Background Patients with relapsed/refractory multiple myeloma (RRMM) and high-risk genetic abnormalities such as del(17p) and TP53 mutation have poor response to standard therapies and shorter survival compared to patients without these aberrations. Here, we investigated the activity and mechanism of action of peptide-drug conjugate melphalan flufenamide (melflufen) in TP53 wild type (TP53wt) and mutant (TP53mut) myeloma models and assessed the efficacy of melflufen in patients with del(17p) and/or TP53 mutation. Methods We evaluated melflufen activity ex vivo in 24 myeloma bone marrow (BM) samples and explored indicators of response from single cell RNA sequencing (scRNAseq) profiles. The efficacy of melflufen vs. control treatments was further investigated in TP53-/- and parental TP53wt myeloma cell lines. DNA damage, apoptosis kinetics, mitochondrial function, plus transcriptomic and metabolic data were analyzed to understand the mechanisms responsible for melflufen activity in the absence of p53. Patient outcome data from the OCEAN phase III clinical trial (NCT03151811), which investigated the clinical activity of melflufen in RRMM, were statistically analyzed to assess the impact of del(17p) and TP53 mutation on clinical response. Results BM plasma cell (PC) response to melflufen was independent of TP53 mutation status, with melflufen active in del(17p), TP53mut, and TP53wt samples. Differential analysis of scRNAseq data demonstrated that melflufen sensitive PCs had lower expression of p53 target genes and higher expression of genes associated with DNA damage repair and cell cycle checkpoints. Analysis of TP53-/- and TP53wt cell lines showed superior efficacy of melflufen in comparison to melphalan or cyclophosphamide. In the presence and absence of functional p53, melflufen robustly induced apoptosis, DNA damage, and mitochondrial dysfunction. In TP53-/- cells, melflufen treatment led to distinct changes in expression of genes associated with cell cycle checkpoint and apoptosis, which were not observed with melphalan treatment. Notably, post-hoc analysis of the OCEAN trial del(17p) patient population demonstrated favorable progression free survival in the del(17p) subgroup treated with melflufen plus dexamethasone compared to the pomalidomide plus dexamethasone arm. Conclusions Our insights into the molecular mechanisms of melflufen activity in TP53mut myeloma support its clinical efficacy and application in the del(17p) and TP53mut patient population.
