Journal: Nature Communications

Article Title: Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL

doi: 10.1038/s41467-017-02688-6

Figure Lengend Snippet: Pharmacologic exploitation of the defective ATM/p53 axis. a Summary of genomic lesions (sCNAs, mutations) and gene expression changes affecting TP53 and its direct regulatory network detected in this series of T-PLL. b ATM Ser1981 , KAP1 Ser824 , and p53 Ser15 phosphorylation upon 10 Gy ionizing irradiation in 9 T-PLL; controls: ATM/P53-competent HEK293 cells. Robust pKAP1 induction (e.g., TP052; ATM-wt/CN = 2), as well as reduced activation is observed (e.g., TP007; ATM-mut/CN = 1.5). Despite at least weak pATM/pKAP induction for most cases, none showed a pP53 response (irrespective of genomic ATM status). All cases lacked 17p sCNAs and TP53 mutations. Median purity: 97.5% T cells; lanes separated for genotype-based arrangement (* denotes nonsense mutation). For results on CHEK2 phosphorylation in a subset of cases see Supplementary Fig. . c Idasanutlin (0.3 µM) reinstated phospho- and acetyl-marks of p53 activity in T-PLL cells. Co-treatment with Bendamustine (1 µM) or Panobinostat (0.01 µM) further enhanced this, including the apoptotic response (cleaved PARP). Immunoblot of case TP104 (representative of 3) at 24 h. There was no p53 phospho-activation by Prima-1 met , a selective reactivator of mutated p53 (Supplementary Fig. ). d In vitro screen for interactions of Idasanutlin (I), Panobinostat (P), Bendamustine (B), and Olaparib (O) across 13 T-PLL. Each of the 6 combinations was plated in a matrix of 7 concentrations covering a 1000-fold range. Incubation of samples in duplicates for 72 h and cell viability as per luminescent CellTiter-Glo assay; positive control: 100 μM Benzethonium-chloride; negative control: DMSO. A delta score reflects positive (synergistic) and negative (antagonistic) drug interactions. It is calculated based on a zero-interaction potency (ZIP) reference model and is computed using the synergyfinder R-package (details in Online Supplements). Waterfall plot: ranking of each combination based on the delta scores of the low dose ranges (1–300 nM) across all samples. Rank-sums over 13 cases significantly differed among combinations (I + P:63, I + B:60, I + O:56, P + B:51, O + B:24, O + P:19; p < 0.0001, one-way Friedman Anova, highest ranks for highest score values). Combinations of I + P ( p = 0.015) and I + B ( p = 0.04) were superior to P + B (Wilcoxon rank-sum test). O + B or O + P showed antagonistic relationships in most T-PLL. Four selected 2D plots illustrate the distribution of synergistic (red) or antagonistic (green) interactions over the entire dose range of all 13 cases (positive controls in red in upper right corners)

Article Snippet: The MDM2 inhibitor Idasanutlin (Hycultec), the mutant p53 reactivator Prima-1 met (GENTAUR), the HDAC inhibitor Panobinostat (Hycultec), the ATM inhibitor KU55933 (Selleckchem), the alkylating agent Bendamustine (Astellas Pharma), and the PARP inhibitor Olaparib (Selleckchem) were solved in dimethyl sulfoxide (DMSO).

Techniques: Gene Expression, Phospho-proteomics, Irradiation, Activation Assay, Mutagenesis, Activity Assay, Western Blot, In Vitro, Incubation, Glo Assay, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL

doi: 10.1038/s41467-017-02688-6

Figure Lengend Snippet: Proposed model of T-PLL development. Top: Projections from an ‘unaffected’ thymic emigrant (left) compared to the scenario of a post-thymic T-PLL precursor (right box) with inappropriate expression of TCL1 and deficient ATM. Effects of this postulated initiating core lesion of TCL1 up / ATM def on the key signaling branches and functions of ATM are highlighted by differential arrows. It includes perturbation of some of ATM’s safeguarding tasks by TCL1 resulting in cell-death evasion. The ‘TCL1’-lesion refers to the deregulation of any TCL1 family member. TCL1 up / ATM def jointly confer a functional signature of ATM to be inefficient in counteracting oxidative damage, to maintain telomere and genome integrity, and to activate protective p53 programs. Timeline: Chronology assumptions are based on identified frequencies in sCNA data and tumor fractions in sequencing data. Constitutive TCL1 expression and loss-of-negative regulators (e.g., CTLA4) cause amplifications of TCR-derived survival signals. In the context of dysfunctional ATM this entertains ROS accumulation and genomic instability, which in turn entails further alterations of oncogenes like MYC , of epigenetic modifiers, and of miR processing (e.g., AGO2). Overt-stage autonomous proliferation, including escape from niche-defined homeostatic control relies on independence from milieu input, as potentially conveyed by JAK/STAT mutations. Emerging data, e.g., on the impact of JAK/STAT signaling on non-canonical functions of histone modulators like EZH2 indicate yet unrecognized cross-talks between the affected functional branches

Article Snippet: The MDM2 inhibitor Idasanutlin (Hycultec), the mutant p53 reactivator Prima-1 met (GENTAUR), the HDAC inhibitor Panobinostat (Hycultec), the ATM inhibitor KU55933 (Selleckchem), the alkylating agent Bendamustine (Astellas Pharma), and the PARP inhibitor Olaparib (Selleckchem) were solved in dimethyl sulfoxide (DMSO).

Techniques: Expressing, Functional Assay, Sequencing, Derivative Assay, Control