To target the untargetable: elucidation of synergy of APR-246 and azacitidine in TP53 mutant myelodysplastic syndromes and acute myeloid leukemia

Mutations of the tumor suppressor gene TP53 represent a common mutation in myeloid malignancies, occurring in 10-20% of patients with de novo myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with profound negative impact on outcomes and a median overall survival (OS) of 6-12 months. 3 Critically, the clonal burden of TP53, that is the variant allele frequency (VAF) and/or allelic state of TP53, is intimately tied with the clinical trajectory of these patients and is a robust, independent predictor of survival. Given the poor OS and lack of therapeutic options for TP53 mutant MDS/AML patients, a number of novel agents are being investigated in this patient group. Of these, APR246 has evoked considerable excitement based on its robust clinical efficacy in combination with azacitidine in TP53 mutant MDS/AML patients. In this issue of Haematologica, Maslah et al. describe compelling preclinical synergy of APR-246 in combination with azacitidine in TP53 mutated MDS and AML and, more importantly, identify a novel molecular mechanism underlying the observed synergy. Recent elegant work has definitively identified that TP53 missense mutations in myeloid malignancies result in a dominant-negative effect without evidence of neomorphic gain-of-function activities, ultimately leading to a selection advantage when exposed to DNA damage. Thus, restoring wild-type function in TP53 mutant clones would be of profound beneficial impact. APR-246, a methylated PRIMA-1 analog, is a novel, first-in-class, small molecule that selectively induces apoptosis in TP53 mutant cancer cells. Mechanistically, APR-246 is spontaneously converted into the active species methylene quinuclidinone (MQ), which is able to covalently bind to cysteine residues in mutant p53 thereby producing thermodynamic stabilization of the protein and shifting equilibrium toward a functional conformation. APR-246 monotherapy was originally investigated in a phase I trial including AML patients with clinical activity and correlative data identifying activation of p53-dependent pathways. Maslah et al. identified in TP53 mutant cell lines, in vivo models, and primary patient samples that the combination of APR-246 and azacitidine results in a synergistic pro-apoptotic effect as well as a dramatic reduction in cell proliferation via cell cycle arrest (Figure 1). As the majority of TP53 mutations are missense and located in the DNA binding domain, synergy experiments were performed with the SKM1 cell line, which harbors a homozygous hotspot mutation of TP53 (p. R248Q), and thus is an appropriate representation of clinical disease. Combination therapy of APR-246 and azacitidine resulted in a doubling of apoptotic cells versus azacitidine alone as well as 83% of cells undergoing cell cycle arrest in G0/G1. This synergistic effect was confirmed in a xenotransplantation model where combination therapy resulted in a pronounced inhibition of disease progression which occurred early and was durable. Subsequently, the authors interrogated differential gene expression profiles of SKM1 cells treated with either drug alone versus the combination of APR-246 and azacitidine. As expected, Gene Set Enrichment Analysis (GSEA) and DAVID analyses of APR246 treated cells showed robust induction of p53-target genes including CDKN1A, CASP1, BAX and FAS, which was confirmed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR), resulting in activation of an early apoptotic program. Furthermore, GSEA analysis of “synergistic only” genes (i.e. genes differentially expressed only with combination treatment) drug combinations that can simultaneously target the undifferentiated leukemic cell populations as well as the more mature myeloid lineages (especially monocytes) may represent a powerful way to prioritize the most promising drug combinations for pre-clinical study and for clinical development. Clearly, this report demonstrates just the beginning of utility of this exciting drug screening platform.