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S. Demir et al.
associated with APR-246 sensitivity in cancer cell lines,38,49,50 consistent with our observation of high APR- 246 activity in TP53mut ALL with high p53 expression. However, evaluation of larger cohorts of patients together with outcome data would be required to explore the value of the level of expression of p53 as an indicator of APR- 246 responsiveness in TP53mut ALL.
APR-246 activity has been reported to be mediated independently of p53 by induction of oxidative stress in other types of cancer, including acute myeloid leukemia and multiple myeloma.30-33 However, we only observed cell death in TP53mut ALL, although TP53mut and TP53wt ALL showed no differences in induction of and sensitivity to oxidative stress. Interestingly, APR-246 activity in TP53mut ALL was partially inhibited by ROS neutralization. This suggests that induction of oxidative stress contributes to APR-246-mediated cell death in ALL, in line with reports on a dual mode of action of APR-246,30- 32 which might vary between tumor types and cellular con- text.
The presence of p53 in a mutated, dysfunctional form, as typically is the case for missense mutations in the DNA-binding core domain, enables binding of the active moiety of APR-246, leading to activity in BCP-ALL.11,28 This is of clinical relevance, since the majority of TP53 mutations in BCP-ALL are missense hot spot mutations in the DNA-binding domain15,43 resulting in accumulation of misfolded p53 protein, which is targeted by APR-246. However, the precise mechanism of activity on DNA con- tact mutations is not yet known. Importantly, we and oth- ers 27 have demonstrated antitumor activity on structural and contact mutants including clear preclinical
antileukemia activity on TP53mut ALL carrying either a structure (R175H) or contact (R248Q) mutation.
Taken together, our study shows that the small molecule APR-246 exhibits profound antileukemia activity in TP53mut BCP-ALL, targeting non-functional mutant p53 resulting from missense mutations in the DNA-binding domain of TP53, the most frequent mutation type reported throughout different malignancies. Mechanistically, we showed that APR-246 led to restoration of p53’s wildtype conformation, pathway activation with expression of tran- scriptional targets and induction of apoptosis in TP53mut ALL. Moreover, we found a clear synergism between APR- 246 and doxorubicin treatment, strongly suggesting that the combination of p53 reactivation and DNA-damage induction could be an effective antileukemia strategy for BCP-ALL patients with TP53 missense mutations. Hence, targeting mutant p53 appears to be a promising, directed treatment for this high-risk subgroup of TP53mut ALL.
Acknowledgments
The authors would like to thank Aprea Therapeutics (Stockholm, Sweden) for kindly providing APR-246 for the study, S. Volk and S. Essig for excellent technical assistance, the Ulm University Sorting and Animal Facilities and Pharmacy of the Ulm University Medical Center, and the INFORM study group. The authors would also like to thank the International Graduate School in Molecular Medicine Ulm (SD, EB), Madeleine-Schickedanz- Stiftung and “Förderverein für Krebskranke Kinder Tübingen” (ME, RH), Swedish Research Council and Swedish Childhood Cancer Society (GS), EU COST Action CA16223 (GtK), the German Research Foundation, SFB 1074 B6 (LHM, KMD) and B1 (SS) for supporting the work.
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