Idasanutlin overcomes the deficiency of T-PLL cells to evoke P53 responses under cytotoxic stress by facilitating the liberation of P53 from its MDM2-bound state. (A) P53^Ser15 phosphorylation upon in vitro treatment with cladribine (100 nM; 24 hours) comparing primary T-PLL cells (n = 9 cases) with CD3⁺ pan–T-cell isolates from 4 age-matched healthy donors. P53 phospho-activation is diminished in 4 of 9 primary T-PLL cases. (B) P53 coimmunoprecipitation was conducted using primary T-PLL cells (n = 2 cases) treated in vitro with idasanutlin and cladribine (each 10 nM). Densitometric quantification involved ratios to the untreated condition based on the expression of the housekeeping protein β-actin. Reduction in the interaction between P53 and MDM2 was observed only in conditions including idasanutlin. (C) P53 phosphorylation/acetylation, PARP cleavage, and Histone 3 acetylation in primary T-PLL cells upon treatment with idasanutlin, cladribine, venetoclax, romidepsin, and respective paired combinations (10 nM; 24 hours; n = 3 cases; exemplary immunoblot shown). Densitometric quantification of phosphorylation was done relative to the untreated condition based on the expression of β-actin. By combining idasanutlin with cladribine, a higher phospho-p53^Ser15 induction was achieved than with the single substances. Acetylation of Histone 3 was observed in all romidepsin conditions, but only the combination of cladribine and romidepsin induced detectable p53^Lys382 acetylation. Venetoclax + romidepsin did not induce p53^Ser15 phosphorylation, but p53 stabilization, and showed the strongest PARP cleavage. (D) Relative cytochrome-c release upon treatment with idasanutlin (red, 0.5 μM), cladribine (green, 0.5 μM), and the combination of both (light-blue, 0.25 μM each; dark blue, 0.5 μM each) in primary T-PLL cells (n = 10 cases). Cytochrome-c release was measured by flow cytometry (mean with SEM; 1-way ANOVA, Bonferroni correction for multiple comparisons, ∗P < .05; ∗∗P < .01; ∗∗∗P<.001). Cytochrome-c release was significantly elevated when cells were treated with the combination of cladribine with idasanutlin. (E-F) ATAC-seq was performed on ex vivo–treated primary T-PLL cells (n = 6 cases; cladribine, idasanutlin, and combination; each 10 nM; 48 and 72 hours). (E) Venn diagram showing differentially accessible regions (FDR < 0.05) upon idasanutlin and the combination treatment after 48 hours (left) and 72 hours (right). Promoter regions of PHLDA3 (48 hours, idasanutlin-only treatment FDR = 0.003; combination treatment FDR = 0.0007; 72 hours, combination treatment FDR = 0.03) and BBC3 (72 hours combination treatment FDR = 0.03) were found to be more accessible preferentially in treated cells. Graphical representation depicting the sliding window sum of counts per million (CPM) for the specified regions of PHLDA3 (left) and BBC3 (right), upon treatment with cladribine, idasanutlin, and their combination for 72 hours (each 10 nM). The ATAC-seq protocol outlined a 500-nucleotide span around the respective peaks within the promoters of PHLDA3 and BBC3, with an additional 400 nucleotides extended to exhibit baseline levels before and after the peak region. Despite notable interindividual variations, both PHLDA3 and BBC3 exhibited significantly enhanced accessibility following treatment with idasanutlin and cladribine. treatment. See supplemental Figure 8 for P53 and MDM2 co-immunoprecipitations in Molt4 and Mac2a T-cell tumor lines, quantification of the immunoblot of panel C, and ATAC-seq data on PHLDA3 accessibility after 48 hours of treatment. Cladri, cladribine; combi, combination; FDR, false discovery rate; Ida, idasanutlin; Romi, romidepsin; Veneto, venetoclax.