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S. Demir et al.
We also addressed whether induction of oxidative stress would increase the antileukemia activity of APR-246. In contrast to clearly increased cell death upon treatment with APR-246 together with the DNA-damaging agent doxorubicin, combining APR-246 with the ROS inducers 3-morpholinosydnonimine and tert-butyl hydroxyperox- ide did not lead to clearly increased cell death (Online Supplementary Figure S7). Thus, APR-246 effectively syner- gizes with doxorubicin and re-sensitizes TP53mut ALL to DNA-damage-induced cell death, while additional ROS induction did not increase APR-246-mediated leukemia cell death.
Preclinical antileukemia activity of APR-246 and in vivo synergy with genotoxic therapy
Based on our findings, we investigated the antileukemia activity of APR-246 in a preclinical setting in vivo. Mice were transplanted with the TP53mut primograft (TP53mut-4; R175H). Upon manifestation of leukemia, as indicated by 5% or more human CD19+ ALL cells in the recipients’ peripheral blood, mice were treated with APR- 246 (25, 50 or 100 mg/kg) or vehicle until control-treated animals showed signs of leukemia-related morbidity (3 weeks, days 1-5) (Figure 6A). Upon APR-246 treatment, a clear dose-dependent reduction of leukemia loads was observed in all three organ compartments: spleen, bone marrow and central nervous system (Figure 6B-D). Moreover, in leukemia cells isolated from these APR-246- treated animals, dose-dependent increases in mutant p53 with wildtype conformation and expression of PUMA and p21 were detected (Figure 6E, F), indicating restoration of wildtype p53 conformation and function in vivo.
Furthermore, APR-246 demonstrated strong in vivo antileukemia activity in another TP53mut ALL primograft sample (TP53mut-1; R248Q/R213X) leading to significant- ly reduced leukemia loads in the spleen, bone marrow and central nervous system upon therapy of leukemia-bearing recipients (Figure 6G-I).
We also addressed the effects of APR-246 in combina- tion with doxorubicin in vivo. Recipients with manifest ALL (TP53mut-1; R248Q/R213X; 5% or more human ALL cells in the peripheral blood) were treated with APR-246, doxorubicin, or the combination of both for 3 weeks. After treatment, the animals were followed up and the time until onset of ALL-related morbidity was analyzed for each animal (Figure 6J). Upon sacrifice, high loads of human ALL were detected in the spleen and bone marrow of all recipients, confirming recurrence of manifest leukemia at clinical onset. Importantly, in addition to clear antileukemia activity as a single agent, leading to increased post-treatment survival (P<0.0001), APR-246 synergized strongly with doxorubicin and re-sensitized TP53mut ALL to genotoxic therapy in vivo, resulting in sig- nificantly prolonged survival as compared to APR-246 alone (P=0.0005) (Figure 6K). In all treatment experiments, application of APR-246 was well tolerated and no side effects were observed in the recipients.
Taken together, our findings in ALL carrying TP53 mis- sense mutations in the DNA-binding domain, which lead to accumulation of dysfunctional p53, indicate that target- ing mutant p53 with APR-246 results in refolding of mutant p53 into its native wildtype conformation, induc- tion of p53 transcriptional targets, involvement of oxida- tive stress, induction of apoptosis, sensitization to DNA damage and, most importantly, preclinical antileukemia
activity with significant reduction of leukemia loads, re- sensitization to genotoxic therapy and clearly prolonged survival in vivo. Thus, application of APR-246 can provide an effective strategy for directed therapeutic intervention in the high-risk subtype of TP53mut BCP-ALL.
Discussion
Investigating a large cohort of 62 patient-derived BCP- ALL samples, all identified TP53mut cases showed mis- sense mutations leading to alterations in the DNA-binding domain of p53, high levels of p53 protein and insensitivity to doxorubicin. Interestingly, APR-246 demonstrated robust antileukemia activity in these cases, including induction of apoptosis, effective reduction of leukemia loads, and sensitization to doxorubicin in an in vivo model of TP53mut ALL. Both in vitro and in vivo experiments showed that treatment with APR-246 led to restored con- formation and activation of mutant p53, and induction of transcriptional targets.
Alterations in TP53 have been described in diverse can- cers at high frequencies of up to 95%.7 In our cohort, TP53 mutations were identified in four out of 62 cases (6.5%), in line with reported rates in ALL of 6-16%.14-16,18 All muta- tions identified in the primograft and cell line samples were missense mutations localized in the DNA-binding domain, with additional loss of the second allele in some of the cases, consistent with mutational patterns reported throughout different cancer types.10,43 One TP53mut sam- ple showed hypodiploidy, in line with reported associa- tions of hypodiploidy with germline and somatic TP53 mutations.19-21
Mutated dysfunctional p53 results in resistance to ther- apy-induced DNA damage48 and poor patient outcome.14,16- 18,22 Correspondingly, increased numbers of TP53 alter- ations are seen at ALL relapse16 and all TP53mut leukemias were insensitive to the DNA-damaging agent doxoru- bicin. Importantly, these TP53mut leukemias were sensi- tive to APR-246, likely by reactivation of high levels of dysfunctional p53 accumulated in the cells. Most impor- tantly, in line with reports in ovarian cancer,32,39 APR-246 clearly synergized with doxorubicin in vitro, ex vivo and in vivo, re-sensitizing initially resistant TP53mut ALL to DNA damage. Therefore, combining functional p53 restoration with genotoxic therapies triggering the p53-mediated DNA-damage response would be the rationale to apply APR-246 together with doxorubicin, a classical DNA- damage-inducing agent used in ALL treatment regimens. Importantly, a favorable pharmacological profile and anti- tumor effects were observed upon first clinical use of APR-246 in patients with refractory cancers40 and APR-246 is being tested in combination with anticancer agents, including doxorubicin, in ongoing phase II trials (ClinicalTrials.gov).41
We addressed the molecular mechanism of action of APR-246 and demonstrated restoration of p53 wildtype conformation, p53 pathway activation with induction of downstream transcriptional targets, and a contribution of oxidative stress leading to apoptosis of TP53mut BCP-ALL cells. Importantly, this antileukemia effect was also observed in vivo in TP53mut ALL, but not in TP53wt ALL, upon p53 knockdown or in a patient’s sample with a splice site mutation and loss of p53 protein expression. High levels of misfolded mutant p53 were described to be
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haematologica | 2020; 105(1)