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.31 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.74 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.8 Of these, APR-246 has evoked considerable excitement based on its robust clinical efficacy in combination with azacitidine in TP53 mutant MDS/AML patients.109 In this issue of Haematologica,11 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.12 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 thermo dynamic stabilization of the protein and shifting equilibrium toward a functional conformation.1413 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.1615
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.17 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 APR-246 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) identified activation of p53 pathway, induction of apoptosis, and downregulation of MYC expression, thus functionally demonstrating restoration of wild-type p53 function. Notably, transcriptome analysis with confirmation by RT-qPCR also identified a novel synergistic mechanism of FLT3 pathway downregulation. Importantly, the inhibition of cell proliferation with combination therapy could be overcome in a dose-dependent fashion in the presence of FLT3 ligand, highlighting a novel therapeutic mechanism of APR-246 that could potentially be exploited in combination with FLT3 inhibitors in future clinical study.
Of importance, synergy was most robust in the presence of TP53 missense mutations where there is accumulation of misfolded p53 protein, strongly supporting the primary mechanism of APR-246. However, APR-246 also has p53-independent function via MQ binding to thioredoxin reductase and glutathione, leading to depletion of glutathione and accumulation of reactive oxygen species (ROS), which can feed forward p53 activation (Figure 1).1918 Indeed, the authors also show synergy in TP53 knockout mutant cell lines where there is absence of p53, albeit with less synergy than in the missense mutant model. Accordingly, there was significant enrichment of ROS-induced genes with APR-246 treatment. The authors also show data whereby both cell proliferation and clonogenic capacity were strongly inhibited, both in the presence and absence of mutant p53 protein.
Perhaps the most compelling data regarding the synergy of APR-246 and azacitidine originates from the clinical activity in TP53 mutant MDS/AML patients, where recent data report an overall and complete remission rate of 87% and 53%, respectively (clinicaltrials.gov identifier: NCT03072043).9 Similarly, preliminary results from a phase II study of APR-246 and azacitidine by the Groupe Francophone des Myélodysplasies (clinicaltrials.gov identifier: NCT03588078) showed comparable response rates.10 Accordingly, the US Food and Drug Administration has recently granted breakthrough therapy designation for the treatment of patients with TP53 mutant MDS with the combination of APR-246 and azacitidine and the randomized phase III study of APR-246 and azacitidine versus azacitidine is ongoing in MDS patients (clinicaltrials.gov identifier: NCT03745716). As TP53 mutations are strong drivers of negative outcomes in multiple hematologic malignancies, as exemplified by relapsed pediatric acute lymphoblastic leukemia, APR-246 may likely have more broad clinical implications including synergy with traditional cytotoxic agents, as has been recently described.20 Together, shedding light on the synergistic mechanisms underlying APR-246 and azacitidine therapy as presented in this study are critical to continue to advance this novel therapeutic option for patients with the poorest outcomes to traditional treatments.
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