DAPK3 participates in the mRNA processing of immediate early genes in Chronic Lymphocytic Leukaemia.

Cross‐linking of the B‐cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post‐translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death‐associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti‐IgM‐dependent manner. DAPK inhibition mimics ibrutinib‐induced repression of both IEG mRNA and histone H3 phosphorylation and has anti‐proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti‐tumour effect than ibrutinib, by repressing both anti‐IgM‐ and CD40L‐dependent activation.


Introduction
Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has shown extremely positive results in the treatment of chronic lymphocytic leukaemia (CLL; Burger et al., 2015;Byrd et al., 2014) and is now being tested in combination with other therapies (Collett et al., 2017;Hillmen et al., 2019;Kater et al., 2019). However, cases of resistance are emerging (Ahn et al., 2017;Kaur and Swami, 2017) and the long-term outcome is generally poor after treatment discontinuation (Jain et al., 2017). Ibrutinib-mediated relapse in CLL is mostly associated with genomic alterations in BTK and its downstream target PLCc2, but cases of progressive disease in which targeted sequencing is unable to explain resistance have also been reported (Landau et al., 2017).
Chronic lymphocytic leukaemia can be divided into two main subsets, depending on whether the leukaemic cells arise from a B cell before somatic hypermutation (U-CLL) or after (M-CLL), the latter being generally Abbreviations (U/M)-CLL, (unmutated/mutated)-chronic lymphocytic leukaemia; BCR, B-cell receptor; BTK, Bruton's tyrosine kinase; Co-IP, coimmunoprecipitation; CpG ODN, CpG oligodeoxynucleotides; DAPK1, death-associated protein kinase 1; DAPK3, death-associated protein kinase 3; DAPKi, DAPK inhibitor; DLBCL, diffuse large B-cell lymphoma; DUSP2, dual-specificity phosphatase 2; EGR1, early growth response 1; GAIT, interferon-gamma-activated inhibitor of translation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3T11-P, histone H3 threonine 11 phosphorylation; H3T6-P, histone H3 threonine 6 phosphorylation; IEG, immediate early gene; IFNc, interferongamma; IgHV, immunoglobulin heavy chain variable (region); Kb, kilobase; MYD88, myeloid differentiation primary response 88; NF-jB, nuclear factor-kappa B; NLC, nurse-like cell; PLCc2, phospholipase Cc2; PPP6C, protein phosphatase 6 catalytic subunit; PTM, posttranslational modification; qPCR, quantitative polymerase chain reaction; RNA pol II S2-P, RNA polymerase II serine 2 phosphorylation; RPMI, Roswell Park Memorial Institute (media); siRNA, small interfering ribonucleic acid; TBP, TATA-box binding protein; TLR, Toll-like receptor; TSS, transcription start site; ZIPK, zipper-interacting protein kinase. less aggressive than the former (Damle et al., 1999;Hamblin et al., 1999). The tissue microenvironment, a distinct anatomical proliferation centre in the lymph node, plays an important role in the pathogenesis of CLL (Burger, 2011). Within these centres, CLL cells thrive from the external pro-survival signals they receive from stromal, nurse-like cells (NLCs) and T cells leading to unique gene expression profiles with substantial activation of nuclear factor-kappa B (NF-jB) and pro-survival signalling cascades from the Bcell receptor (BCR) (Burger, 2011;Choi et al., 2016;Herishanu et al., 2011). The importance of the microenvironment is highlighted by the clinical efficacy of ibrutinib which inhibits BCR signalling to disrupt trafficking of CLL cells to the lymphoid organs (Choi et al., 2016). A significant number of cases of both primary and secondary resistance to this treatment have been described (Ahn et al., 2017;Kaur and Swami, 2017). However, activation of alternative signalling pathways to bypass BTK inhibition may also play a role in the development of resistance to ibrutinib. For example, it has been reported that Toll-like receptor (TLR) signalling in the lymph node is only partially repressed by ibrutinib (Dadashian et al., 2019). Furthermore, combining ibrutinib with the BCL-2 inhibitor venetoclax is a proven and successful treatment option in CLL Kater et al., 2019), yet resistance can still arise from strong activation of the NF-jB pathway via microenvironmental agonists such as CD40L and CpG oligodeoxynucleotides (Jayappa et al., 2017). Similarly, WNT5A, a secreted glycoprotein belonging to the WNT family, can activate RAC1 and lead to proliferation in CLL and mantle cell lymphoma, a process which is not repressed by ibrutinib (Yu et al., 2017(Yu et al., , 2018. Therefore, intensive efforts have been undertaken to better understand the mechanism of response to ibrutinib and the development of drug resistance. We have previously reported an important epigenomic plasticity of CLL cells in response to ibrutinib treatment (Holmes et al., 2019). In the present analysis, focussing on two immediate early genes (IEG) stimulated in response to BCR signalling activation with anti-IgM, we observed that ibrutinib-dependent transcriptional repression of these genes correlated with a block in histone H3 threonine 6 and 11 (H3T6 and H3T11) phosphorylation. We identified death-associated protein kinase 3 (DAPK3), also known as zipper-interacting protein kinase (ZIPK), as the enzyme mediating these histone H3 post-translational modifications (PTMs). DAPK inhibition (DAPKi) impairs mRNA processing and CLL cell proliferation. Finally, in contrast to ibrutinib, DAPKi prevents transcriptional activation induced by both anti-IgM and CD40L suggesting that targeting this kinase might have a wider effect than ibrutinib. In conclusion, targeting DAPK3 might be a suitable alternative to BTK inhibitors in the treatment of CLL, including in patients carrying BTK or PLCc2 mutations.

Cell culture and siRNA knockdown
Chronic lymphocytic leukaemia cells were obtained from the St James's University Hospital (Leeds) Haematological Malignancy Diagnostic Service (HMDS) from patients with no previous treatment for their disease. The experiments using these cells were undertaken with the understanding and written consent of each patient and the study methodologies conformed to the standards set by the Declaration of Helsinki. These experiments were performed under ethical approval granted by the Leeds Teaching Hospital NHS Trust REC: 14/WS/0098.

cDNA preparation, qPCR and RT-PCR
Total RNA was prepared using TRIzol Ò reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. RNA was prepared with Direct-zol TM RNA MiniPrep kit (Zymo, Irvine, CA, USA). cDNA was synthesised with Random Primers (Invitrogen) or Oligo(dT) (Invitrogen), 59 FS buffer (Invitrogen), MLV-reverse transcriptase (Invitrogen), RNase-Out (Invitrogen) and dNTPs (Invitrogen). qPCR reactions were carried out using Luna Ò Universal qPCR Master Mix (NEB, Ipswich, MA, USA) on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher). Relative expression was calculated as a ratio of specific transcript to one/several housekeeping genes: TATA-box binding protein (TPB), protein phosphatase 6 catalytic subunit (PPP6C) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as indicated. RT-PCR reactions were carried out using a thermal cycler (Bio-Rad). RT-PCR products were run on 1.5% agarose gels for 30-40 min at 100 V and visualised with a ChemiDoc system. See Table S1 for primer sequences.
Sonicated chromatin from 10 7 cells was used for each immunoprecipitation. The volume was adjusted to give 100 lL per IP with fresh immunoprecipitation buffer. Immunoprecipitation was achieved using Dynabeads TM Protein G (Thermo Fisher) with 2.4 µg of anti-histone H3T11-P (Abcam, Cambridge, UK; ab5168), anti-histone H3T6-P (Diagenode; C15410282), anti-RNA polymerase II (Abcam; ab817) or anti-RNA polymerase II S2-P (Abcam; ab5095) per 10 µL of beads. Beads were washed twice with ice cold immunoprecipitation buffer prior to use. Beads and antibodies were incubated for 2 h at 4°C with gentle agitation. After 2 h, the bead-antibody mixture was pelleted using a magnet and the beads were washed once with ice cold immunoprecipitation buffer. About 100 µL of sonicated chromatin was added to achieve a bead to chromatin ratio of 1 : 10. Chromatin was incubated for 2 h at 4°C with gentle agitation. After 2 h, beads were pelleted using a magnet and washed once with washing buffer A [20 mM Tris/HCl (pH 8), 2 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate and 150 mM NaCl supplemented with 19 protease inhibitor cocktail), once with washing buffer B (20 mM Tris/HCl (pH 8), 2 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate and 500 mM NaCl supplemented with 19 protease inhibitor cocktail], once with LiCl buffer [0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris/ HCl (pH 8) supplemented with 19 protease inhibitor cocktail], and twice with TE buffer [10 mM Tris/HCl (pH 8.0) and 1 mM EDTA]. The immune complexes were eluted by adding 50 lL of elution buffer (1% sodium dodecyl sulfate, 100 mM NaHCO 3 ). The crosslink was reversed at 65°C overnight. Inputs were treated with RNAse A for 30 min at 37°C and reverse cross-linked at 65°C overnight with proteinase K.
Reverse cross-linked DNA was purified using Axy-Prep TM MAG PCR clean-up kit (Appleton Woods, Birmingham, UK) according to manufacturer's instructions. qPCR reactions were carried out using Luna Ò Universal qPCR Master Mix (NEB) on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher). See Table S1 for primer sequences.

Flow cytometric analysis
Cells were analysed on a 3 laser BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). CLL cell apoptosis analysis was carried out using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions. CLL cell proliferation assays utilised the antibody FITC Mouse Anti-human Ki-67 (BD Pharmingen TM , Franklin Lakes, NJ, USA; 556026 clone B56). CLL cells were cultured on a CD40L-expressing feeder layer prior to analysis. Analysis was performed with BD FACSDIVA Software 5.0 (BD Biosciences) and FLOWJO (Ashland, OR, USA).

Primers for qPCR, RT-PCR and ChIP-qPCR
Primers were designed online using PRIMER3PLUS (University of Heidelberg, Germany) and the NCBI PRIMER-BLAST (Bethesda, MD, USA) tool in conjunction with the in silico PCR function of the UCSC genome browser (Table S1). The number following a primer indicates the distance from the transcription start site (TSS) in kilobases (kb) of the corresponding gene where negative values are upstream of the TSS and positive values are downstream of the TSS.

Phosphorylation of histone H3 is induced by BCR cross-linking at IEGs loci
The link between signalling-dependent chromatin alterations and changes in gene expression is poorly understood. To better understand, how chromatin structure responds to BCR cross-linking, we performed a screening of histone H3 phosphorylation marks induced after anti-IgM stimulation in primary CLL cells and identified two phospho-marks (H3T6p and H3T11p) enriched at IEGs and correlated with expression of these genes. To further analyse these chromatin structure alterations, we chose two genes as markers of response to anti-IgM in U-CLL (EGR1 and DUSP2) and investigated the changes in mRNA level and chromatin structure in response to BCR cross-linking. These two genes were selected after an initial screening for genes responding to anti-IgM stimulation in primary CLL, and for which the response was repressed by addition of ibrutinib looking at EGR1, DUSP2, MYC, DUSP4, CCND2, BCL2A1, PRDX1, NCL, CDC25B, TLR10, PIM1, CXCR4, CXCR5, CCR6 and CCR7. From these genes, only the first 5 were convincingly stimulated and repressed without or with ibrutinib, respectively. However, EGR1 and DUSP2 mRNA level was maximum after 30-min stimulation whereas, for MYC, DUSP4 and CCND2, this peak occurs between 2 and 4 h post-stimulation. The IEG early growth response 1 (EGR1) controls B-cell proliferation in response to BCR cross-linking but not after CD40 ligation or TLR4 activation (Gururajan et al., 2008;McMahon and Monroe, 1996) and correlates with survival in CLL (Stratowa et al., 2001). Dualspecificity phosphatase 2 (DUSP2) is another IEG induced by anti-IgM stimulation in B cells (Grumont et al., 1996). DUSP2 regulates MAPK activity and plays an essential role in cell proliferation and cancer (Wei et al., 2013). Expression of both genes was activated 30-60 min post-anti-IgM stimulation (Fig. 1A) and correlated with enriched phosphorylation of histone H3T6 and H3T11 within the body of these genes (Fig. 1B). In addition, both gene expression and histone H3 phosphorylation were impeded by ibrutinib (Fig. 1A,B). Consequently, this approach represented a reliable and simple method to assessing the cellular response to BCR cross-linking and ibrutinib-mediated repression of this signal.
To determine which kinase downstream of the BCR signalling pathway might mediate histone H3 phosphorylation at the indicated threonine residues, we used the in silico kinase prediction software GPS (Xue et al., 2008) (Fig. S1A). Among the identified kinases, MEK2, RSK1/2 and PKCs are known downstream elements of the BCR signalling pathway. Consequently, inhibitors targeting these kinases were tested for their ability to inhibit expression of EGR1 and DUSP2 and phosphorylation of H3T6 and H3T11 along these genes. The inhibitors used were G€ o6983, a pan-PKC inhibitor, RSK inhibitor II, a pan-RSK inhibitor, and U0126, a MEK1/2 inhibitor. None of the three inhibitors tested significantly inhibited the induction of EGR1 or DUSP2 expression or prevented histone H3 phosphorylation after 30-and 90-min anti-IgM stimulation (Fig. S2). In contrast, the death-associated protein kinase inhibitor (DAPKi) was the only compound which showed repression of both anti-IgM stimulation and histone H3 phosphorylation (Fig. 1A, B). DAPKi is an inhibitor specific against DAPK1 and DAPK3, two enzymes of the DAPK family (Fig. S1B). Epigenetic silencing of DAPK1 by promoter methylation is a characteristic of sporadic CLL (Raval et al., 2007), indicating, therefore, that DAPKi was mainly targeting DAPK3 in these cells. This enzyme was originally identified as a chromatin-associated enzyme (K€ ogel et al., 1998) mediating histone H3T11 phosphorylation (Preuss et al., 2003). co-IP experiments demonstrated that DAPK3 and histone H3 were interacting in vivo independently of anti-IgM stimulation (Fig. 1C), and in vitro kinase assay confirmed that DAPK3 could phosphorylate histone H3 at T6 and T11, both effects being repressed by DAPKi (Fig. 1D, E). Our data were therefore in line with the original observations identifying DAPK3 as a chromatin-associated enzyme inducing mitosis-specific H3T11 phosphorylation (K€ ogel et al., 1998;Preuss et al., 2003). In contrast, ibrutinib had no significant impact on DAPK3 activity apart from T11 at 60 min (Fig. 1D, E), which may be due to an indirect effect arising from the addition of ibrutinib to the reaction (e.g. changes in stoichiometry). Altogether, these results showed that DAPK3 was activated downstream of BTK in response to BCR cross-linking.

DAPK inhibition impacts on mRNA processing
To further decipher the role of DAPK3 in the transcriptional activation of our two IEGs, we performed co-IP experiments looking at DAPK3 interaction with total RNA polymerase II (RNA pol II) and the elongating form of the polymerase phosphorylated at serine 2 (RNA pol II S2P) ( Fig. 2A). DAPK3 interacted with both forms of the polymerase, and this interaction is enhanced by anti-IgM stimulation and marginally impaired by addition of DAPKi ( Fig. 2A). In addition, looking at RNA pol II recruitment to EGR1 and DUSP2 loci in response to anti-IgM stimulation, we detected an accumulation of total and elongating polymerases at promoters and gene bodies, which is prevented by ibrutinib (Fig. 2B,C). In contrast, no effect was observed after addition of DAPKi, suggesting that transcription elongation was unaffected by this inhibitor. Some alterations of chromatin structure during transcription favour the recruitment of the RNA processing machinery (Brown et al., 2012). Since DAPKi did not repress transcription elongation, we decided to assess the role of DAPK3 in mRNA processing. For this, we performed conventional RT-PCR looking at mature mRNAs as well as primary transcripts.
Dual anti-IgM/CD40L stimulation induced increased level of mature mRNA as well as primary transcript for both EGR1 and DUSP2 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 ( Fig. 3A). Addition of ibrutinib had no effect on this transcriptional activation (Fig. 3A). DAPKi repressed mature mRNA processing and primary transcript but only if the reverse transcription was performed using an oligo-dT but not a random primer (Fig. 3A). These data suggested that DAPK3 was participating in mRNA processing and not transcription elongation. Having shown that DAPKi was altering mRNA processing in CLL, we decided to test if its effect was reproducible on other ibrutinib-sensitive cells. For this, we selected a diffuse large B cell lymphoma (DLBCL) cell line, HBL1, which carries a myeloid differentiation primary response 88 (MYD88) mutation and which is sensitive to ibrutinib (Wilson et al., 2015). EGR1 and DUSP2 expression was activated by anti-IgM and/or CD40L stimulation in these cells, with this activation being repressed by the addition of DAPKi (Fig. S3A).
Interestingly, ibrutinib did not prevent the expression of these IEGs in response to dual anti-IgM/CD40L stimulation and could only marginally reduce the expression of DUSP2 after anti-IgM stimulation alone (Fig. S3A). At the same time, ibrutinib showed the strongest repressive effect in the absence of any stimulation (Fig. S3A).
Moreover, the analysis of RNA pol II recruitment in response to dual anti-IgM/CD40L stimulation showed a similar profile compared to CLL for EGR1 ) 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. b-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 ΔC 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.
but not DUSP2 (Figs S3B and S4). The reason why ibrutinib appeared to prevent the recruitment of the polymerase without having any effect on the expression of the studied genes was unclear. In these cells, MYD88 mutation induces chronic NF-jB activation, which could compensate for BTK inhibition. We have shown previously that NF-jB-dependent activation of TNFa is accompanied by an accumulation of RNA pol II at the transcription end site of the gene (Thorne et al., 2012), as seen for EGR1 (Fig. S3B). Therefore, it was possible that the dynamics of RNA polymerase II recruitment to the coding region induced by NF-jBdependent transcription are different when compared to other transcription factors. However, further investigations would be necessary to answer this question and are beyond the scope of this study. In HBL1, the production of mature mRNAs and primary transcripts was altered after stimulation in presence of DAPKi (Fig. 3B). Levels of mRNAs were decreased for both IEGs and increased or unaffected for EGR1 or DUSP2 primary transcripts, respectively (Fig. 3B). These results confirmed that DAPK3's function in the transcription of these IEGs was the same in CLL and HBL1 cells. To confirm that DAPKi-dependent impact on mRNA processing was not due to an off-target effect, we performed siRNA-mediated knockdown in HBL1 cells using two DAPK3-specific siRNAs (Fig. 3C,D, Fig. S5). In these cells, DAPK3 knockdown was correlated with a global loss of H3T11 and H3T6 phosphorylation (Fig. 3C) and a significant loss of processed mRNA and gain of primary transcript for EGR1 (Fig. 3D). We could not see any significant effect on DUSP2 expression (Fig. S5), potentially due to the partial knockdown and the moderate induction of this gene in response to anti-IgM and/or CD40L stimulation compared to EGR1. Nevertheless, these experiments confirm that DAPK3 control on IEG expression was post-transcriptional.

DAPK inhibition blocks proliferation and both anti-IgM-and CD40L-mediated IEG expression
Ibrutinib blocks CLL cell proliferation in vitro and in vivo. To compare ibrutinib and DAPKi efficiency, we incubated primary CLL cells with or without these inhibitors and measured the rate of proliferation as well as the pro-apoptotic effect of the two compounds (Fig. 4, Fig. S6). First, we pretreated CLL cells with increasing doses of DAPKi and assessed the level of EGR1 protein and phospho-DAPK3 after anti-IgM stimulation. DAPK3 phosphorylation at threonine 265 is essential for its full auto-phosphorylation and enzymatic activity (Graves et al., 2005). We determined that DAPKi was having an important repressive action on both proteins beyond 20 µM (Fig. 4A). Similarly, the reduction in CLL cell proliferation was comparable between ibrutinib and DAPKi for 20 µM and beyond (Fig. 4B, Fig. S6A). Moreover, combining both drugs had a synergistic effect on cell proliferation (Fig. 4B, Fig. S6A). Impact on CLL cell apoptosis correlated with the effect on proliferation, but with some variability from one experiment to another due to the small duration of the experiments performed with primary CLL (Fig. 4C, Fig. S6B). As observed for CLL cells, both ibrutinib and DAPKi blocked HBL1 cell proliferation (Fig. 4D). Finally, as suggested by the experiments performed with conventional RT-PCR (Fig. 3A), ibrutinib did not repress EGR1 and DUSP2 after CLL stimulation with both anti-IgM and CD40L compared to DAPKi, indicating that DAPKi had a broader repressive effect than ibrutinib (Fig. 4E).

Discussion
DAPK3, like other members of the DAPK family, is a pro-apoptotic kinase with a reported tumour-suppressor activity (Brognard et al., 2011;Kawai et al., 1998). DAPK3-induced apoptosis involves both caspase-dependent and independent pathways (Kogel et al., 2001) as well as an alternative mechanism through autophagic cell death (Inbal et al., 2002). However, this protein was initially identified as a chromatin-associated enzyme phosphorylating the core histones H3, H4 and H2A (K€ ogel et al., 1998) and more specifically H3T11 (Preuss et al., 2003), suggesting that this enzyme might be involved in a wide range of functions. In line with its association with chromatin, we have shown that DAPK3 recruitment to the transcriptional machinery is enhanced in response to anti-IgM stimulation. This kinase then targets histone H3 at threonine 6 and 11 within the body of IEGs, these PTMs being correlated with gene transcription. This mechanism does not appear to impact on transcription per se, but DAPK inhibition induces a defect in mRNA processing.
Transcription and mRNA processing are two interconnected processes, and one of the functions of the chromatin is to coordinate them (Brown et al., 2012;Jimeno-Gonzalez and Reyes, 2016). However, any potential role for the two identified histone marks in controlling mRNA processing is still unknown. In addition, it is worth considering that IEGs show rapid pre-mRNA induction and RNA polymerase recruitment compared to secondary/delayed response genes (Bahrami and Drablos, 2016). IEGs have a distinct genomic architecture and chromatin structure to facilitate their rapid transcription characterised by bivalent promoters poised for rapid activation (Tullai et al., 2007). Consequently, DAPK inhibition may be particularly effective against the pre-mRNA processing of this class of gene. Alternatively, it cannot be excluded that DAPK3 could directly target proteins of the processing machinery.
Ibrutinib blocked DAPK3-dependent chromatin PTMs induced by anti-IgM stimulation by possibly preventing RNA polymerase II recruitment to the IEG promoters and gene bodies. Ibrutinib presents a breakthrough in CLL treatment, but cases of resistance which cannot be explained by genetic alterations are emerging (Ahn et al., 2017;Kaur and Swami, 2017;Landau et al., 2017), and recent studies indicate that alternative pro-proliferative signalling pathways, mainly NF-jB, were only partially blocked by this small molecule (Dadashian et al., 2019;Jayappa et al., 2017). Interestingly, DAPK inhibitor, like ibrutinib, prevents IEG expression and CLL and HBL1 proliferation. Off-target effects cannot be completely ruled out at the tested inhibitor concentrations; however, ERK (IC50 > 10 lM for ERK2), the most relevant alternative target (Ten Hacken et al., 2016), shows no effect on EGR1 and DUSP2 expression as well as on H3 phosphorylation after directly targeting MEK the enzyme activating ERK (Fig. S2) suggesting that the latter might not mediate DAPKi effect.
DAPK3 is activated downstream of BTK and PLCc2, suggesting that it could be an attractive alternative to ibrutinib, particularly in CLL cells for which relapse is correlated with mutations of both upstream proteins. In addition, DAPKi appears to have a broader repressive function by blocking both the BCR and CD40 signalling pathways. Ibrutinib does not prevent transcription of IEGs in response to activation with CD40L as seen in our work and as described in other studies (Woyach et al., 2014). Activation of CD40, a key regulator of B-cell proliferation, augments NF-jB activity and prolonged CLL cell survival in vitro (Furman et al., 2000). Therefore, DAPK3 controlling the mRNA processing of NF-jB target genes downstream of CD40 activation suggests it might also regulate other signalling pathways activating NF-jB. In this respect, it has been shown that DAPK3 also regulates pro-inflammatory genes in response to TNFa-dependent activation (Usui et al., 2012) as well as in response to interferon-gamma (IFNc) (Mukhopadhyay et al., 2008). Remarkably for the latter, DAPK3 acts first as a repressor by activating the interferon-gamma-activated inhibitor of translation (GAIT), which phosphorylates the ribosomal protein L13a, a key component of this repressive complex, before GAIT-dependent inactivation of DAPK3 will reactivate gene expression (Mukhopadhyay et al., 2008). IFNc activates a signalling cascade in which DAPK1 targets DAPK3 before DAPK3-dependent phosphorylation of L13a (Mukhopadhyay et al., 2008). DAPK1, the first member of the death-associated protein kinase family, can induce apoptosis in response to INFc, TNFa and FAS pathways. Silencing of DAPK1 via hypermethylation of its promoter has been suggested to influence tumour progression and metastasis in cancers such as CLL (Raval et al., 2007) and DLBCL (Kristensen et al., 2014) and is a marker of poor survival. Consequently, DAPKi anti-proliferative function in CLL and HBL1 is unlikely to be mediated by DAPK1 repression. In addition, our data suggest that, depending on the cellular context, DAPK3 could induce either proliferation and survival or cell death, the latter function being DAPK1-dependent. By extension, DAPKi could have a repressive function of the NF-jB pathway restricted to DAPK1-silenced cells. Constitutive activation of NF-jB is known to mediate cancer development as well as resistance to anti-cancer therapies. However, targeting NF-jB is also seen as problematic because of its key role in many physiological processes. An attractive possibility suggested by our data is that DAPK3 might repress this transcription factor only in cells with aberrant epigenetic silencing of DAPK1, therefore having a limited impact on 'normal' cells.
At this stage, the link between ibrutinib-and DAPKi-mediated repression of IEGs and the anti-proliferative potential of these molecules is not entirely clear. CLL cells tend to undergo rapid and spontaneous apoptosis when cultured in vitro (Collins et al., 1989) which has been attributed to a dramatic decrease in the levels of the anti-apoptotic protein Bcl-2 during in vitro culture of CLL cells (Pepper et al., 1997). Recreating the complex microenvironmental signalling network which is vital to CLL cell proliferation and survival in tissue niches in vivo is challenging to the extent that ex-vivo stimulation of the BCR, one of the most important receptors for these cells in vivo, cannot induce CLL cell proliferation in culture (Schleiss et al., 2019). Co-culture of CLL cells on a CD40L-expressing feeder layer can promote their proliferation to some extent, and some studies suggest addition of IL-4 and IL-21 (Schleiss et al., 2019) or CpG ODN (Purroy et al., 2015) can aid proliferation, but neither method allows indefinite culture or a true representation of in vivo conditions. We believe the discrepancies in our data may be explained somewhat by the limited culture conditions and suggest that more elaborate and developed models would be necessary to more fully recapitulate and understand CLL cell proliferation and apoptosis in response to ibrutinib and DAPK inhibition in vitro.

Conclusion
In conclusion, we have identified an enzyme activated downstream of the BCR signalling pathway, known as death-associated protein kinase 3 (DAPK3). DAPK inhibition represses CLL cell proliferation in vitro and prevents expression of genes transcribed in response to activation of the BCR pathway. We have determined that DAPK3 is closely associated with these genes during transcription and that it participates in the processing of their mRNA. Moreover, DAPK3 inhibition appeared to have a broader effect in repressing pathways involved in cancer proliferation than ibrutinib. Our data suggest that DAPK3 inhibition could be an alternative to ibrutinib treatment in CLL, particularly in cases where resistance is associated with BTK/ PLCc2 mutations, but also more generally in cancers characterised by DAPK1-silencing, which is a common marker of poor prognosis.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. DAPK3/ZIPK was a candidate kinase for H3T6 and H3T11 phosphorylation. Fig. S2. MEK1/2, RSK1/2 and PKCs inhibitors do not significantly inhibit IgM-induced expression of EGR1 and DUSP2 in CLL cells. Fig. S3. qPCR data analysis of EGR1 and DUSP2 gene expression at 30-60 minutes post-anti-IgM/ CD40L stimulation in HBL1 cells. Fig. S4. ChIP-qPCR data from CLL cells assessing levels of RNA polymerase II (left) and RNA polymerase II S2-P (right) binding across the EGR1 and DUSP2 loci. Fig. S5. qPCR data analysis of DUSP2 primary transcript in HBL1 cells transfected with siRNAs against DAPK3. Fig. S6. Effects of DAPK3 inhibition on CLL cell proliferation and viability. Table S1. Primers list.