Fig 2: DAPK3‐mediated histone H3 threonine phosphorylation is required for faithful co‐transcriptional splicing. (A) EGR1/DUSP2 gene schematics showing introns and exons. Primer locations for qPCR and RT‐PCR indicated by vertical arrows and blue boxes. Numbers after gene names correspond to the position of the primers in and around the gene relative to the TSS, where + denotes 3′ of the TSS and − denotes 5′ of the TSS. Reverse transcription PCR (RT‐PCR) analysis comparing levels of processed EGR1/DUSP2 mRNA (top left) with EGR1/DUSP2 primary transcript levels (bottom left, top right, bottom right). CLL cells were pretreated with either 1 µm ibrutinib or 25 µm DAPKi for 1 h as indicated and then stimulated with anti‐IgM and sCD40L to activate IEG expression. Extracted RNA was reverse transcribed with either random primer (RP) or oligo‐dT, subjected to RT‐PCR and analysed by agarose gel electrophoresis. (B) qPCR analysis of EGR1/DUSP2 primary transcript (shades of blue) vs processed mRNA (red) from HBL1 cells pretreated with either 1 µm ibrutinib or 25 µm DAPKi for 1 h and then stimulated with or without anti‐IgM and CD40L for 1 h as indicated. Error bars represent the SD of n = 3 independent experiments. (C) Western blots of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, cells were harvested and lysates probed with antibodies against DAPK3, DAPK3‐T265‐P, histone H3T6‐P and histone H3T11‐P. β‐actin was used as a loading control and for normalisation to siCtrl using image lab software (right). Blots are representative of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test. P values for DAPK3 expression normalised to siCtrl = 0.0036 and 0.0004 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. (D) qPCR analysis of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, HBL1 cells were stimulated with anti‐IgM for 1 h and then harvested for analysis. Expression changes were quantified using the ∆C t method with TBP, GAPDH and PPP6C as control genes. Error bars represent the SD of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM stimulated siCtrl as control. P values for EGR1, EGR1 +0.55 kb and EGR1 +1.0 kb = 0.0082 and 0.0009, 0.1163 and 0.2759, 0.0047 and 0.0270 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Fig 3: Effects of DAPK3 inhibition on CLL and lymphoid cell proliferation and cellular function. (A) Representative western blots displaying levels of EGR1 and DAPK3‐T265‐P in CLL cells pretreated with 0–120 μm DAPKi for 1 h and with or without anti‐IgM stimulation for 1 h. β‐actin was used as a loading control. Blots are normalised to unstimulated sample (unstimulated = 1.0) with image lab software (below). Error bars represent the SD of three independent experiments. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM stimulated 0 µm sample as control. P values = 0.0076, 0.0256 and 0.0087 for unstimulated 0 and 80 and 120 µm samples, respectively. (B) Flow cytometric analysis of CLL cells cultured on a CD40L‐expressing cell feeder layer. CLL cells were pretreated with either 1 µm ibrutinib (green), increasing concentrations (5–80 µm) of DAPKi (red), both inhibitors (green/red), or DMSO (white; negative control) for 1 h as indicated. Proliferation was assessed via the percentage of cells positive for Ki‐67. Data shown are representative of n = 3 CLL patients. (C) Flow cytometric analysis of CLL cells cultured on a CD40L‐expressing cell feeder layer. CLL cells were pretreated with either 1 µm ibrutinib, increasing concentrations (5–80 µm) of DAPKi or DMSO (negative control) for 1 h. Apoptosis was assessed using the Annexin V Apoptosis Detection Kit, as indicated. Proliferation was assessed via the percentage of cells positive for Ki‐67. Data shown are representative of n = 3 CLL patients. (D) Cell survival assay in untreated HBL1 cells compared with HBL1 cells treated with either 25 or 0.4 μm ibrutinib. Cell count was determined by trypan blue staining at 2–12 days. Error bars are representative of three independent counts. (E) qPCR data analysis of EGR1 and DUSP2 gene expression at 30–60 min post‐anti‐IgM/sCD40L stimulation in CLL cells (n = 5 patients). CLL cells were pretreated with 1 µm ibrutinib or 25 µm DAPKi for 1 h as indicated below the graphs. Expression changes were quantified using the ∆C t method with TBP and PPP6C as control genes. Bars represent the grand mean of the samples for n = 5 patients. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM as control. EGR1 (30 m) P values = 0.0626 and 0.0054 for anti‐IgM vs Ibr and DAPKi, respectively. DUSP2 (30 m) P values = 0.1713 and 0.0086 for the same comparisons. EGR1 (60 m) P values = 0.1893 and 0.0245 for the same comparisons. DUSP2 (60 m) P values = 0.1507 and 0.0064 for the same comparisons. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Fig 4: H3T6 and H3T11 phosphorylation at IEGs is catalysed by DAPK3 and is similarly abrogated by both BTK and DAPK3 inhibition. (A) qPCR data analysis of EGR1 and DUSP2 gene expression at 30 (n = 6 patients) and 60 min (n = 4 patients) post‐anti‐IgM stimulation in U‐CLL cells. CLL cells were pretreated with 1 µm ibrutinib or 25 µm DAPKi for 1 h as indicated below the graphs before anti‐IgM stimulation. Expression changes were quantified using the ∆C t method with TBP and PPP6C as control genes. Bars represent the grand mean of the samples. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM as control. EGR1 (30 m) P values = 0.0009, 0.0015 and 0.0009 for anti‐IgM vs Unstim, Ibr and DAPKi respectively. DUSP2 (30 m) P values = 0.0013, 0.0023 and 0.0011 for the same comparisons. EGR1 (60 m) P values = 0.0039, 0.0052 and 0.0052 for the same comparisons. DUSP2 (60 m) P values = 0.0204, 0.0269 and 0.0205 for the same comparisons. (B) ChIP‐qPCR data assessing levels of H3T6‐P and H3T11‐P at the EGR1 and DUSP2 gene loci. CLL cells pretreated with either 1 µm ibrutinib or 25 µm DAPKi for 1 h before stimulation with anti‐IgM for 30 min. Values on the x axis refer to specific gene regions relative to the TSS in kilobases (kb) as indicated on the gene schematics below (not to scale). Error bars represent the SD of three independent experiments. CTCF1/3 (C1/C3) were used as negative control regions which are not indicated on the gene schematics. (C) co‐IP of CLL cells stimulated with anti‐IgM for 1 h as indicated. Immunoprecipitates from histone H3 pulldown were analysed by SDS/PAGE followed by western blot probing for DAPK3‐T265P. Untreated, crude cell lysate was used as positive control (input), and IgG beads were used for negative control (Ctrl IgG). Blot is representative of three independent co‐IP experiments. Western blot is cropped due to different exposure times for input and co‐IP lysates. (D) Kinase assay quantified by dot‐blot with recombinant histone H3 and recombinant DAPK3 to measure DAPK3 kinase activity over time (5–60 min) with or without (negative control) ATP and in the presence of 1 µm ibrutinib and 25 µm DAPK inhibitor. (E) Kinase assay displaying histone H3 phosphorylation quantified relative to untreated assay with error bars representing the SD of three independent experiments. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with the untreated sample as control for each time point. H3T6‐P P values = 0.3075 and 0.0139 (5 m), 0.4157 and 0.0021 (20 m), 0.1583 and 0.0001 (60 m) for untreated vs. Ibr and DAPKi, respectively. H3T11‐P P values = 0.6679 and 0.6844 (5 m), 0.0672 and 0.0095 (20 m), 0.0043 and 0.0001 (60 m) for the same comparisons. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.