Cladribine and inhibitors of MDM2 lead to activation of p53 and subsequent cell death in primary T-PLL cells. (A) Bar chart showing the viability of primary T-PLL cells after treatment with the alkylating agents 4-OOH-cyclophosphamide (n = 10) and bendamustine (n = 24) for 48 hours and the purine analogs pentostatin (n = 19), nelarabine (n = 14), fludarabine (n = 10), clofarabine (n = 10), and cladribine (n = 16) for 72 hours (means with SEM). After thawing, 1 × 106 T-PLL cells/mL were cultured in the presence of 1 μM or 10 μM of indicated substances. Cladribine was most effective in reducing T-PLL cell viability (eg, viability at 1 μM = 24.4% ± 7.7%). (B) P53 phosphorylation and PARP cleavage upon treatment with cladribine, fludarabine, nelarabine, bendamustine, 4-OOH-cyclophosphamide (all at LD50 concentrations), pentostatin, and clofarabine (10 μM each) for 24 hours. An exemplary immunoblot of 1 primary T-PLL sample of 3 cases is presented. Densitometric quantification of phosphorylation was performed relative to the untreated condition based on the expression of the housekeeping protein β-actin; quantification of cPARP included correction for expression of the uncleaved protein. Phospho-activation of P53 was highest after exposure to cladribine, fludarabine, and clofarabine. (C) Bar chart showing the viability of primary T-PLL cells after treatment with the direct p53 reactivator Prima-1MET and the MDM2 inhibitors serdemetan, MI-773, KRT-232, APG-115, idasanutlin, and siremadlin (mean with SEM). After thawing, 1 × 106 T-PLL cells per mL were treated with 0.1 μM and 1 μM of indicated substances for 48 hours. Idasanutlin (viability at 0.1 μM = 67.8% ± 4.1%) and siremadlin (viability at 0.1 μM = 44.3% ± 4.6%) were most effective. (D) P53 phosphorylation, PARP cleavage, and MDM2 protein expression upon treatment with the direct p53 reactivator Prima-1MET and the MDM2 inhibitors idasanutlin and siremadlin using LD50 concentrations. An exemplary immunoblot of 1 primary T-PLL sample of 3 cases is presented. Densitometric quantifications as in panel B. Induction of phospho-p53, cleavage of PARP, and expression of MDM2 was highest after exposure to idasanutlin and siremadlin. In the experiments of panel A-D, apoptotic cells were quantified by flow cytometric analysis of differential annexin V (AnnV)/7AAD staining. (E) A total of 38 T-cell leukemia/lymphoma lines, representing 7 entities, including anaplastic large cell lymphoma (ALCL; yellow), cutaneous T-cell lymphoma (CTCL; light gray), γδ-T-cell (GD; dark gray), NK-cell (dark green), peripheral T-cell lymphoma not-otherwise-specified (PTCL-NOS; brown), T-cell acute lymphoblastic leukemia (T-ALL; orange), and T-large granular lymphocytic leukemia (T-LGLL; light green) were subjected to single-agent cell-viability testing. The screening included multiple drugs (rows) assigned to specific classes (see corresponding color codes) such as 4 MDM2 inhibitors and others. Incubation of 1 × 105 cells per mL in 25 μL medium ± drug was done for 72 hours. Cell viability was assessed by CTG luminescent assays. Cell line–specific DSSs (red, higher efficacy; blue, lower) were calculated. Unsupervised hierarchical clustering revealed 2 distinct groups of cell lines, predominantly aligned with their TP53 mutation status, implicating a link between TP53 mutations and resistance to MDM2 inhibition. See supplemental Figure 4 for quantification of immunoblots on T-PLL cases, as well as for data on phosphorylation of p53 and PARP cleavage in TP53 WT vs mutated cell lines upon idasanutlin treatment. BTKi, Bruton tyrosine kinase inhibitor; ctrl, control; Cycloph., cyclophosphamide; NK, natural killer; rel., relative; WT, wild-type.
