Reactivating p53 responses and inhibiting T-PLL inherent survival signals emerge as efficient and selective treatment strategies in T-PLL. (A) Schematic overview showing functional points of attack of established (blue represents alkylating agents, purine analogs, and antibodies) and experimental (red) treatment options in T-PLL. Considering the genomic and transcriptomic landscape of T-PLL cells as well as published data on molecular vulnerabilities, including unbiased drug screens,7,21 8 compounds were chosen for an ex vivo single-agent cell-viability testing: bendamustine (alkylating agent),31,32 cladribine (ribonucleotide reductase inhibitor with additional epigenetic mode of action),33,34 dinaciclib (inhibitor of CDK1/2/5/9),35 ibrutinib (BTK/ITK inhibitor),36,37 idasanutlin (MDM2 inhibitor),21 romidepsin (HDAC1/2 inhibitor),21 ruxolitinib (JAK1/2 inhibitor),38 and venetoclax (BCL2 inhibitor).21,37,39 (B) Single-agent cell-viability testing of 20 primary T-PLL samples: after thawing, 5 × 105 primary T-PLL cells per mL were treated in 25 μL for 72 hours (respective concentrations are presented below the substance name; see also supplemental Table 2). Cell viability was assessed using CellTiter-Glo (CTG) luminescent assay. sDSS was calculated as a normalized version of the area under the dose-response curve. sDSS values were calculated by subtracting the average DSS of PBMCs of 2 age-matched healthy donors from the DSS of the patient samples. Higher sDSS values indicate increased selective sensitivity of the compound. Heat map (left) showing color-coded sDSSs, presented per patient (red, DSS > 0; white, sDSS = 0; blue, sDSS < 0). Box plots (middle/right) showing DSSs/sDSS values of the respective compounds (mean with first and third quartile). Compounds with the highest scores are at the top. (C) Dose-response curves of primary T-PLL cells, together with age-matched, healthy donor–derived PBMCs and CD3+ T cells, treated with increasing concentrations of idasanutlin (lower panel) and cladribine (upper panel). After thawing, 1 × 106 primary cells per mL were treated with indicated concentrations of idasanutlin for 48 hours or cladribine for 72 hours (mean with standard error of the mean [SEM]; 2-way ANOVA, Bonferroni correction for multiple comparisons; ∗∗∗∗P < .0001). (D) Dose-response curves of primary T-PLL cells, with (light-blue) and without (dark-blue) coculturing with NKtert BMSCs, treated with increasing concentrations of idasanutlin (continuous line, n = 15 cases) and cladribine (dotted line, n = 16 cases). After thawing, 1 × 106 primary cells/mL were cocultured with NKtert cells and subsequently treated with indicated concentrations of idasanutlin (48 hours) or cladribine (72 hours; mean with SEM; 2-way ANOVA, Bonferroni correction for multiple comparisons; ∗∗∗∗P < .0001). Phase-contrast microscope image (background) showing T-PLL cells in contact with NKtert BMSCs after 72 hours of incubation (original magnification, 220×). See supplemental Figure 1 for the respective DSSs, presented per patient, and the effect of indicated drugs on NKtert cell viability. AnnV, annexin V; ctrl, control; neg., negative; rel., relative.
Figure 1.

Reactivating p53 responses and inhibiting T-PLL inherent survival signals emerge as efficient and selective treatment strategies in T-PLL. (A) Schematic overview showing functional points of attack of established (blue represents alkylating agents, purine analogs, and antibodies) and experimental (red) treatment options in T-PLL. Considering the genomic and transcriptomic landscape of T-PLL cells as well as published data on molecular vulnerabilities, including unbiased drug screens,7,21 8 compounds were chosen for an ex vivo single-agent cell-viability testing: bendamustine (alkylating agent),31,32 cladribine (ribonucleotide reductase inhibitor with additional epigenetic mode of action),33,34 dinaciclib (inhibitor of CDK1/2/5/9),35 ibrutinib (BTK/ITK inhibitor),36,37 idasanutlin (MDM2 inhibitor),21 romidepsin (HDAC1/2 inhibitor),21 ruxolitinib (JAK1/2 inhibitor),38 and venetoclax (BCL2 inhibitor).21,37,39 (B) Single-agent cell-viability testing of 20 primary T-PLL samples: after thawing, 5 × 105 primary T-PLL cells per mL were treated in 25 μL for 72 hours (respective concentrations are presented below the substance name; see also supplemental Table 2). Cell viability was assessed using CellTiter-Glo (CTG) luminescent assay. sDSS was calculated as a normalized version of the area under the dose-response curve. sDSS values were calculated by subtracting the average DSS of PBMCs of 2 age-matched healthy donors from the DSS of the patient samples. Higher sDSS values indicate increased selective sensitivity of the compound. Heat map (left) showing color-coded sDSSs, presented per patient (red, DSS > 0; white, sDSS = 0; blue, sDSS < 0). Box plots (middle/right) showing DSSs/sDSS values of the respective compounds (mean with first and third quartile). Compounds with the highest scores are at the top. (C) Dose-response curves of primary T-PLL cells, together with age-matched, healthy donor–derived PBMCs and CD3+ T cells, treated with increasing concentrations of idasanutlin (lower panel) and cladribine (upper panel). After thawing, 1 × 106 primary cells per mL were treated with indicated concentrations of idasanutlin for 48 hours or cladribine for 72 hours (mean with standard error of the mean [SEM]; 2-way ANOVA, Bonferroni correction for multiple comparisons; ∗∗∗∗P < .0001). (D) Dose-response curves of primary T-PLL cells, with (light-blue) and without (dark-blue) coculturing with NKtert BMSCs, treated with increasing concentrations of idasanutlin (continuous line, n = 15 cases) and cladribine (dotted line, n = 16 cases). After thawing, 1 × 106 primary cells/mL were cocultured with NKtert cells and subsequently treated with indicated concentrations of idasanutlin (48 hours) or cladribine (72 hours; mean with SEM; 2-way ANOVA, Bonferroni correction for multiple comparisons; ∗∗∗∗P < .0001). Phase-contrast microscope image (background) showing T-PLL cells in contact with NKtert BMSCs after 72 hours of incubation (original magnification, 220×). See supplemental Figure 1 for the respective DSSs, presented per patient, and the effect of indicated drugs on NKtert cell viability. AnnV, annexin V; ctrl, control; neg., negative; rel., relative.

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