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Targeting mutant p53 in ALL
TP53mut ALL but no effect in TP53wt cells (Figure 4G-J and Online Supplementary Figure S6). Together with our observations on APR-246 insensitivity in TP53wt ALL (Figure 1E, F) and the absence of p53 transcriptional target expression upon APR-246 treatment in TP53wt ALL (Figure 3D, E), these findings indicate that the activity of APR-246 is associated with the presence of mutant p53. Accordingly, distinct sensitivities to APR-246 were found in four primary ALL samples obtained from patients with therapy-resistant disease or relapse carrying different TP53 mutations (Figure 4L, Table 2). Robust dose-depen-
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dent cell death induction was observed in leukemia cells from patients 2, 3, and 4 carrying missense mutations resulting in expression of mutant p53 (Figure 4L, N-P), whereas APR-246 did not induce cell death in ALL cells of patient 1 carrying a hemizygous splice site mutation with- out detectable expression of p53 protein (Figure 4L, M).
APR-246 re-sensitizes TP53-mutated acute lymphoblastic leukemia to doxorubicin
TP53mut cancer cells show resistance to DNA damage. Therefore, we analyzed whether reactivation of mutant
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Figure 3. Conformational and functional restoration of mutant p53 by APR-246. (A) Increased levels of p53 with wildtype conformation in TP53mut ALL (KOPN-8, mutation R248Q; RS4;11, mutation I254T) upon exposure to APR-246 (5 mM, 24 h). Immunoprecipitation (IP, anti-wt p53 specific antibody PAb1620) and western blot analysis (WB, anti-p53 anti- body DO-7, light chain-specific goat anti- mouse peroxidase conjugated binding protein), GAPDH expression in input lysates and absence in precipitates, NALM-6 serves as a wildtype p53 positive control. (B-E) Expression of p53 transcrip- tional targets PUMA, p21 and NOXA in (B, C) TP53mut ALL upon APR-246 treatment and in (D, E) TP53wt ALL upon doxoru- bicin treatment. Western blot, exposure to solvent (CTRL), doxorubicin (DOX, 15 ng/mL), or APR-246 (APR, 5 mM) for the indicated times, with GAPDH as a loading control. The results of one representative out of two independent experiments are shown. See also Online Supplementary Figures S2 and S3.
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