Page 181 - 2020_01-Haematologica-web

Targeting mutant p53 in ALL
affect the structural integrity and DNA-binding ability of p53, leading to accumulation of dysfunctional p53 protein and increased oncogenic potential.10-13
TP53 mutations are found frequently, in up to 95% of carcinomas, typically in older patients.7,8 In ALL, recent studies identified alterations of TP53 in subsets of up to 16%, with higher rates in T-ALL, at relapse, and in elderly patients.14-18 Moreover, more than 90% of ALL cases with a low hypodiploid karyotype (including loss of chromo- some 17) carry somatic TP53 alterations19,20 and TP53 germline mutations confer a high risk for hypodiploid ALL.21 In pediatric ALL, TP53 alterations are associated with poor response to chemotherapy and an inferior out- come, particularly at relapse, identifying TP53-mutant B- cell precursor (BCP)-ALL patients as a high-risk subgroup with a particular need for alternative therapies.14,16-18,22
Different strategies to interfere with the p53 pathway have been evaluated. Inhibition of the interaction of p53 and its negative regulator, mouse double minute 2 (MDM2), leads to sustained p53 transcriptional activity, but requires the presence of wildtype p53.23 Therefore, direct targeting of mutant p53 has been investigated, identifying small molecules that reactivate p53 function.24 In line, anti-tumor activity has been observed in murine lymphoma and liver cancer models upon genetic restora- tion of p53, supporting the principle of p53 reactivation as a therapeutic strategy.25,26 APR-246 (PRIMA-1Met), the structural analog of PRIMA-1 (p53 reactivation and induc- tion of massive apoptosis) is a small molecule, identified in a screen for mutant p53-dependent growth suppression in sarcoma cells, showing activity on both structural and DNA-binding mutants.27 APR-246 is a prodrug that is con- verted into methylene quinuclidinone, which binds cova- lently to the core domain of mutant p53 interacting with thiol groups of cysteines, restoring p53 wildtype confor- mation and function.28,29 In addition, induction of oxida- tive stress has been reported as a second activity of APR- 246, deriving from glutathione depletion, thioredoxin reductase inhibition and other effects.30-33
APR-246 demonstrated preclinical antitumor activity and synergism with DNA-damaging drugs in different cancers32,34-39 and showed very moderate side effect pro- files in a first-in-human phase I/IIa clinical trial in patients with refractory prostate cancer, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma and lymphoma.40 Accordingly, APR-246 is currently being investigated in ovarian and esophageal cancer, myeloid neoplasms and melanoma in phase II clinical trials (ClinicalTrials.gov).41 However, mutant p53 has so far not been addressed as a target for therapeutic intervention in ALL.
In this study, we investigated a large cohort of patient- derived pediatric BCP-ALL primograft samples identify- ing TP53-mutated cases and analyzed the effects of APR- 246 in TP53-mutated (TP53mut) and TP53-wildtype (TP53wt) BCP-ALL. We identified strong and selective antileukemia activity of APR-246 in TP53mut ALL pro- viding the basis to develop personalized therapy regi- mens for this high-risk subgroup of ALL.
Methods
Additional detailed information is provided in the Online Supplementary Data.
Sixty-two patient-derived xenograft samples established by transplantation of patients’ ALL cells onto NOD.CB17-Prkdcscid/J mice42 and six BCP-ALL cell lines were studied. Leukemia samples were obtained from pediatric BCP-ALL patients at diagnosis or relapse upon informed consent from the children and/or their legal guardians in accordance with the institution’s ethical review boards. All animal experiments were approved by the appropriate authority (Regierungspräsidium Tübingen) and carried out follow- ing the national animal welfare guidelines. TP53 mutations were analyzed by denaturing high-performance liquid chromatography and confirmed by Sanger sequencing, 17p deletions were assessed by fluorescence in situ hybridization. Mutation information was matched to the IARC-TP53 database.43 The sensitivity of leukemia samples to doxorubicin, APR-246 (kindly provided by Aprea Therapeutics, Stockholm, Sweden) or the combination was assessed after incubation of ALL cells with increasing drug concen- trations, analyzing cell death by flow-cytometry according to for- ward- and side-scatter criteria. Data from three independent exper- iments performed in triplicate (cell lines) or of one experiment per- formed in triplicate (primografts) were analyzed by t-test, and dif- ferences of half maximal inhibitory concentrations (IC50) titrations by F-test. P values ≤0.05 were considered statistically significant. Synergies of drug combinations were assessed calculating combi- nation indices (CI), indicating strong synergism (CI 0.1-0.3), syner- gism (CI <1), an additive effect (CI=1) or antagonism (CI>1). Apoptosis was analyzed assessing annexin-V-FLUOS positivity and caspase-3 activity. Proteins (p53, PUMA, p21, NOXA, GAPDH) were detected by western blot analysis using the respec- tive antibodies. The wildtype conformation of p53 was detected by immunoprecipitation using a conformation-specific anti-p53 wildtype antibody (PAb1620) followed by western blot analysis with an anti-p53 (total) antibody (DO-7). An immunoglobulin light chain-specific peroxidase conjugated binding protein was used for western blot analyses carried out following immuno-pre- cipitation. Depletion of p53 was achieved by lentiviral shRNA- mediated knockdown or siRNA-mediated downregulation in TP53mut or TP53wt ALL cells. For in vivo treatment, transplanted recipients showing >5% human ALL cells in peripheral blood were randomized and treated (for 3 weeks) with solvent, APR-246 (days 1-5), doxorubicin (day 1), or the combination (APR-246 days 1-5, doxorubicin day 5) and sacrificed at the end of treatment for analy- sis of leukemia loads. For survival analyses, recipients were fol- lowed up after treatment until onset of leukemia-related morbidity and sacrificed. High loads of human ALL cells were detected in bone marrow and spleen in all cases, confirming reoccurrence of manifest leukemia.
Results
Identification of TP53 mutations in B-cell precursor acute lymphoblastic leukemia
We investigated 62 patient-derived pediatric BCP-ALL samples, which were established in our NOD/SCID/huALL xenograft model from patients at diagnosis (n=53) or relapse (n=9). TP53mut cases were identified by denaturing high-performance liquid chromatography and confirmed by Sanger sequencing (exons 4-10). Four TP53mut cases were found, one derived from a patient at second relapse (TP53mut-1) and three at diagnosis (TP53mut-2, -3, -4) (Online Supplementary Table S1). In parallel, we character- ized six BCP-ALL cell lines and identified two TP53mut (RS4;11, KOPN-8) and four TP53wt (MUTZ-5, EU-3, UoCB-6 and NALM-6) lines. All samples carried missense mutations previously described (p53.iarc.fr),15,43 localized
haematologica | 2020; 105(1)
171