Page 82 - Haematologica - Vol. 105 n. 6 - June 2020

N. Maslah et al.
shown to be a strong predictor of poor overall survival.12 Mutations of p53 proteins generally result in a loss of their normal functions (including cell cycle and apoptosis con- trol), but can also result in a deleterious “gain of function”. In addition, at least in case of complex karyotypes, TP53- mutated MDS/AML generally acquire del17p, i.e. loss of the remaining TP53 allele. TP53 mutations correlate with poor overall outcomes in MDS and AML, independent- ly4,5,13 or not independently14 of a complex karyotype. TP53- mutated MDS/AML are associated with resistance to treat- ment, including anthracycline + aracytine combinations, low-dose cytarabine,14 and allogeneic bone marrow trans- plantation (BMT),15 while the hypomethylating agents (HMA) 5-azacitidine (AZA)7,15 and decitabine16 yield some- what better, albeit transient, results. For this reason, HMA are generally considered to be the first-line treatment of MDS/AML with a TP53 mutation.
As with many other types of cancer, mutated p53 pro- tein, therefore, appears to be one of the most important therapeutic targets in MDS and AML, and restoration of its biological function could prove to be highly beneficial. PRIMA-1Met (APR-246, APR) is a methylated derivative of PRIMA-1, a compound that induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53.17 Mechanistically, APR is a prodrug that forms an active moiety that cova- lently binds to thiol groups of the core domain of mutated p53 protein,18 thereby resulting in a structural change that restores its active conformation. APR induces dose-depen- dent apoptosis in various tumor models, mostly in combi- nation with other drugs.19-28 A phase I/II clinical trial com- bining AZA and APR in TP53-mutated MDS/AML is ongoing at US centers and at our center.29-31
In this study, we show that APR is efficient on its own, while it also synergizes with AZA in TP53-mutated MDS/AML cell lines and in TP53-mutated primary cells from MDS/AML patients. We also identified a functional pathway involved in the synergy between these two drugs.
Methods
Reagents and drugs
APR was provided by Aprea Therapeutics AB, Stockholm, Sweden, and kept frozen (-20°C) as a stock solution. AZA was provided by Celgene Corporation (Summit, NJ, USA) and was kept frozen at -80°C as a stock solution. As previously described,32 AZA was added once at day 0 of cell culture and APR was used in the same manner. FLT3 ligand (FLT3-L) was from PeproTech (Neuilly-sur-Seine, France).
Cell lines and primary cells
The MDS cell line SKM1 (kindly provided by Thomas Cluzeau, University Hospital of Nice, France) is an MDS-derived cell line that has undergone detailed characterization in terms of its pheno- type and genotype.33 We confirmed the presence of a homozygous mutation of TP53 (p.R248Q). SKM1 cells were grown in RPMI 1640 medium (GIBCO; Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (GIBCO), 1% Penicillin/Streptomycin (P/S), and 1% GlutaMAX. All of the other leukemic cell lines (K562, KG1a, THP-1, and HL60) were cultured under the same conditions. Cells in the logarithmic phase of growth that had been seeded at a density of 2x105/mL were used for all of the experiments.
Primary cells from MDS and AML patients with complex kary- otypes, del5q, or known or suspected to have mutated TP53 were provided by the Service d‘Hématologie of the Hôpital Saint-Louis, Paris, France, after informed consent had been received. Finally, primary cells were isolated from healthy blood donors as controls. This study was approved by the Groupe Francophone des Myélodysplasies (GFM) review board.
Cytotoxicity assay
The following concentrations were used for each drug: APR from 10-8 M to 10-5 M and AZA from 10-8 M to 10-5 M. Proliferating cells were plated, incubated for three days, and then counted. Experiments were performed at least in triplicate. FLT3-ligand was added at final concentrations varying from 0.1 to 100 ng/mL for specific experiments.
Clonogenic assays
Due to partial blastic infiltration in the majority of samples, as generally reported in MDS/AML with a complex karyotype and TP53 mutation,9 liquid cultures were not consistently successful. We used semi-solid cultures to test for the impact of drugs on pro- genitor clonogenicity. Briefly, 3x105 peripheral blood mononuclear cells (PBMC) or 1x105 bone marrow-derived mast cells (BMMC) were cultured in cytokine-containing methylcellulose (MethoCultTM, 84434, STEMCELL Technologies) in the presence of the IC50 of each drug (0.5 μM APR or 3 μM AZA) on their own or in combination. After 14 days at 37°C, the erythroid and myeloid colonies were counted. For patients with TP53 mutations, isolated colonies were picked, and the DNA was extracted and Sanger sequenced to determine the TP53 genotype at the single progeni- tor level.
Statistical analysis
All of the results are expressed as means±standard deviation (SD). All of the single-parameter measurement comparisons were determined using the Mann-Whitney test (PRISM 18964 software, GraphPad, La Jolla, CA, USA). All tests were two-sided; P<0.05 was considered statistically significant. Combination indices were calculated by CompuSyn software according to the Chou-Talalay method for drug synergy studies.
Results
APR inhibits the proliferation of myeloid cell lines
To evaluate the efficacy of APR-mediated growth inhi- bition of MDS/AML cells, we tested increasing doses of the drug on a series of MDS/AML cell lines with mutant forms of the TP53 gene (i.e. SKM1, K562, KG1a, THP-1, and HL60 cells). We first verified that all of these cell lines harbor TP53 mutations. The SKM1 cell line derived from an MDS patient carries the recurrent homozygous TP53 mutation p.R248Q (c.743G>A). The four other cell lines carry a frameshift or a splicing mutation (K562: c.406dupC, HL60: c.1_1182del1182, KG1a: c.672+1G>A, or THP-1: c.520_545del26). The p53 protein was detectable in the SKM1 cell line only (Online Supplementary Figure S1). Treatment with APR led to a pronounced and dose-dependent reduction in cell proliferation of all of the cell lines tested (Figure 1A), with 50% inhibitory concen- trations (IC50) that were in the micromolar range (Figure 1B). Of note, the IC50 was at least 2-fold lower for the SKM1 cell line compared to the other AML lines (Figure 1B and Online Supplementary Table S1).
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