Fig 2: Ibrutinib synergizes with venetoclax in T-prolymphocytic leukemia via inhibition of ITK and enhances Bcl2-dependent apoptotic priming. (A) Single cell suspensions of blood or bone marrow samples were subjected to combinatorial drug screens with venetoclax and 24 potential combination partners. Deviation from the Bliss Independence score was evaluated for each combination. For BH3-profiling single cells were stained for cytochrome and analyzed by flow cytometry. (B) Heatmap demonstrating deviation from the Bliss independence score for each combination and individual patients (n=7). Synergy is denoted in red while antagonism is shown in blue. Clear boxes represent missing data for Patient 5 whose material was only screened for a subset of drugs due to sample availability. The bar plot in the lower half shows the synergy score as a mean over all patients. Drug synergy was calculated using the Synergy finder R package by integrating data of three independent runs (Online Supplementary Figure S1B). (C) A representative three-dimensional drug synergy plot (data from Patent 7) for ibrutinib and cisplatin with venetoclax. (D) Annexin V assay showing viability compared to that with a DMSO control for ibrutinib (n=12 different T-PLL patient samples), ITK-inhibitor BMS-509744 (n=6), and BTK-inhibitor acalabrutinib (n=12, Wilcoxon-Mann-Whitney test, ***P<0.001, *P<0.05, all compounds tested at 10 µM with drug exposure for 24 h under NKtert co-culture). (E) Annexin V assay demonstrating viability of T-PLL samples under NKtert co-culture treated with venetoclax alone (n=10), or in combination with ibrutinib (n=10), BMS-509744 (n=6), and acalabrutinb (n=10) compared to DMSO-Ctrl. (t-test,*P=0.05, ***P<0.001, ibrutinib, acalabrutinib and BMS-509744 were used at a dose of 10 mM with drug exposure for 24 h, venetoclax was used at a dose of 100 nM with drug exposure for 4 h both as a single agent and in combination. After annexin V/Hoechst staining, cells were fixed with paraformaldehyde and analyzed using a BD Fortessa with a 96-well HTS plate-reader. NKTert cells were excluded using forward and side scatter parameters. Primary antibodies were directed against cytochrome c (Alexa Fluor 488-labeled, 6H2.B4/612308, Biolegend), CD19 (PE/Cy7-labeled, H13B19/302216, BioLegend), anti–CD5 (PE-labeled, UCHT2/300608, Biolegend). The analysis was performed on the CD5+CD19– cell fraction. (F) BH3-profiling in primary T-PLL samples treated with ibrutinib: cytochrome C release compared to control for overall mitochondrial priming and specific BCL2-dependence is shown for samples treated with either DMSO or ibrutinib 10 mM for 24 h (t-test,**P=0.01). T-PLL: T-prolymphocytic leukemia; DMSO: dimethylsulfoxide; Ctrl. Control.

Fig 3: Impaired production of IFN-? and TNF by ITK-deficient T lymphocytes. (A and B) PBMCs from P1 and P2 (obtained at the ages of 19 and 18 yr, respectively) and their father were stimulated for (A) 18 h or (B) 3 or 5 d with IL-2. Secreted cytokine levels were determined in LEGENDplex assays. Technical duplicates were prepared for (A) P1 or (B) P1, P2, and their father (except P2 for 3 d). (C and D) Expanded T cell blasts (T-blasts) from P1 and P2 were stimulated for 2 h with various reagents and then for an additional 5 h in the presence of a secretion inhibitor. Secreted cytokines were determined with the LEGENDplex assay, and intracellular cytokine levels were determined by flow cytometry. Technical duplicates were prepared for the unstimulated and anti-CD3 mAb-conjugated bead-stimulated samples. (E) Lentiviral rescue. P2’s T-blasts underwent lentiviral transduction with the EV or the WT ITK CDS. Cells were selected on puromycin. Cells were either left unstimulated or stimulated with anti-CD3 mAb-conjugated beads for 5 h. Cytokine production was assessed by flow cytometry. (F) Pharmacological ITK inhibition. Magnetically enriched CD4+ T-blasts from four healthy donors were incubated for 1 h with DMSO or an ITK inhibitor (BMS509744) and were then stimulated for 6 h. Secreted cytokines were determined in LEGENDplex assays. In A–D and F, bars represent the mean and SEM. In A–D, statistical significance was determined for differences between the two ITK-deficient patients on the one hand, and local and familial controls on the other. *, P < 0.05; **, P < 0.01; ***, P < 0.001, and two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Fig 4: The effect of BTK and ITK phosphorylation on Cx43 regulation in lymphocytes as observed through changes in electrophoretic mobility. Western blot of (A) Daudi cells with and without BTK activation using the a-IgM antibody or (B) Jurkat cells with and without ITK activation using a-CD3/a-CD28 antibodies. Antibodies used were labeled on the right of each blot. Arrows point to the different migrating Cx43 bands from the electrophoresis. Data were quantified using ImageJ (all units measured in pixels) and GraphPad Prism by Student’s t-test (n = 3, ** p < 0.01; **** p < 0.0001).

Fig 5: Effect of BTK and ITK phosphorylation on Cx43 regulation in the HEK-293T cells as observed through changes in electrophoretic mobility. Western blot of HEK-293T parental (HEKPar) and (A) stably expressing BTK (HEKBTK) or (B) stably expressing ITK (HEKITK) cell lysates. Antibodies used are labeled on the right of each blot. Arrows are pointing to the different migrating Cx43 bands from the electrophoresis. Data were quantified using ImageJ (all units were measured in pixels) and GraphPad Prism by the Student’s t-test (n = 3, ** p < 0.01).