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Apoptotic Fragmentation of Tricellulin

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell–cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. Methods: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. Results: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. Conclusions: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Introduction
Tight junctions (TJs) represent the most apical cell-cell contacts in polarized epithelial cell layers. They form essential barriers for solutes including nutrients, metabolites and toxins as well as ions, thereby separating luminal and external compartments from the interior of multicellular bodies. Accordingly, TJs are also important to exclude pathogens such as bacteria and viruses from organisms. Break-down of this barrier function is associated with different diseases and is involved in inflammatory bowel diseases [1] or in organ failure during sepsis [2,3]. However, it turned out that TJ components may also represent targets of specific bacteria and viruses to invade cells or tissues resulting in a deregulation of the cytoskeleton or of signaling pathways required for barrier maintenance [4]. Primarily, claudins and occludin form the apical sealing belt between two opposing cells where they are arranged in a network of TJ strands that restricts and regulates paracellular flux depending on the specific claudin composition. The situation is different at tricellular contacts where apical TJs strands of three neighboring cells meet and thus are assumed to represent weak points for the paracellular TJ network [5]. At these tricellular tight junctions, bicellular TJs converge, and TJ strands turn basally and form the so-called central sealing element [6]. These tricellular TJs (tTJs) differ from bicellular TJs (bTJs) by a specific set of proteins including tricellulin [7] and one of the three angulins [8].
Tricellulin (marvelD2) together with occludin and marvelD3 form the TJ-associated MARVEL protein (TAMP) family of 4-transmembrane-domain proteins with long N-and C-terminal domains both located in the cytosol. The tricellulin C-terminus interacts with the cytosolic adapter protein zona occludens-1 (ZO-1) and is thereby linked to the actin cytoskeleton [9][10][11]. Knock-down of tricellulin impairs epithelial barrier function and results in disorganized bTJs, indicating an essential role of tricellulin in maintenance of overall TJ structure and function [7]. Tricellulin at tTJs was shown to restrict macromolecular passage and strong overexpression contributes to reduced paracellular permeability [12,13]. Interestingly, in occludin knock-down cells, tricellulin is mislocated to bTJs preferentially at edges of elongating bTJs [14]. How homomeric or heteromeric tricellulin/occludin complexes may be involved in this process or in the transport of tricellulin to the TJs [15] is currently not understood. However, it is obvious that tricellulin and occludin do not form heterophilic trans-interactions within established TJs [11,16].
With the identification of LSR (lipolysis-stimulated lipoprotein receptor)/angulin-1 as a tTJ-located receptor that recruits tricellulin to tTJs, it became clear that a specific anchor-protein is responsible for the defined localization of tricellulin. Knock-down of LSR/angulin-1 impaired barrier function in reducing transepithelial resistance (TER) probably as a consequence of mislocalization of tricellulin to bTJs [17]. In addition, mislocalization of LSR was associated with cancer progression and metastasis [18]. Based on sequence homology, two LSR/angulin-1-related proteins including immunoglobulin-like domain-containing receptor (ILDR) 1/angulin-2 and ILDR 2/angulin-3 were identified. The three members show tissue-specific expression patterns and at least one family member is located at tTJs [8,19]. However, it is still open regarding how angulins are directed to tTJs.
The physiological importance of tricellulin for hair cells is emphasized by mutations in the tricellulin C-terminal domain found in patients with nonsyndromic deafness (DFNB49). All mutations result in tricellulin variants with premature stop codons and, consequently, structurally impaired C-terminal ends and limited binding to ZO-1 [20,21]. Moreover, truncation of the tricellulin C-terminal domain diminishes its correct localization to tricellular contacts [22]. Tricellulin knock-out mice show a progressive hearing loss due to apoptotic death of hair cells [23]. It is currently not clear why tricellulin knock-out mice show this limited and hair cell-specific phenotype although tricellulin was deleted in all other tissues too. Interestingly, ILDR1/angulin-2 deficiency causes a similar phenotype with progressing hearing loss by outer hair cell degeneration [19,24,25]. Whether compensatory effects or regulatory roles of tricellulin and/or angulins may play a role still has to be solved.
Little is known in respect to mechanisms involved in regulation of tricellulin function. Like occludin, tricellulin can be phosphorylated [7]. However, in occludin, a multitude of kinases and corresponding phosphosites as well as functional consequences of these modifications have been studied in detail [26]. A phosphorylation hotspot region [27] that seems to be involved in the regulation of occludin mobility, apical junctional complex dynamics and cell migration [28] has been identified. In contrast, tricellulin phosphorylation-sites have been found by mass spectrometry, but, to our knowledge, none of them has been functionally characterized. However, there is the first evidence that occludin and tricellulin are differentially targeted by specific kinases [29]. Interestingly, JNK1/JNK2-dependent phosphorylation of LSR/angulin-1 was reported to be crucial for its exclusive localization to tTJs [30]. In addition, tricellulin was shown to be targeted by the ubiquitin ligase Itch, which might affect its stability and trafficking [31].
To maintain epithelial functionality, old or damaged cells have to be removed by apoptosis and replaced by new cells. Apoptosis is induced by extrinsic and intrinsic signals. Both pathways in an initial step lead to activation of initiator caspases, which subsequently activate effector caspases. These finally hydrolyze essential structural and housekeeping proteins by cleavage after aspartate residues [32]. Caspases have multiple functions also in inflammation and immunity [33]. Extrusion of apoptotic cells from the cell layer ideally occurs without loss of barrier function and disruption of the cytoarchitecture [34]. In consequence, contacts between the dying and neighboring cells have to be disassembled. Previous studies have analyzed bTJs in this respect [35], but, to our knowledge, tTJs have not been investigated in more detail. Here, we induced apoptosis with staurosporine or camptothecin and observed a caspase-dependent cleavage of the C-terminus of tricellulin. We identified two specific caspase cleavage-sites and mutation of these sites protected tricellulin from fragmentation. In addition, we provide evidence that LSR/angulin-1, which anchors tricellulin to tTJs, is also targeted by caspases.

Caspase-Dependent Cleavage of Tricellulin upon Apoptotic Stimuli
To investigate the fate of tricellulin during apoptosis, MDCKII cells were treated with staurosporine as a known inducer of apoptosis [36]. At different time points after addition of staurosporine, both floating and adherent cells were collected and lysed for subsequent Western blot analysis. Already three hours after induction of apoptosis, a time-dependent decline of endogenous tricellulin was detectable compared to DMSO-treated control cells ( Figure 1A,B). Induction of apoptosis was confirmed by detection of cleaved poly(ADP-ribose)-polymerase (PARP), a well-known effector caspase substrate. The typical PARP cleavage fragment was detectable only in staurosporine-treated cells but not in control cells ( Figure 1A). Moreover, when camptothecin was used as an alternative apoptosis inducer, the loss of endogenous tricellulin in MDCKII cells was detectable between 8 h and 24 h (Supplementary Figure S1). The produced tricellulin cleavage fragments were not detectable using the anti-tricellulin monoclonal antibody (clone 54H19L38). Immunofluorescence microscopy confirmed degradation of tricellulin. Signals for endogenous tricellulin disappeared with time ( Figure 1C). camptothecin and observed a caspase-dependent cleavage of the C-terminus of tricellulin. We identified two specific caspase cleavage-sites and mutation of these sites protected tricellulin from fragmentation. In addition, we provide evidence that LSR/angulin-1, which anchors tricellulin to tTJs, is also targeted by caspases.

Caspase-Dependent Cleavage of Tricellulin upon Apoptotic Stimuli
To investigate the fate of tricellulin during apoptosis, MDCKII cells were treated with staurosporine as a known inducer of apoptosis [36]. At different time points after addition of staurosporine, both floating and adherent cells were collected and lysed for subsequent Western blot analysis. Already three hours after induction of apoptosis, a time-dependent decline of endogenous tricellulin was detectable compared to DMSO-treated control cells ( Figure 1A,B). Induction of apoptosis was confirmed by detection of cleaved poly(ADP-ribose)-polymerase (PARP), a wellknown effector caspase substrate. The typical PARP cleavage fragment was detectable only in staurosporine-treated cells but not in control cells ( Figure 1A). Moreover, when camptothecin was used as an alternative apoptosis inducer, the loss of endogenous tricellulin in MDCKII cells was detectable between 8 h and 24 h (Supplementary Figure S1). The produced tricellulin cleavage fragments were not detectable using the anti-tricellulin monoclonal antibody (clone 54H19L38). Immunofluorescence microscopy confirmed degradation of tricellulin. Signals for endogenous tricellulin disappeared with time ( Figure 1C). From our observations, we hypothesized that tricellulin like occludin [35] is a caspase target during apoptosis. To verify that the loss of tricellulin is indeed the consequence of caspase cleavage MDCKII cells were pre-treated with the pan-caspase inhibitor Z-VAD-FMK or the caspase-3 inhibitor Z-DEVD-FMK before adding staurosporine. Both caspase inhibitors significantly reduced degradation of endogenous tricellulin ( Figure 1D). Inhibition of caspase activity was confirmed by the reduced formation of the PARP cleavage-fragment. A similar effect was obtained in RT-112 cells (bladder carcinoma) ( Figure 1E) showing that the observed effect is not restricted to MDCKII cells.

Mapping of Potential Caspase Cleavage-Sites in Tricellulin
For prediction of possible caspase cleavage-sites in human tricellulin, the online software tool CaspDB [37] was applied. Two potential caspase cleavage-sites with the highest scores were predicted in the C-terminal domain of human tricellulin after amino acid aspartate 487 (D487) and/or aspartate 441 (D441) (Figure 2A). To confirm caspase-3-mediated cleavage in the C-terminal domain of tricellulin, we expressed and purified a GST-TricC fusion protein as reported previously [29,31] and applied in vitro digestion using recombinant active caspase-3. In the presence of caspase-3, two additional fragments with a molecular mass of about 40 and 35 kDa were detectable by Western blot analysis, representing potential caspase-3 cleavage-products. The molecular mass of these fragments coincides with the predicted molecular weight of GST-TricC fragments when cleaved at D487 (frag1,~41 kDa) and D441 (frag2,~35 kDa) ( Figure 2B). Remarkably, fragment 1 is generated earlier and more efficiently than fragment 2, suggesting that D487 is the preferred caspase-3 cleavage-site and D441 appears to be used as a sequential second site after cleavage at D487. Addition of the pan-caspase inhibitor Z-VAD-FMK inhibited the generation of both GST-TricC fragments ( Figure 2C). As control, purified recombinant GST alone was analyzed for caspase-3 cleavage, but no fragmentation was detectable ( Figure 2D). Finally, we generated GST-TricC-D487N, GST-TricC-D441N and GST-TricC-D441N/D487N double mutant constructs and subjected them to in vitro cleavage-assays with recombinant caspase-3. Consistent with our previous results, caspase cleavage of GST-TricC-D487N only resulted in formation of fragment 2 (frag2), whereas cleavage of GST-TricC-D441N only generated fragment 1 (frag1). The GST-TricC-D441N/D487N double-mutated construct was no longer cleaved by recombinant caspase-3 ( Figure 2E).  To validate the in vitro results, N-terminally FLAG 3 -tagged human tricellulin was transiently transfected into MDCKII cells. Cells were subsequently treated with or without staurosporine for 6 h in the presence or absence of pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVD-FMK, respectively. After induction of apoptosis, two bands with a molecular weight of about 55 kDa and 65 kDa were detectable ( Figure 3A). Fragmentation was abrogated in the presence of each of the caspase inhibitors. In contrast to wildtype FLAG 3 -Tric, transient transfection of a mutated FLAG 3 -Tric-D441N construct abolished the generation of caspase-3 cleavage product frag 2 (~55 kDa) upon induction of apoptosis with staurosporine. Generation of cleavage-product frag 1 (~65 kDa) was not affected. Transfection of mutated FLAG 3 -Tric-D487N revealed no fragment 1 and only to a very limited amount fragment 2. When the double-mutated FLAG 3 -Tric-D441N/D487N was transfected, none of the fragments was detectable. These observations suggest that cleavage at D487 supports caspase-3-mediated cleavage at D441 ( Figure 3B). Taken together, these results confirm D487 and D441 as potential caspase-sites in human tricellulin that are targeted in apoptotic cells. To validate the in vitro results, N-terminally FLAG3-tagged human tricellulin was transiently transfected into MDCKII cells. Cells were subsequently treated with or without staurosporine for 6 h in the presence or absence of pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVD-FMK, respectively. After induction of apoptosis, two bands with a molecular weight of about 55 kDa and 65 kDa were detectable ( Figure 3A). Fragmentation was abrogated in the presence of each of the caspase inhibitors. In contrast to wildtype FLAG3-Tric, transient transfection of a mutated FLAG3-Tric-D441N construct abolished the generation of caspase-3 cleavage product frag 2 (~ 55 kDa) upon induction of apoptosis with staurosporine. Generation of cleavage-product frag 1 (~ 65 kDa) was not affected. Transfection of mutated FLAG3-Tric-D487N revealed no fragment 1 and only to a very limited amount fragment 2. When the double-mutated FLAG3-Tric-D441N/D487N was transfected, none of the fragments was detectable. These observations suggest that cleavage at D487 supports caspase-3-mediated cleavage at D441 ( Figure 3B). Taken together, these results confirm D487 and D441 as potential caspase-sites in human tricellulin that are targeted in apoptotic cells.

The Functional Interaction of Tricellulin and LSR Is Disrupted during Apoptosis
Tricellulin is recruited to tTJs by lipolysis-stimulated lipoprotein receptor (LSR/angulin-1) in epithelial and endothelial cells [38,39]. This interaction is mediated by the cytosolic C-terminus of tricellulin [17]. In this context, the question arises if caspase-mediated cleavage within the cytosolic C-terminus of tricellulin affects its interaction with LSR. Therefore, co-immunoprecipitation experiments were performed using cell lysates obtained from HEK-293 cells transiently transfected with LSR together with either full-length tricellulin or deletion constructs lacking amino acids 487-558 (FLAG 3 -Tric∆487-558), amino acid 441-558 (FLAG 3 -Tric∆441-558) or complete cytosolic C-terminus (FLAG 3 -Tric∆C) ( Figure 4A). Confirming literature, co-transfection of FLAG 3 -Tric and green fluorescent protein GFP-tagged LSR in HEK-293 cells revealed an interaction of both proteins in co-immunoprecipitation experiments, whereas FLAG 3 -Tric∆C did only show a weak signal for GFP-LSR ( Figure 4B). Similar to FLAG 3 -Tric∆C, an interaction between GFP-LSR and FLAG 3 -Tric∆487-558 or FLAG 3 -Tric∆441-558 protein lacking the cytosolic C-terminal parts released by caspases was not detectable ( Figure 4B). In this context, it is interesting to note that, in a Western blot experiment, using the anti-tricellulin (clone 54H19L38) ABfinity TM rabbit monoclonal antibody generated against amino acids 369-558 of human tricellulin did no longer detect neither the FLAG 3 -Tric∆487-558 nor the FLAG 3 -Tric∆441-558 protein in transiently transfected cells, thus suggesting that the epitope of this antibody is located between amino acids 487-558 (Supplementary Figure S2).  These results indicate that caspase-mediated cleavage of tricellulin liberates it from angulin and thus from tTJs. However, this does not exclude that LSR/angulin-1 itself is targeted during apoptosis. Therefore, we next analyzed the fate of LSR/angulin-1 after induction of apoptosis in MDCKII cells. Indeed, staurosporine treatment led to a fragmentation of endogenous LSR/angulin-1 with time. Already 4 h after induction of apoptosis, four fragments of LSR were detectable using an anti-LSR antibody targeting the cytosolic part of LSR ( Figure 5A). LSR/angulin-1 cleavage during apoptosis was inhibited by both caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. This indicates that LSR fragmentation is a consequence of caspase activation during apoptosis ( Figure 5B). These results indicate that caspase-mediated cleavage of tricellulin liberates it from angulin and thus from tTJs. However, this does not exclude that LSR/angulin-1 itself is targeted during apoptosis. Therefore, we next analyzed the fate of LSR/angulin-1 after induction of apoptosis in MDCKII cells. Indeed, staurosporine treatment led to a fragmentation of endogenous LSR/angulin-1 with time. Already 4 h after induction of apoptosis, four fragments of LSR were detectable using an anti-LSR antibody targeting the cytosolic part of LSR ( Figure 5A). LSR/angulin-1 cleavage during apoptosis was inhibited by both caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. This indicates that LSR fragmentation is a consequence of caspase activation during apoptosis ( Figure 5B).

Discussion
Release of apoptotic cells from epithelial and endothelial layers is critical for the maintenance of the barrier function and is often accompanied with a corresponding local decrease of transepithelial resistance [40]. In the gut, it is of importance that apoptotic cells, which are continuously extruded from the epithelium, do not generate microbial entry sites. Thus, it was not surprising that, in epithelial cell layers, there are mechanisms that efficiently break-down cell-cell contacts between apoptotic and neighboring cells. In previous studies, it was shown that components of the cadherincatenin adhesion complex are targets of caspases. The adherens junction proteins E-and VE-cadherin are both cleaved by metalloproteinases, resulting in a release of the extracellular domain and thereby disrupting their trans-interactions with cadherin molecules on the surface of neighboring cells. Caspases are responsible for the cleavage of the cytosolic domains, thus disconnecting the interaction with the actin cytoskeleton [36,41]. Moreover, β-catenin as a linker between E-cadherin and the actin cytoskeleton is a caspase target itself [42]. Along with the adherens junctions, desmosomal cadherins

Discussion
Release of apoptotic cells from epithelial and endothelial layers is critical for the maintenance of the barrier function and is often accompanied with a corresponding local decrease of transepithelial resistance [40]. In the gut, it is of importance that apoptotic cells, which are continuously extruded from the epithelium, do not generate microbial entry sites. Thus, it was not surprising that, in epithelial cell layers, there are mechanisms that efficiently break-down cell-cell contacts between apoptotic and neighboring cells. In previous studies, it was shown that components of the cadherin-catenin adhesion complex are targets of caspases. The adherens junction proteins E-and VE-cadherin are both cleaved by metalloproteinases, resulting in a release of the extracellular domain and thereby disrupting their trans-interactions with cadherin molecules on the surface of neighboring cells. Caspases are responsible for the cleavage of the cytosolic domains, thus disconnecting the interaction with the actin cytoskeleton [36,41]. Moreover, β-catenin as a linker between E-cadherin and the actin cytoskeleton is a caspase target itself [42]. Along with the adherens junctions, desmosomal cadherins and components of the cytosolic desmosomal plaque are inactivated similarly during apoptosis [43]. In addition, occludin, ZO-1 and ZO-2 were identified as caspase substrates [35]. Here, we extended this study to tricellulin and show that the C-terminal cytosolic tail of tricellulin is also targeted by caspases independent from whether apoptosis is induced by staurosporine or camptothecin and also independent from the cell system, in that case MDCKII or RT-112 cells. We identified two cleavage sites in tricellulin and mapped them C-terminal to Asp441 and Asp487. Specificity of cleavage was verified by treatment with caspase inhibitors and by mutation of the corresponding aspartate to asparagine, preventing the formation of cleavage fragments both in apoptotic cells as well as in in vitro fragmentation assays.
From our experiments, we conclude that D487 is the preferred cleavage-site. This is based on the observation that the corresponding cleavage-fragment was detectable far earlier as compared to the product generated by cleavage at D441 in in vitro cleavage-assays ( Figure 2B). Moreover, it seems that initial cleavage at D487 is prerequisite for efficient cleavage at D441. This was confirmed by analyzing FLAG-tagged tricellulin where caspase cleavage-sites were mutated. The induction of apoptosis in cells transfected with wildtype FLAG 3 -tricellulin revealed two cleavage fragments. However, in cells transfected with FLAG 3 -tricellulin-D487N, a fragment generated by cleavage at D441 was more or less not detectable, most likely due to the lack of the initial cleavage at D487 ( Figure 3B).
Caspase cleavage of tricellulin liberates a fragment of 71 C-terminal amino acids containing a region known to form a coiled-coil dimer [10]. This fragment was not detectable, even not when using high percentage SDS-PAGE gels, either because this small fragment escaped detection on Western blots or due to further degradation. Based on our observations, the antibody used in this study appears to bind to this fragment since truncated tricellulin where the C-terminus is deleted after D487 was no longer detectable. Definitively, it is worth testing by other methods if this fragment is detectable and of functional relevance. Not only due to its size can it easily translocate to the nucleus where it might be involved in transcriptional regulation, as shown for the amyloid precursor protein intracellular domain (AICD) [44] or the Notch intracellular domain (NICD) [45]. It has also been reported that nuclear localization of full-length tricellulin in pancreatic cancer promotes cell proliferation and invasiveness [46].
Based on our studies, we cannot exclude that the remaining tricellulin N-terminal part undergoes further processing steps as observed for occludin. There, the induction of apoptosis leads to caspase cleavage in the cytosolic C-terminus and to further fragmentation by metalloproteinases probably in the first extracellular loop [35]. First evidence for a potential contribution of metalloproteinases is provided by a study in Caco2 cells, where MMP-2-and MMP-3-catalyzed degradation of tricellulin is induced by N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) [47].
A consequence of caspase cleavage and the concomitant release of the tricellulin C-terminal fragment is the disruption of the ZO-protein-mediated linkage of tricellulin to the actin cytoskeleton. Moreover, loss of the C-terminal amino acids might impair localization of tricellulin to tTJs by LSR/angulin-1. However, we observed that also LSR/angulin-1 is fragmented after induction of apoptosis. We conclude that, during apoptosis, tTJs are targeted at more than one level.
Taken together, here we showed that tricellulin is efficiently targeted by caspases in apoptotic cells within its C-terminal coiled-coil domain at D487 and D441. This together with concomitant cleavage of LSR/angulin-1 may contribute to a coordinated release of apoptotic cells from cell layers as it occurs continuously in the gastrointestinal tract at high frequency.

Plasmids, Site-Directed Mutagenesis and Transient Transfections
Plasmids were cloned by standard procedures and verified by sequencing. The cloning of p3xFLAG-CMV10-Tric and p3xFLAG-CMV10-Tric∆C constructs was described previously [15]. To generate caspase cleavage-site mutated tricellulin variants, site-directed mutagenesis of the potential caspase cleavage-sites at positions D441 and D487 to asparagine was performed by SBOE (splicing by overlap extension)-PCR. Oligonucleotides used for the SBOE-PCR reactions are summarized in Table 2. The mutated tricellulin cDNAs were cloned into the vector p3xFLAG-CMV10 (Sigma-Aldrich, Taufkirchen, Germany) using BamHI restriction site. The C-terminal tricellulin deletion constructs Tric∆441-558 and Tric∆487-558 were generated by PCR using the oligonucleotides 5 -CGG ATC CTC AAA TGA TGG AAG ATC CAG-3 as forward primer and 5 -CGG ATC CTT AGG GCA TCA CGA TAG GTT TAG-3 or 5 -GGA TCC TTA CAG CTC ATC AAA CTT CCT CA-3 as reverse primers, respectively, and cloned into the BamHI restriction site of p3xFLAG-CMV10 (Sigma-Aldrich, Taufkirchen, Germany). For recombinant expression of the tricellulin C-terminal cytosolic domain, pGEX4T1-TricC described previously [31] was used. The caspase-site mutated variants of GST-TricC were constructed by PCR employing oligonucleotides 5 -GCG GGA TCC ATG TGG AGG CAT GAG GCA GCT C-3 and 5 -GCG GGT ACC GGA TCC TTA AGA ATA ACC TTG TAC ATC-3 using p3xFLAG-CMV10-Tric-D441N, -D487N or -D441N/D487N as templates. After digestion with BamHI, the amplified products were ligated into BamHI-cleaved and dephosphorylated pGEX4T1 (GE Healthcare, Freiburg, Germany). pCAGGS-LSR for expression of GFP-tagged LSR was obtained from Mikio Furuse and described in [17].
MDCKII and HEK-293 cells were transiently transfected with the indicated plasmids using a DNA:PEI (poyethylenimine) ratio of 1:4 as described previously [48]. Cells were seeded in 6-well plates (2 × 10 5 cells for MDCKII and 5 × 10 5 HEK-293 cells per well one day before transfection. For transiently transfected HEK-293 or MDCKII cells, one or two 6-wells were lysed per sample. Empty vectors were transfected as a control. Table 2. Sequences of oligonucleotides used to generate mutated tricellulin constructs. The bases marked in red represent those bases that differ from the wild-type sequence to generate the indicated mutations.

Mutation
Product Sequence Oligonucleotides

In Vitro Caspase Cleavage
Recombinant glutathione S-transferase (GST)-tagged c-cytosolic (amino acids 366-558) domain of tricellulin wild type and mutant (D441N; D487N; D441N/D487N) proteins were expressed and purified as reported elsewhere [49]. In total, 4 µg of recombinant protein was digested with 150 ng of recombinant, active caspase-3 (BD Biosciences, Heidelberg, Germany) in 20 mM Pipes pH 7.2, 100 mM NaCl, 1 mM EDTA, 10% (w/v) sucrose, 10 mM DTT and 0,1% (w/v) CHAPS at 37 • C for the indicated incubation times. For inhibition of the in vitro caspase cleavage, 10 µM of pan-caspase inhibitor Z-VAD-FMK or DMSO as a control was added. Finally, samples were analyzed by SDS-PAGE and Western blot analysis.

Cell Lysis and Co-Immunoprecipitation
HEK-293 cells were seeded in 6-well plates and transfected (1µg DNA each construct) with PEI as described above. Cells (two 6-wells) were harvested 48 h after transfection and lysed in 300 µL ice-cold modified RIPA-buffer including Complete™ protease inhibitor mix (Roche Life Science, Mannheim, Germany) for 20 min on ice. After lysis, samples were centrifuged (10 min, 20,800× g, 4 • C) to remove insoluble material. For co-immunoprecipitation experiments, 200 µL lysate was incubated with 2 µg of anti-FLAG ® M2 antibody for 1 h at 4 • C under constant rotation. Subsequently, 30 µL of equilibrated Protein A Sepharose CL-4B beads (GE Healthcare, Freiburg, Germany) were added and incubated for further 30 min. Finally, beads were washed for three times with lysis buffer and protein complexes were eluted using 2 × SDS-loading buffer at 95 • C for 5 min. Samples were separated by SDS-PAGE and subsequently analyzed by Western blotting. For each experiment, 10 µL of cell lysate (5% of input) was loaded as a control.

Western Blot Analysis
Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane by tank blotting. After blocking with 5% (w/v) dry milk in Tris-buffered saline/Tween20 (TBS-T) for 1 h, membranes were incubated with the primary antibodies overnight at 4 • C and with horseradish peroxidase (HRP)-labeled secondary antibody at least for 1 h at room temperature (Table 1).

Immunofluorescence Microscopy
For immunofluorescence microscopy, MDCKII cells were seeded on transwell filter supports (Millicell Cell Culture Insert, 12 mm, 0.4 µm, Merck Millipore, Darmstadt, Germany) with a density of 150,000 cells/cm 2 . At day 7, cells were treated with 1 µM staurosporine or DMSO for 4 h and 7 h. Subsequently, the cells were fixed with 2% paraformaledhyde in PBS and stained with 1 µg/mL anti-tricellulin (clone 54H19L38) antibody for 18 h at 4 • C followed by secondary antibody incubation (donkey anti-rabbit, Alexa-594 conjugated) for 1 h at room temperature. Nuclei were labeled using DAPI (Sigma-Aldrich, Taufkirchen, Germany). Images (z-stacks) were acquired with a laser scanning system TC/SP5 (Leica Microsystems, Wetzlar, Germany) and were maximum intensity projected. Acknowledgments: We thank Mikio Furuse for the GFP-tagged LSR expression vectors. Expert technical assistance by Manuela Neumann is gratefully acknowledged.

Conflicts of Interest:
The authors declare no conflict of interest.


extfieldsofstudy: Medicine Biology

text:
Characterization of complete mitochondrial genome of seven-eleven crab Carpilius maculatus (Linnaeus, 1758)

Abstract In this study, we first determined and characterised the complete mitochondrial genome of seven-eleven crab Carpilius maculatus from South China Sea. The C. maculatus mitogenome is 15,761 bp long, and consists of 22 tRNA genes, two rRNA genes, 13 protein-coding genes (PCGs), and one control region. The nucleotide composition of C. maculatus mitogenome is significantly biased (A, G, T, and C was 35.9%, 9.8%, 35.5%, and 18.9%, respectively) with A + T contents of 71.3%. Three PCGs used an unusual initiation codon, and four PCGs were terminated with an uncomplete or abnormal stop codon. Six microsatellites were identified in C. maculatus mitogenome sequences. Phylogenetic tree showed that C. maculatus formed monophyletic clade with other Heterotremata species.

Carpilius maculatus; mitochondrial genome; phylogenetic analysis Carpilius maculatus, common names seven-eleven crab or spotted reef crab, belongs to the family Carpiliidae. It is probably the most prominent representative of its genus, on its surface with 11 symmetrically disposed red blots (Raju et al. 2015). It is a reef crab, and found on coral and rocky reefs in Indo-West Pacific moving slowly along sandy bottoms (Zacharia et al. 2008). They feed nocturnally on marine snails. Although occasionally offered on the Southeast Asian markets as food, and there are reports that it is poisonous, but so far it could also by biochemical tests have not been confirmed (Holthuis 1968;Halstead and Cox 1973). It is considered that the crab possible by eating of poisonous snails will also be toxic, but even this is not confirmed. In addition, the first-stage zoaea of C. maculatus has also been studied (Clark et al. 2004).
The samples were collected from Huanqiu wharf of Wenchang, China (N19 33 0 51.12 00 , E110 49 0 27.98 00 ), and stored in the marine crustacean specimen room (C20190616CM) in Qionghai research base of Hainan Academy of Ocean and Fisheries Sciences for reference, Muscle samples of C. maculatus were preserved in absolute ethanol for total DNA extraction.
The whole mitogenome of C. maculatus is 15,761 bp in size (GenBank Accession No. MN381805). The base content was 35.9% A, 9.8% G, 35.5% T, and 18.9% C. The 71.3% of (A þ T) showed great preference to AT. It consists of 22 tRNA genes, two rRNA genes, 13 protein-coding genes (PCGs), and one control region (D-loop). Four PCGs (ND1, ND4, ND4L and ND5), eight tRNA genes and two rRNA genes were located on the light strand, the others were encoded by the heavy strand.
The 22 tRNA genes in mitogenome of C. maculatus vary in length from 63 bp to 74 bp. tRNA-Leu and tRNA-Ser both have two type copies respectively. The 12S rRNA is 843 bp and located between tRNA-Val and D-loop, and the 16S rRNA is 1342 bp, located between tRNA-Val and tRNA-Leu. Except ND1 using an unusual GTG as the start codon, the others use a normal initiation codon ATN. Simultaneously, most PCGs were terminated with a usual codon TAA or TAG in addition to COX2 and CYTB genes using an incomplete stop codon TT or T respectively. The control region is 758 bp, located between 12S rRNA and tRNA-Ile. Interestingly, we identified six microsatellites (SSRs) in C. maculatus mitogenome using MISA. A (A) 10 is located in ND1 genes, a (TA) 6 in ND4 genes, a (T) 10 in 16S rRNA, a (A) 10 and (AT) 7 both in D-loop region, and a (T) 10 is sited in non-coding sequences.
A maximum likelihood phylogenetic analysis with 36 crab mitogenomes placed C. maculatus among Heterotremata species with 1000 bootstrap replicates. The result (Figure 1) showed that Carpilioidea represented by C. maculatus formed monophyletic clade with other Heterotremata species, and further clarified the phylogenetic relationships of the superfamily in Heterotremata compared with the previous work (Wetzer et al. 2003;Lai et al. 2014).
CONTACT Yugui He yghgood@163.com Hainan Academy of Ocean and Fisheries Sciences, Haikou, China


s2fieldsofstudy: Biology Chemistry Medicine

text:
Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

GSK3α has been identified as a new target in the treatment of acute myeloid leukemia (AML). However, most GSK3 inhibitors lack specificity for GSK3α over GSK3β and other kinases. We have previously shown in lung cancer cells that GSK3α and to a lesser extent GSK3β are inhibited by the advanced clinical candidate tivantinib (ARQ197), which was designed as a MET inhibitor. Thus, we hypothesized that tivantinib would be an effective therapy for the treatment of AML. Here, we show that tivantinib has potent anticancer activity across several AML cell lines and primary patient cells. Tivantinib strongly induced apoptosis, differentiation and G2/M cell cycle arrest and caused less undesirable stabilization of β-catenin compared to the pan-GSK3 inhibitor LiCl. Subsequent drug combination studies identified the BCL-2 inhibitor ABT-199 to synergize with tivantinib while cytarabine combination with tivantinib was antagonistic. Interestingly, the addition of ABT-199 to tivantinib completely abrogated tivantinib induced β-catenin stabilization. Tivantinib alone, or in combination with ABT-199, downregulated anti-apoptotic MCL-1 and BCL-XL levels, which likely contribute to the observed synergy. Importantly, tivantinib as single agent or in combination with ABT-199 significantly inhibited the colony forming capacity of primary patient AML bone marrow mononuclear cells. In summary, tivantinib is a novel GSK3α/β inhibitor that potently kills AML cells and tivantinib single agent or combination therapy with ABT-199 may represent attractive new therapeutic opportunities for AML.

which is approved for the treatment of epilepsy and bipolar disorder 7,8 . However, given the narrow therapeutic index of LiCl, the lack of GSK3α specificity, and its limited kinome-wide selectivity 9,10 , its utility as an AML therapy is questionable. There are a number of GSK3 inhibitors in development, but current compounds are either highly unselective featuring various off-targets in addition to GSK3α/β, lack isoform selectivity or have not yet advanced to clinical studies 11,12 . We have previously identified GSK3α/β as novel targets of tivantinib (ARQ197) 13 , an advanced clinical drug candidate, which was initially thought to be a highly specific MET inhibitor 14 . We observed that tivantinib, compared to other GSK3 inhibitors, has remarkable kinome-wide selectivity for GSK3α/β, as well as a slight preference for GSK3α over GSK3β. Considering the identification of GSK3α as a potential pro-tumorigenic signaling protein, we hypothesized that tivantinib may be an effective, novel therapeutic option for AML. In the current study, we therefore characterized tivantinib's anticancer activity in AML cell lines, identified a synergistic drug combination with the BCL-2 inhibitor ABT-199, and demonstrated its efficacy in primary AML samples. The results presented herein suggest that tivantinib, either as a single agent or in combination with ABT-199, may be a novel and attractive targeted therapy option for AML.
Viability assays and synergy calculations. Cell viability assays were conducted according to manufacturer's specifications for CellTiter-Glo Luminescent Cell Viability Assay (Promega). Cells were seeded at 1000 or 3000 cells/well in a 384 well microtiter plate and treated after 24 hours with drug diluted in the respective culture medium at the indicated concentrations. Cells were treated for 72 hours before the addition of CellTiter-Glo reagent and read on a M5 Spectramax plate reader (Molecular Devices). Raw data was normalized to DMSO controls and a three-parameter log-logistic function was fit to the data for an IC 50 estimation using R. For the synergy screen, tivantinib was plated at 0 µM and 0.25 µM, respectively. Each library drug was tested at 0.5 µM and 2.5 µM, respectively. Subsequent drug combination effects were evaluated by the Bliss method.
Flow cytometry. Cells were treated as indicated with DMSO, NaCl, LiCl, or tivantinib. For cell cycle experiments, cells were harvested following incubation, fixed with 70% cold ethanol and stored at −20 °C until analyzed. Cells were washed with PBS and cell cycle was determined by incubating in a 1 μg/mL DAPI (4′,6-Diamidino-2-phenylindole, Sigma)/0.1% Triton X-100/PBS solution. For apoptosis experiments, treated cells were stained with Annexin V-APC (BD Biosciences) and 100 ng/mL DAPI according to the manufacturer's instructions. PE Mouse Anti-Human CD11b/Mac-1 (BD Biosciences) was used to monitor cell differentiation. Analyses were conducted using a FACSCanto II benchtop analyzer (BD Biosciences). Cell cycle was analyzed using ModFitLT V3.2.1 (Verity Software House). Apoptosis and differentiation data was analyzed using Flowjo (Treestar, Inc.). Analyses represent data for singlet cells using a standard aggregate gating strategy.
Colony formation assays. AML patient bone marrow mononuclear cells (BMNCs) were seeded into 6-well plates and treated overnight at the indicated concentrations of drug using IMDM (10% FBS) as the diluent. Gene expression profiling data analysis. GSK3α and GSK3β expression levels across different human myeloid lineages were queried using the Bloodpool aggregation of hematopoietic expression profiles from numerous studies catalogued in the manually curated BloodSpot database 15 , which provides gene expression profiles of a number of mouse/human hematopoietic cells (normal and AML).
Gene silencing sensitivity profiling data analysis. shGSK3α and shGSK3β sensitivity data was downloaded from the Project DRIVE database (https://oncologynibr.shinyapps.io/drive/) 16 and imported into python for analysis. The redundant siRNA activity (RSA) score was used as the sensitivity measurement as the RSA sensitivity is calculated using all shRNA reagents against a given gene to determine a score 17 . Tivantinib sensitivity prediction. In order to predict sensitivity of AML cell lines to tivantinib, we built a regularized linear regression model (elastic net) to select gene features that can predict a tivantinib response vector. Elastic net regularization is a machine learning algorithm that is specially suited for the case of many more input features (genes) than samples (cell lines). Candidate predictive features were selected from 18989 genes with normalized measures of gene expression in CCLE for cell lines that have tivantinib sensitivity data in CTRPv2 (n = 297) 18 . Data was split into training (0.75) and test sets (0.25). Let  ∈ X nxp be the matrix of predictive features, where n is the number of cell lines included in the training set and p is the number of features. Let  ∈ y n be the vector of sensitivity values for the same cell line panel. The elastic net attempts to find the weighted (β) linear combination of columns of features (genes) that can best approximate tivantinib AUC (y) or by solving the following: where λ and α are tunable parameters where λ controls the overall penalty and α controls the mixing ratio of L1-and L2-norm. We optimized λ and α for the model with a tuning grid of 1000 values of λ from 10e-10 to 10e10 and 10 values of α from 0 to 1 using 10000 iterations of 10-fold cross validation. The values of λ and α were chosen to be those that minimized the root mean square error for each fold. The trained model was then used to predict AML cell line sensitivity (n = 34) to tivantinib (for which data does not exist in CTRPv2 and thus not used in model training). Statistically significant differences in tivantinib sensitivity between AML and non-AML cell lines was determined by Kolmogorov-Smirnov test.
Proteomics and bioinformatic analysis. Drug affinity chromatography experiments were conducted essentially as described previously 13 . Briefly, c-(−)-tivantinib, c-(+)-tivantinib, and ampicillin were immobilized on NHS-activated Sepharose for Fast Flow resin (GE Healthcare). Non-coupled resin (blocked beads) and coupled resin were then blocked with ethanolamine for 4 hours. HL60 and U937 cells were lyzed and total cell lysate containing 10 mg of protein were added to the affinity matrix for 6 hours. Competition experiments were conducted by incubating total cell lysates with 20 μM BIO for 2 hours prior to affinity chromatography. Blocked beads were incubated with lysate without immobilized compound. A nanoflow ultra high performance liquid chromatograph (RSLC, Dionex) coupled to an electrospray bench top orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher) was used for tandem mass spectrometry peptide sequencing experiments. Samples were first loaded onto a pre-column (2 cm × 100 µm ID packed with C18 reversed-phase resin, 5 µm, 100 Å) and washed for 8 minutes with aqueous 2% acetonitrile (ACN) and 0.04% trifluoroacetic acid. The trapped peptides were eluted onto the analytical column, (C18, 75 µm ID × 50 cm, 2 µm, 100 Å, Dionex). The 120-minute gradient was programmed as: 95% solvent A (2% ACN + 0.1% formic acid) for 8 minutes, solvent B (90% ACN + 0.1% formic acid) from 5% to 50% in 90 minutes, then solvent B from 50% to 90% B in 7 minutes and held at 90% for 5 minutes, followed by solvent B from 90% to 5% in 1 minute and re-equilibrate for 10 minutes. The flow rate on the analytical column was 300 nl/min. Sixteen tandem mass spectra were collected in a data-dependent manner following each survey scan. Both MS and MS/MS scans were performed in the Orbitrap to obtain accurate mass measurements using 60 second exclusion for previously sampled peptide peaks. Mascot searches were performed against the Swiss-Prot human database downloaded on June 12, 2014 19 . Two trypsin missed cleavages were allowed, the precursor mass tolerance was 10 ppm. MS/MS mass tolerance was 0.05 Da. Dynamic modifications included carbamidomethylation (Cys) and oxidation (Met). Mascot search results were summarized in Scaffold 4.3.
Subsequently data was imported into Galaxy for analysis with APOSTL 20,21 . Data was formatted into inter, prey and bait files using total spectral counts as a measure of abundance and ampicillin drug affinity chromatography experiments as negative controls. Data was then analyzed by SAINTexpress and the CRAPome within APOSTL to determine the probability of selective interactions of proteins with tivantinib 22,23 . The resulting files were merged and summarized in APOSTL's interactive environment for analysis and visualization (Table S1).

Results
GSK3α is a drug target in AML cells. GSK3α has been described to be a novel target in AML 5 . Supporting this report, analysis of publically available expression levels of GSK3α and GSK3β using the BloodSpot database (which contains more than 2000 AML and normal samples assembled from six independent studies on AML) revealed that GSK3α is overexpressed across multiple AML subtypes as compared to normal hematopoietic lineages (Fig. S1a). Interestingly, GSK3β expression in AML differs little from normal hematopoiesis (Fig. S1b). In order to evaluate AML sensitivity to GSK3α/β gene silencing, we analyzed the publically available shRNA screening data in Project DRIVE 16 , which contains the cell viability data following shRNA gene silencing of various genes across 384 cancer cell lines. Consistent with GSK3α being overexpressed in AML, this analysis suggested that AML cell lines are significantly more sensitive (low RSA Sensitivity score) to GSK3α silencing as compared to GSK3β gene silencing (Fig. S1c). Furthermore, AML cell lines constitute the most sensitive population of hematopoietic cell lines with regard to GSK3α gene silencing (Fig. S1d). Taken together these analyses support that GSK3α is an actionable target in AML cell lines.

Tivantinib potently inhibits viability of AML cells.
Since we had previously identified GSK3α as a prominent tivantinib target 13 , we wanted to determine tivantinib's efficacy in AML cells. To the best of our knowledge, tivantinib has never been tested across multiple AML cell lines, including the various large drug screening efforts such as the Cancer Therapeutic Response Portal v2 (CTRPv2) 18 . Therefore, to evaluate tivantinib's efficacy across all AML cell lines in the Cancer Cell Line Encyclopedia (CCLE) 24  sensitivity data in CTRPv2 (which does not include tivantinib sensitivity information for AML cell lines) using the gene expression profiles of these cell lines (CCLE) as features. Our model had good accordance between predicted and experimental values (r = 0.71, AUC = 0.83) which is comparable to similar models (Figs 1a, S2a) 25 . Interestingly, many of the gene features selected through regularization are known to associate with GSK3 signaling (STRING) 26 , such as the TCF7 cofactor MLLT11, which had the most highly weighted coefficient (Fig. S2b) 27 .
We then applied this model to all AML cell lines in CCLE using their gene expression profiles, which predicted AML cell lines to be sensitive to tivantinib treatment. Interestingly, AML cell lines were predicted to be significantly more sensitive to tivantinib than non-AML cell lines (Fig. 1b).
In order to validate the predicted AML sensitivity to tivantinib, we treated HL60 and U937 AML cell lines with (−)-tivantinib, which is currently in advanced clinical development, its enantiomer (+)-tivantinib, which is a much weaker GSK3 inhibitor, the bona fide pan-GSK3α/β inhibitor LiCl and the MET inhibitor PF-04217903 as indicated. Intriguingly (−)-tivantinib, but not (+)-tivantinib, displayed nanomolar efficacy in HL60 and U937 cell lines (Fig. 1c,d). This is in accordance with our previous results in MLL-rearranged acute lymphoblastic leukemia (ALL) cell lines, which are known to be sensitive to GSK3 inhibition 13,28 . As expected, LiCl also showed strong activity (10-20 mM is a widely used concentration relevant for GSK3 inhibition by LiCl 5 , which as a salt has a different mechanism of action) while the potent and selective MET-inhibitor PF-04217903 was essentially inactive suggesting that GSK3, not MET, inhibition is responsible for tivantinib's activity in AML cells (Fig. 1c,d).
Since tivantinib has previously been suggested to elicit anticancer activity in NSCLC through disruption of microtubule dynamics 29,30 , we further evaluated the relative contribution that inhibition of MET, GSK3 or microtubule polymerization plays in tivantinib's mechanism of action in these cells. We trained additional elastic net regularized regression models to predict paclitaxel (microtubule inhibitor), SGX253 (MET inhibitor), and ML320 (highly selective GSK3 inhibitor) sensitivity across cell lines present in CTRPv2. We then applied these models to predict AML sensitivity, and performed pairwise comparisons (Spearman) of the model predictions. As expected, tivantinib's sensitivity profile was uncorrelated with SGX253 further supporting that MET is not involved in tivantinib's mechanism in these cells. Interestingly, whereas tivantinib was only weakly correlated with paclitaxel, it was highly correlated with ML320 suggesting the GSK3 inhibition is the primary mechanism in which tivantinib elicits activity in these cells (Fig. S2c). In summary, this data demonstrates that tivantinib harbors potent anticancer activity in AML cell lines and this activity can likely be explained by GSK3 inhibition.

Tivantinib binds GSK3α/β in AML cells.
To confirm tivantinib's ability to bind and inhibit GSK3α/β in these cells, we performed drug affinity chromatography using a couplable (−)-tivantinib analog as previously described (Fig. 2a) 13 . Pulldowns were of high quality with good reproducibility between biological replicates (Fig. S3a). To identify selective and potent interactions, we performed Significance Analysis of Interactomes (SAINT) 23,31 analysis and determined the relative protein abundance in the sample eluates using the Normalized Spectral Abundance Factors (NSAF) 32 , respectively. In addition, we filtered the data set for common binders using the Contaminant Repository of Affinity Purification-Mass Spectrometry Data (CRAPome) 22 . We prioritized targets with a SAINTScore ≥ 0.95, a CRAPomePCT ≥ 0.95 and an -ln(NSAF) ≥ −7 (Fig. S3b). These criteria suggested two major kinase targets of tivantinib in these cells, namely GSK3α and GSK3β (Fig. 2b,c). Tivantinib selectivity was confirmed by Western blot, where (−)-c-tivantinib much more prominently enriched GSK3α/β as compared to (+)-c-tivantinib. Importantly, the GSK3 inhibitor BIO was able to compete away GSK3α/β suggesting a specific interaction (Fig. 2d). MET protein was not observed by proteomics in these cells.

Tivantinib inhibits GSK3α/β signaling in AML cells.
To gain further insight into the downstream effects of GSK3 inhibition by tivantinib, we investigated the cellular outcome following drug treatment. Treatment of HL60 cells with tivantinib decreased GSK3α/β phosphorylation on Tyr279/216, which as an autophosphorylation site is directly correlated with GSK3 kinase activity (Fig. 3a) 33 . Furthermore, upon treatment with tivantinib we observed an increase in total β-catenin levels, which is characteristic for GSK3 inhibitors 6 . A larger and more prolonged increase in β-catenin was observed with LiCl than with tivantinib (Fig. 3a). Since β-catenin stabilization requires inhibition of both GSK3α and GSK3β 34 , this is consistent with LiCl strongly targeting both GSK3α/β isoforms and tivantinib being more selective for GSK3α, as we have shown previously by in vitro kinase assay 13 . Previous studies have suggested that tivantinib causes G2/M arrest through inhibition of microtubule polymerization 30 , an observation which could also be explained by GSK3 inhibition. We therefore investigated the effects of tivantinib and LiCl on cell cycle arrest. Tivantinib caused a pronounced and rapid increase in phosphorylation of histone H3 Ser10 (Fig. 3b), which is indicative of cell cycle arrest. Detailed flow cytometry analysis showed a strong accumulation of cells in G2/M phase upon tivantinib treatment (Figs 3c, S4). This was similarly prominent with LiCl suggesting the observed G2/M arrest is mediated through inhibition of GSK3α/β. Furthermore, after 24 h of tivantinib treatment, we observed a strong and dose-dependent induction of apoptosis as assessed by PARP-1 and caspase 3 cleavage (Fig. 3b). Consistent with previous reports 5 , this was also apparent for LiCl although less pronounced than for tivantinib, even at relatively low doses. We next assessed the timing and magnitude of the induction of apoptosis by Annexin V staining followed by flow cytometry. Similar increases in early apoptosis were observed over time between tivantinib and LiCl; however, a much larger late apoptotic population was observed with tivantinib treatment (Figs 3d, S5).
Given that GSK3α silencing by RNAi has been described to induce cell differentiation 5 , we stained HL60 cells with α-CD11b to assess the ability of tivantinib and LiCl to differentiate AML cells by flow cytometry. Interestingly, while tivantinib treatment for 96 hours resulted in a significant increase of cell differentiation, LiCl caused a much stronger effect (Figs 3e, S6), which is consistent with previous studies 5 . Thus, tivantinib and LiCl have largely similar effects on AML cells as they both induce apoptosis, G2/M arrest, and differentiation. However one notable distinction is that tivantinib more potently induces apoptosis while LiCl has a markedly larger effect on cell differentiation.
Tivantinib displays drug synergy with the BCL-2 inhibitor ABT-199. Resistance against single drug therapy with targeted agents can often be delayed or suppressed by potent drug combinations. In the case of tivantinib, we hypothesized that drug combinations may allow for a reduction of the tivantinib dose and thereby a less pronounced stabilization of β-catenin. In order to further amplify tivantinib's anticancer activity in AML cells, we conducted a drug combination screen in HL60 cells using a collection of 240 clinically relevant targeted agents. The majority of these (90+%) were in clinical development so that identification of a drug that synergizes with tivantinib would have the potential for clinical translation. The data was highly reproducible with good correlations between biological replicates (Fig. 4a). One of the strongest hits from this screen for potential synergy with tivantinib in HL60 cells was the BCL-2 inhibitor navitoclax (ABT-263) (Fig. 4a). This was interesting as cancer cell lines with activating mutations in β-catenin or increased β-catenin levels as the result of GSK3 inhibition have been shown to exhibit increased sensitivity to BCL-2 inhibitors 35 . In addition to navitoclax, we identified its newer structural analogue ABT-199 (venetoclax) as a potentially synergistic drug (Fig. 4a) 36 . We posited that since navitoclax has shown acute toxicity in patients and ABT-199 has been recently approved by the FDA for chronic lymphocytic leukemia, combination of tivantinib with ABT-199 may be a safer alternative with higher translational potential 37 . Importantly, ABT-199 has already been shown to be effective in AML cells 38,39 , leading to a recent FDA designation as a breakthrough therapy and multiple AML specific clinical trials currently recruiting patients to test the safety and efficacy of ABT-199 alone or in combination with chemotherapy. We therefore selected ABT-199 for detailed synergy analysis. In addition to a clear shift of the dose response curve for the combination treatment, synergy analysis using the Bliss model of independence across a range of concentrations suggested pronounced synergy within physiologically relevant concentrations between tivantinib and ABT-199, but antagonism with the standard of care agent cytarabine (Ara-C) (Fig. 4b).
As we had observed a strong induction of apoptosis with tivantinib treatment and as ABT-199 inhibits the anti-apoptotic protein BCL-2, we hypothesized that combining tivantinib and ABT-199 would also further increase apoptotic signaling. Indeed, upon combination treatment of tivantinib and ABT-199, we observed an increase in cleavage of caspase 3 and a complete cleavage of PARP-1 protein, in which no native 116 kDa PARP-1 remained, suggesting a large increase in apoptosis (Fig. 4c). The pronounced G2/M arrest as indicated by pS10 histone H3 following tivantinib treatment was reversed by the drug combination. ABT-199 by itself did not affect GSK3 tyrosine phosphorylation, but the combination with tivantinib caused complete loss of pY GSK3 (Fig. 4d). Importantly, addition of ABT-199 completely abrogated the increase of β-catenin that is observed with single agent tivantinib treatment (Fig. 4d). Since MCL-1 and BCL-XL expression have been shown to cause resistance to ABT-199 39,40 , we next hypothesized that the observed synergy with tivantinib was a result of altered MCL-1 and BCL-XL protein levels. Interestingly tivantinib single agent and, more pronouncedly, ABT-199 combination caused a loss of anti-apoptotic MCL-1 and BCL-XL protein levels while maintaining pro-apoptotic Bak levels (Fig. 4e). In summary, these results suggest that tivantinib and ABT-199 combination greatly increases the already strong apoptotic effects of tivantinib in AML cells by down regulating anti-apoptotic proteins while simultaneously suppressing activation of β-catenin.

The combination of tivantinib and ABT-199 is effective in primary AML patient samples.
In order to better evaluate the potential for clinical translation of our observations with tivantinib in AML, we next tested the efficacy of tivantinib in primary AML patient Bone Marrow Mononuclear Cells (BMNCs) as a single agent, as well as in combination with ABT-199. Using several different primary AML patient samples, tivantinib displayed a strong ability to inhibit colony formation at the clinically relevant concentration of 5 μM across all patients with only a few colonies remaining (Figs 5a, S7). Single drug ABT-199 treatment showed slight variations in efficacy, but on average reduced colony formation to approximately 30-40 percent consistent with previous reports (Figs 5a, S7) 39,40 . The combination of tivantinib and ABT-199 exerted synergy in 4 of 7 patients with moderate (patients 1 and 2) to strong synergy (patients 3 and 4) values ( Figure S7). Patients 5 and 7 were exquisitely sensitive to ABT-199 single agent treatment and therefore a Bliss value could not be accurately calculated. Consistent with GSK3α being overexpressed across all major AML karyotypes ( Figure S1a), tivantinib efficacy did not show any obvious relationship with mutational status or karyotypes ( Fig. 5a; Table S2) although the number of patient samples was low. Overall, these data suggest that tivantinib is highly effective in inhibiting the colony forming capacity of primary AML patient samples as a single agent or in combination with ABT-199.

Discussion
GSK3 plays a central role in a broad range of cellular processes, including glycogen metabolism, insulin signaling, apoptosis and microtubule function. Accordingly, it is under investigation as a potential target in Alzheimer's disease and diabetes 6,41 . In the context of cancer, GSK3 is best known in its function as a tumor suppressor, which is deactivated by AKT or Wnt signaling 6,41 . However, most studies have focused on GSK3β, whereas significantly less is known about GSK3α. Moreover, it is increasingly appreciated that GSK3 signaling is context-dependent. For instance, there have been several reports that describe tumor supporting roles of GSK3α in glioblastoma, pancreatic cancer, multiple myeloma, and MLL-rearranged leukemia 28,[42][43][44] . GSK3α has been identified by Banerji et al. through a functional genomic screen as a promising target in AML 5 . However, targeting GSK3 in hematological malignancies does have theoretical challenges in that GSK3 is known to phosphorylate β-catenin thereby marking it for subsequent proteasomal degradation. Upon GSK3 inhibition, β-catenin accumulates, translocates to the nucleus and activates transcriptional pathways 6,45 . Such increased β-catenin signaling has been implicated in a number of leukemogenic effects, such as self-renewal of leukemic stem cells 46 . As β-catenin stabilization requires inhibition of both kinases and most GSK3 inhibitors target GSK3α and GSK3β with similar potency 34 , these compounds may possess some significant limitations. Accordingly, the nonspecific pan-GSK3 inhibitor LiCl, which is currently FDA approved for the treatment of epilepsy and bipolar disorder 7,8 , has met limited success in clinical studies of AML [47][48][49] . In addition to isoform selectivity, various other aspects, such as the binding mode, magnitude of inhibition, and kinome-wide target specificity influence the overall cellular outcome of inhibiting GSK3α. For this reason, not all GSK3 inhibitors should be treated equally.
Tivantinib was originally developed as an inhibitor of the receptor tyrosine kinase MET 14 , but its target selectivity and mechanism of action has been a controversial subject. We and other groups have suggested that MET is actually not a significant target of tivantinib in many cancer cells 13,30,50 . We have previously observed that tivantinib, although being indeed a weak MET inhibitor, much more prominently targets GSK3α and GSK3β in NSCLC cells and that inhibition of these targets can explain its potent anticancer activity in NSCLC 13 . Importantly, we also noted remarkable kinome-wide specificity and some moderate selectivity of tivantinib for GSK3α over GSK3β, which are unique features among clinical GSK3 inhibitors 12 . It has also been suggested that tivantinib binds tubulin and inhibits microtubule dynamics resulting in anticancer activity 30,50 . While these observations are compelling, our results suggest that the sensitivity profile of tivantinib in AML cells more closely matches that of a GSK3 inhibitor. Furthermore, we show that pharmacological inhibition of GSK3 by LiCl largely mimics tivantinib's effects in these cells with regard to viability, apoptosis, cell cycle arrest and differentiation. However, as GSK3α/β have well described roles in microtubule regulation through phosphorylation of Tau (MAPT) 51,52 and other microtubule associated proteins, such as MAP2C 53 , it would be difficult to precisely identify the contributions to tivantinib's overall cellular effects that stem from targeting tubulin in addition to GSK3, particularly as histone H3 phosphorylation is induced rapidly, which could indicate further crosstalk between GSK3 and the cell cycle regulation pathway 54 . Impairment of microtubule polymerization however may be translationally beneficial as it may result in synergistic anticancer activity in the context of dual GSK3 and BCL-2 inhibition as has been previously observed in breast cancer 55 . However, additional studies are necessary to elucidate the complex interplay between these pathways in AML.
In light of the suggested role of GSK3α in AML, we investigated the potential for repurposing tivantinib for the treatment of AML, which to the best of our knowledge has not been reported. Consistent with previous reports 5 , analysis of publically available datasets showed that GSK3α is overexpressed in AML and that knockdown of GSK3α has strong effects on the viability of AML cell lines. We also show that tivantinib interacts with and inhibits GSK3α/β in AML cells and that it potently kills these cells by inducing apoptosis. Interaction of tivantinib with its intended target MET, which was observed to a minor extent in NSCLC cells 13 , was not detectable in AML cells. While tivantinib does target both GSK3α and GSK3β, its effects on β-catenin levels were somewhat less pronounced than with LiCl. This was consistent with our previous observation that tivantinib is more selectively targeting GSK3α 13 . Also, β-catenin stabilization was more transient with tivantinib, whereas it is sustained for a longer period of time upon LiCl treatment. Tivantinib may therefore provide an important therapeutic advantage over pan-GSK3 inhibitors, such as LiCl. Banerji et al. have shown that LiCl readily causes differentiation of AML blasts at relatively low concentrations 5 . Our results confirmed these observations and showed that tivantinib also induces differentiation. However, tivantinib was a much stronger inducer of apoptosis than of differentiation; and although LiCl also induces apoptosis, tivantinib is markedly more potent than LiCl in this regard, which might be due to additional effects of tivantinib on microtubules.
In addition to tivantinib exhibiting potent single agent activity in AML, we observed that the BCL-2 inhibitor ABT-199, which displays activity and is in clinical trials in AML 39 , synergizes with tivantinib by further enhancing tivantinib's already potent ability to inhibit cell viability and induce apoptosis. MCL-1 and BCL-XL expression have previously been associated with ABT-199 resistance 39,40 , and it is noteworthy that we observed a dose-dependent decrease in MCL-1 and BCL-XL levels following tivantinib treatment, which was enhanced in combination with ABT-199. This is consistent with previous reports showing that tivantinib downregulates these proteins in hepatocellular carcinoma 56 . It is also known that GSK3 transcriptionally regulates BCL-XL expression and that GSK3 inhibition by BIO or SB-415286 leads to a reduction in BCL-XL levels 57,58 . Interestingly, while BIO similarly downregulated BCL-XL in breast cancer cells, it also reduced MCL-1 expression, not via transcriptional control, but through a proteasome-dependent mechanism 57 . This downregulation of MCL-1 and BCL-XL expression likely contributes to the synergy observed between tivantinib and ABT-199. By downregulating anti-apoptotic MCL-1 and BCL-XL that cause ABT-199 resistance, tivantinib is amplifying the relative apoptotic effect of ABT-199. This synergy is in excellent agreement with a previous study that described cancer cells with increased β-catenin levels, for instance as the consequence of GSK3 inhibition, to be particularly sensitive to inhibition of BCL-2 by the ABT-199 analogue navitoclax 35  anti-apoptotic proteins we observed that the tivantinib/ABT-199 combination completely abrogated β-catenin stabilization seen with tivantinib single drug treatment. This was apparent already after 4 hours and is therefore likely due to cross-talk between the GSK3 and BCL-2 pathways that is independent of altered transcription. This pronounced reduction of β-catenin persisted for 24 hours and could possibly help prevent some of the leukemogenic effects previously associated with β-catenin signaling in AML 46 . Thus by modulating tivantinib's effects on β-catenin levels in conjunction with the amplification of apoptotic signaling, this suggests a superior therapeutic potential of this drug combination in AML. In this context, it is important to note that tivantinib, as a single drug and even stronger in combination with ABT-199, showed potent anti-leukemic activity in AML patient-derived samples within clinically relevant concentrations. This appears to be independent of the mutational status of common prognostic genes although our sample size was too small to allow broader conclusions. A potential correlation with tivantinib sensitivity could be amplification of GSK3α expression levels as GSK3α is more highly expressed in several different subtypes of AML, including 11q23 MLL-rearranged leukemia, which has previously been shown to be sensitive to GSK3 inhibition 13,28 . Interestingly, high expression of GSK3 and BCL-XL has previously been suggested to correlate with poor prognosis in AML 59 . However, a more thorough investigation of GSK3α expression and signaling in AML is necessary to make detailed conclusions. Although tivantinib has been described to cause myelosuppression 60,61 , this is readily managed in most cases and tivantinib is generally considered a well-tolerated compound 60,61 , which has been evaluated in more than 40 clinical studies, including phase III. Considering also that the concentrations required for its activity in AML cells are well within the therapeutically achievable levels and could be potentially even further reduced in combination with ABT-199 61,62 , repurposing tivantinib provides a tangible opportunity for clinical translation into AML.
In summary, repurposing the advanced clinical drug candidate tivantinib based on its off-target GSK3α identified it as a highly potent agent in AML cells. Combination with the BCL-2 inhibitor ABT-199, which is already under clinical investigation for AML, further enhanced tivantinib's potency and eliminated undesirable β-catenin activation. Together, these findings suggest that tivantinib, either as a single agent or in combination with ABT-199, represents a novel and promising therapeutic option for AML, a disease, which is still in high need for new therapies.

Data Availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD010217 and 10.6019/PXD010217 63 .


Development of Liquid Nitrogen Free and Efficient DNA Isolation Method for Plants Rich in Volatile Oil, Secondary Metabolites and Polysaccharides

Isolation of pure and high-quality DNA from plants is crucial step towards molecular biological studies. DNA isolation process requires costly reagents and instrumentation. However, high levels of secondary metabolites and polysaccharides affect the purity and quality of isolated DNA. Generally, the DNA isolation protocol is to be developed or modified according to different plant species having different metabolites. Holy basil or Ocimum sanctum L. (O. tenuiflorum) is a sacred plant of India and belongs to International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 12 (2019) Journal homepage: http://www.ijcmas.com

Introduction
Isolation of pure and high-quality DNA from plants is crucial step towards molecular biological studies. DNA isolation process requires costly reagents and instrumentation. However, high levels of secondary metabolites and polysaccharides affect the purity and quality of isolated DNA. Generally, the DNA isolation protocol is to be developed or modified according to different plant species having different metabolites. Holy basil or Ocimum sanctum L. (O. tenuiflorum) is a sacred plant of India and belongs to Lamiaceae family. This herb is also known as "Tulsi", "The Queen of herbs" and "The Mother Medicine of Nature" (Singh and Hoette, 2002). O. sanctum has been indicated as anti-microbial, anti-spasmodic, antidiabetic, anti-cancer and anti-fertility agent (Pattanayak et al., 2010). The chemical composition of different varieties or accessions of O. sanctum is very complex having various phytochemicals in different proportions.
All herbage parts of this species have high level of secondary metabolites. Specifically, the leaves have peltate glands and other glandular structures which synthesise volatile oils containing eugenol, chavicol, methyl eugenol, ursolic acid, caryophyllene, methyl chavicoletc (Kelm et al., 2010;Shishodia et al., 2003;Iijima et al., 2004). These metabolites affects the quality of DNA isolated from O. sanctum. At present, commercially available kits for DNA isolation or purifications seems the only source for high quality DNA purification. This is also evident in the genomic DNA isolation methodologies used for O. sanctum in recent reports (Rastogi et al., 2015;Upadhyay et al., 2015). But use of such kits increases cost of studies. Similarly, DNA isolation from some plant species rich in polysaccharide like Aloe barbadensis and Plantago ovata using CTAB methods and commercially kits is very difficult. Such species give sticky DNA samples which cannot be used for PCR amplifications. There are no single DNA isolation method which can universally be used to isolate DNA from O. sanctum, A. barbadensis and P. ovata. In present study, we have developed a methodology for the isolation of high-quality DNA from O. sanctum, A. barbadensis and P. ovata amenable to be used in further downstream molecular studies. This newly developed methodology is a cost effective as it eliminates use of CTAB, phenol and liquid nitrogen, rapid and also equally effective in fresh as well as stored leaves. This methodology may be useful in other plant species having high level of contaminating metabolites and polysaccharides in affordable manner.

Plant samples and DNA isolation
Four different accessions of O. sanctum species were used to isolate the DNA (Fig. 1). These different accessions have different degree of phytochemical present. Some accessions have comparative high level of volatile oils and peltate gland and some are known to have different terpenes and phenylpropanoids. These plants were grown in field and leaves were harvested and stored for one year in minus 80 deep freezer. Further, DNA was also isolated from two polysaccharide rich crops Aloe barbadensis and Plantago ovate.
Modified CTAB method: This method was based on method given by Doyle and Doyle (1990) with some modification. 300-400 mg leaf sample was ground in liquid nitrogen in 800 μl of 4% CTAB extraction buffer (100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA with 4% CTAB) containing PVP (up 2%).
Samples were incubated at 64 °C for 1 hr with mixing after each 15 min. intervals.
Centrifuged at 12,000 × g for 5 min. and supernatant was collected.
RNaseA was added and incubated at 37 °C for 20 min.
Emulsions were centrifuged for 15 min. at 14,000 × g. The aqueous supernatants were transferred to separate tubes and repeated the same step.
In aqueous supernatants, ice cold isopropanol was added and mixed gently.
Incubation at minus 20 °C was given overnight.
Samples were centrifuged for 14,000 × g for 10 min.
Pellets were washed with 70 % alcohol by spinning at 12,000 × g for 8 min.
The pellets were air dried until complete loss of alcohol smell. Finally, air dried pellets were suspended in molecular biology grade water. CTAB method followed by purification by Kit: DNA isolated by above method was taken as starting material for the kit supplied by thermo scientific (GeneJET Plant Genomic DNA Purification Kit). Finally, DNA was eluted in elution solution supplied with kit.
Grind 200-300 mg leaves samples in extraction buffer (having a pinch of PVP).
Incubate suspended material at 60 °C for 30 min.
Centrifuge at 12,000 × g for 10 min and discard supernatants.
Mix the suspension and incubate at 64 °C for 45 min. Centrifuge for 12,000 × g for 12 min.
Collect the supernatants and add equal amount of C:I.
Mix the sample to form emulsion and centrifuge for 14,000 × g for 15 min.
Collect upper clear aqueous layer and add 1/10 volume of 3 M sodium acetate (pH 5.2).
After mixing, add 2/3 volume of ice-cold isopropanol and mix gently.
Incubate at minus 20 °C for 1 hr.
Wash the pellets with 70 % alcohol solution by centrifuging at 12,000 × g for 8 min.
Air dry the pellets and dissolve in molecular grade water.
These DNA solutions were directly used in PCR or RNA was removed as described above. Genomic DNA samples were analysed using 0.8 % agarose gel electrophoresis. Quantitative estimations of all DNA samples were performed by Nano Drop spectrophotometer.

PCR amplification
DNA isolated was subjected to PCR amplification. 50 ng DNA was PCR amplified using both gene specific primers and primers developed for SSR in O. sanctum (unpublished). For gene specific primer amplification, the PCR cycles were as: 94 °C for 4 min., 35 cycles of 94 °C for 45 sec., 54.5 °C for 45 sec., 72 °C for 55 sec., followed by final extension of 72 °C for 7 min. For SSR primers the annealing temperature was 53 °C and remaining protocols were same with minor variations. A. barabadensis and P. ovata DNA isolated using newly developed method was amplified using primers designed for SRAP marker analysis (primers: Me1+Em6 and Me2 +Em6) and PCR cycles defined for SRAP analysis (Bhatt et al., 2017). PCR amplified products were analysed by 1.2 % agarose gel with ethidium bromide.

Results and Discussion
Pure and high-quality DNA is starting point for any DNA based molecular study. Secondary metabolites form complexes with DNA which hinders the downstream enzymatic reactions. The use of CTAB and liquid nitrogen generally has their own limitations. CTAB method requires fresh preparations each time as CTAB buffer cannot be stored for long time. Second, liquid nitrogen is not available easily in developing countries but these are main hot spots for medicinal plants. Phenol also associated with its own corrosive nature. Overall, there is always a need to develop a method which is free from all above limitations. Agarose gel electrophoresis of isolated DNA from Ocimum sanctumis shown in Figure 2a. Intensity of bands and absence of smearing indicated that sufficient and intact DNA was isolated using all three methods used in present study. Table  1 shows quantitative estimation of isolated DNA samples. The quantitative estimation suggested that all used methods seem equally efficient to isolate DNA from the accessions of different chemical compositions. After diluting the O. sanctum DNA samples (50 ng/μl) PCR amplifications were carried out and results of amplifications are shown in Figure 2b. Interestingly, despite the good quantity and intact nature all DNA isolated from all methods used, DNA isolated from O. sanctum using CTAB alone failed to amplify gene specific and SSR primers. But DNA isolated by CTAB method followed by purification by kits resulted in to amplifications. These results indicated that some interfering metabolites may be present in DNA which may form complex with DNA and limits the enzymatic reactions. Even after the purification with P:C:I isolated DNA failed to give PCR amplification. Use of kits for further purification of DNA may eliminate these interfering molecules. But contrasting results were obtained when DNA was isolated with SDS based methods. Regardless of chemically different accessions used in the study our new method resulted in to amplification in all samples. The newly developed method is cost effective, rapid and efficient method for the intact and high-quality DNA isolation from the plants which are rich in volatile oils and secondary metabolites. This method does not require CTAB, costly liquid nitrogen and hazardous phenol. Also, above method is equally effective for plant leaves having high degree of polysaccharides.