clonal level. PPM1Dmutlikely arises in pre-leukemic or leukemic cells selected to expand during VEN exposure. Resistance to VEN mediated by PPM1D abnormalities appeared to be TP53 dependent and alleviated by combining VEN with MCL1 inhibition, which may also have clinical rationale in pts harboring concurrent PPM1D and TP53 mutant disease.