Olivier Bailet

Nina Fenouille

Patricia Abbe

Guillaume Robert

Stéphane Rocchi

Nadège Gonthier

Christophe Denoyelle

Michel Ticchioni

Jean-Paul Ortonne

Robert Ballotti

St Phane Rocchi

Nad Ge Gonthier

Marcel Deckert
email: deckert<tartare@unice.fr

Sophie Tartare-Deckert

Spleen tyrosine kinase

Keywords: Cell Aging, physiology, Cell Growth Processes, physiology, Chemotaxis, Cyclin-Dependent Kinase Inhibitor p21, biosynthesis, genetics, DNA Methylation, Humans, Intracellular Signaling Peptides and Proteins, genetics, metabolism, physiology, Melanoma, enzymology, genetics, pathology, Neoplastic Stem Cells, pathology, Promoter Regions, Genetic, Protein-Tyrosine Kinases, biosynthesis, genetics, metabolism, physiology, RNA, Small Interfering, genetics, Signal Transduction, Spheroids, Cellular, Transfection, Tumor Suppressor Protein p53, metabolism, Up-Regulation, src Homology Domains

INTRODUCTION

Cutaneous melanoma is the leading cause of skin cancer-related mortality in Western societies owing to its high capability of invasion and rapid metastasis to other organs ( ). The first step toward transition to melanoma is uncontrolled proliferation of epidermal 1 melanocytes and dysplasia that may arise within a preexisting benign lesion (nevus) or more frequently directly from a new site.

Subsequently, primary melanoma progresses through the radial growth phase (RGP) characterized by horizontal growth of transformed cells within the epidermis and the vertical growth phase (VGP), which is associated with depth invasion of cells into dermis and acquisition of metastatic potential ( ). Melanoma formation and progression is accompanied by activating mutations in , 2 B-RAF N-RAS and genes as well as in gene and loss of tumor suppressor genes such as , and ( , ). Oncogenic KIT CDK4 CDKN2A PTEN E-cadherin 2 3

signaling and failure of tumor suppressor mechanisms are believed to contribute to the molecular pathogenesis of melanoma.

Cellular senescence represents a powerful tumor-suppressive process that constrains malignant transformation, as it controls excessive proliferation driven by oncogenic mutations ( ). B-RAF mutations are the most prevalent oncogenic mutations in human melanocytic 4 -7 skin lesions ( ). B-RAF mutation was found in over two-thirds of human melanoma but also in a high proportion of benign nevi in 8 V600E which melanocytes were though to be senescent. Michaloglou ( ) demonstrated that B-RAF activates a senescence program that et al. 9 V600E induces melanocyte growth arrest, and that senescence in nevus cells is triggered by B-RAF signaling. This work supports the model V600E that nevi represent senescent clones of melanocytes and that senescence is a barrier to melanoma progression ( ). In this context, 10

senescence was shown to be dependent on p16 expression ( ). The B-RAF study, however, has suggested that besides p16 another melanoma suppressor(s) contribute to protection against oncogenic B-RAF signaling ( ). In this regard, the secreted protein 9

IGFBP7 has been recently proposed to contribute to B-RAF -mediated cell senescence ( ).

V600E

Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase that is widely expressed in hematopoietic cells. It contains tandem N-terminal Src homology 2 (SH2) domains, multiple tyrosine phosphorylation sites and a C-terminal tyrosine kinase domain. The SH2 domains bind phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) and hence couple activated immunoreceptors to multiple downstream signaling pathways. Syk is essential for lymphocyte development and function, and signal transduction via a variety of membrane receptors in non-lymphoid cells such as mast cells or platelets ( ). It was believed for years that Syk function was solely 13 linked with hematopoietic cell signaling. However, more recent studies have indicated a ubiquitous pattern of gene expression. Syk is Syk expressed in endothelial cells, fibroblasts, epithelial cells and neuronal cells, but its function in these cells is not yet completely understood ( ). [START_REF] Yanagi | Syk expression and novel function in a wide variety of tissues[END_REF] Accumulating evidence suggests that Syk can function as a tumor suppressor, unlike other tyrosine kinases that generally promote growth-stimulating activity leading to tumorigenicity ( ). The role of Syk as candidate tumor suppressor has been well documented in 15 breast cancer. Loss or reduced expression of Syk in human breast cancers was associated with a higher degree of malignancy and poor

prognosis (

). Consistent with a role for Syk inactivation in tumor progression, re-expression of Syk in invasive breast cancer cells 16 -18 was shown to inhibit tumor growth and reduce metastasis in mouse xenografts ( ). The tumor suppressive activity of Syk in breast 16

cancer cells has been associated with abnormal mitotic progression and cell death ( , ). Epigenetic silencing through 18 19 hypermethylation of critical CpG islands was proposed to be involved in loss of Syk in a significant fraction of breast tumors ( ). More 20 recently, a similar loss of Syk expression has been documented in melanoma cells. Importantly, reintroduction of Syk was shown to restrict tumor growth and metastasis ( , ). However, the molecular events responsible for Syk tumor suppressor effects in in vivo 21 22 melanoma cells have remained completely unknown.

Here we aimed to investigate the tumor suppressor function of Syk in melanoma cells. We present evidence that Syk exerts its antitumor activity through induction of a senescence-like growth arrest and activation of a p53-dependent pathway, suggesting that loss of Syk may contribute to the senescence bypass generally observed in malignant melanoma. Our findings identify a novel link between the tyrosine kinase Syk, p53 and the senescent program in cancer cells.

MATERIALS AND METHODS

Reagents and plasmids

The list of antibodies used is provided in . Pifithrin-(PFT-) was from Calbiochem. p53 small interfering supplementary methods α α RNA (siRNA) was purchased from Santa Cruz Biotechnology. Wild-type and kinase-defective (Syk-KD, K395R) porcine Syk cDNAs as well as SH2 domains-deleted Syk mutant (Syk-SH2) were cloned into pEF6/V5 vector. These mutants have been described elsewhere (

Δ

). The SH2-inactive domains mutant (Syk-SH2D) was produced using a QuikChange site-directed mutagenesis kit supplied by 23

Stratagene. Recombinant adenoviruses expressing wild-type and kinase-defective Syk were kindly provided by Dr Yamamura (Kobe University Japan) and have been described previously ( ).

24

Cell culture, RNAi, and adenovirus infection

Human melanoma cell lines were provided by Drs M. Herlyn (Wistar Institute, Philadelphia, PA) and F. Jotereau (INSERM U601, Nantes, France). Cells were routinely maintained in DMEM supplemented with 7 FBS as described elsewhere ( ). Transfection of % 25 duplex siRNAs (50nM) was carried out using Lipofectamine RNAiMAX (Invitrogen). Adenovirus infection was performed as described previously ( ). 26

Analysis of cell proliferation, colony formation assays and three-dimensional spheroid growth

For BrdU incorporation and DNA-content analysis, subconfluent cultures were pulsed with 10 M BrdU for 4 hr. Cells were harvested μ and DNA stained with 7-amino-actinomycin D (7-AAD) and a APC-conjugated anti BrdU antibody using the APC BrdU Flow Kit (BD -Biosciences). Flow cytometric analyses were performed on a Becton Dickinson FACSCalibur machine and data were analysed using CellQuest software.

For colony formation assays, cells were transfected by using FuGENE 6 (Roche Diagnostics) with DNA mixtures containing 1 g of μ the relevant plasmids plus 0.1 g of pBabe-Puro vector. After 48 hr, cells were selected with 1 g/ml of puromycin and maintained for 12 quantitatively using Image J software.

Melanoma spheroids were generated using the liquid overlay technique as described ( ). Briefly, 24-well culture plates were coated 27 with 1.5 agarose prepared in sterile water. Cells from a single-cell suspension were added at 10,000 per well. The plates were gently % swirled and incubated at 37 C in 5 CO atmosphere until spheroid aggregates were formed. Cells were washed in PBS and fixed with 3 formaldehyde for 15 min at room temperature. Staining for SA--gal activity was % β performed as described previously ( ), utilizing the senescence -galactosidase staining kit (Cell Signaling Technology). Stained cells 28 β were visualized by phase contrast microscopy, photographed and counted.

Immunoblotting and immunofluorescence

Whole cell lysates were prepared by lysing cells in radioimmunoprecipitation assay buffer supplemented with 1 NP40 and subjected % to SDS-PAGE. Immunoblotting was accomplished according to standard procedures using enhanced chemiluminescence detection. For immunofluorescence, cells were fixed with 3 paraformaldehyde, and then incubated with primary and secondary antibodies as described % elsewhere ( , ). [START_REF] Robert | SPARC represses E-cadherin and induces mesenchymal transition during melanoma development[END_REF] 29

Migration and invasion assays

Chemotaxis and Matrigel invasion assays were monitored using modified Boyden chambers as described ( , ). 25 29

Statistical analysis

Unless indicated otherwise, experiments shown were representative of at least three independent experiments. Results were expressed as mean SD. Where appropriate the Student s t test was done and <0.001 was considered statistically significant.

± '

P

RESULTS

Syk expression and Syk promoter methylation status in melanocytes and melanoma cells

Initially, we examined expression, phosphorylation level and cellular localization of Syk in melanocytes. A 72 kDa band corresponding to Syk was readily detected by immunoblotting of lysates from different primary human melanocytes cultures (

). We also observed that Syk is phosphorylated on tyrosine 352, a site that has been proposed to bind signaling Supplementary Fig. S1 proteins ( , ). Consistent with previous reports ( , ), we observed that Syk is absent or nearly undetectable in most human 23 [START_REF] Law | Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk[END_REF] 21 22

primary and metastatic melanoma cells tested ( ). Methylation-specific PCR (MSP) studies showed that transcriptional silencing of Fig. S2 Syk in Sbcl2, A375, 1205Lu and WM9 cells was associated with aberrant DNA methylation of the promoter region that we examined. In contrast, no methylation was observed for Syk-positive cell lines and melanocytes. However, we found that in Syk-negative WM793 cells, the promoter is unmethylated, suggesting that mechanism other than methylation of this region may be involved in gene inactivation. SYK Finally, we found that treatment with the demethylating drug, 5-azadC led to an increase of Syk mRNA levels whereas the histone deacetylase inhibitor TSA had no discernible effect on Syk mRNA expression ( ).

Fig. S2

Syk decreases colony formation, tumor growth in 3D spheroids and chemotaxis

Having shown that Syk was inactivated in several melanoma cell lines, we examined its potential growth suppressor function in monolayer cultures and 3D spheroid model. We transfected a Syk-expression plasmid into melanoma cell lines and colony formation assays were done to evaluate long-term growth on plastic dishes (

). Reintroduction of Syk dramatically reduced colony formation Fig. 1A efficiency by at least 70 in Syk-negative A375 and SKmel28 cells. The same experiment was also carried out in 501mel cells expressing % endogenous Syk protein levels ( , 501mel lanes; see for Syk blot of 501mel showing endogenous Syk level). In this setting Fig. 1A Fig. S2 as well, elevated expression of Syk led to inhibition of colony formation. Transient transfection showed that each of the cell lines were able to express comparable levels of exogenous V5 epitope-tagged Syk ( ). Fig. 1A We also investigated the effect of Syk reintroduction on tumor spheroid growth using adenoviral delivery system. To exclude the possibility that this effect would be the result of overexpression of Syk, we determined the level of Syk re-expression in melanoma cells and we found that level of exogenous Syk in lysates made from infected A375 cells was comparable to endogenous level seen in melanocytes (see ). In all subsequent experiments, we infected cells with a MOI of 5, which is the dose supplemental information Fig. S3 of virus that induces physiological level of Syk expression. Culture of WM793 cells under nonadherent conditions resulted in generation of 3D multicellular tumor spheroids, which may be used as an experimental model to study the tumor biology context. We prepared 3D spheroids from cells infected with control or Syk-expressing adenoviruses. After 3 days, there was significant decrease in spheroids diameter when cells were infected with Ad-Syk compared with vector control. Expression of adenovirus-delivered Syk was confirmed by immunoblot analysis (

). Together, these observations indicate that Syk has growth-suppressing activity in standard monolayer Fig. 1B cultures and 3D spheroids.

Finally, we analyzed the effect of Syk on melanoma cell migration and invasion towards serum using modified Boyden chambers assays. As seen , re-expression of Syk markedly decreased A375 chemotactic migration and invasion. Because no further Fig. 1C reduction was observed in the invasive capacity of cells, we can conclude that Syk primarily inhibits melanoma cell chemotaxis. Inhibition of tumor cell invasion into collagen I matrix by Syk was also evidenced in 3D cultures (

). Fig. S4 Suppression of clonogenic survival by Syk is independent on functional SH2 domains but requires tyrosine kinase activity Because A375 had complete methylation and silencing of Syk (

) and was most sensitive to Syk-induced growth inhibition ( Fig. S2 ), we analyzed this cell line further. We conducted loss-of-function experiments, to define domain(s) of Syk that mediate growth Fig. 1A suppression. We took advantage of a kinase-inactive mutant and generated a mutant that harbors inactivating mutations in the SH2 domains, as well as a mutant with a deletion of the two SH2 domains and interdomain A (

). Cells transfected with plasmids Fig. 2A encoding these mutants or a wild-type control expressed similar levels of the corresponding proteins 3 days after transfection ( ). Fig. 2B Subsequently, these same transfections were subjected to colony-forming assay. As shown in , expression of Syk SH2 mutants Fig. 2C inhibited colony formation relative to vector control. Thus, Syk functions independently of its SH2 domains and interdomain A to suppress colony formation abilities of melanoma cells. However, mutation of the ATP-binding site did not significantly inhibit the ability to form colonies, indicating that enzymatic activity of Syk is required for its effect on growth inhibition.

Expression of Syk inhibits proliferation by modulating cell cycle progression

We undertook to determine how Syk reintroduction suppresses melanoma cell growth and survival. A375 were transduced with adenoviral vector expressing Syk and we analyzed ability of cells to proliferate by proliferation curves, cell cycle analysis and BrdU incorporation assays. Proliferation curves revealed that Syk re-expression markedly slowed growth of A375 cells (

). Levels of Syk Fig. 3A expression in infected cells during the course of the experiment is shown . We also examined cell cycle profiles of Syk-expressing Fig. 3B and control cells 4 days after infection by flow cytometry (

). Restoration of Syk resulted in accumulation of cells in G0/G1 and Fig. 3C decrease in S-phase cells compared with control cells, suggesting that Syk growth suppression results from G1 cell cycle arrest. In support of this, A375 cell population expressing Syk displayed a strong reduction in BrdU incorporation compared to vector-transduced cell population (

). Of note, cells transduced with Syk show no increase in sub-G1 phase and no caspase 3 activation, indicating that Fig. 3D Syk expression did not result in cell death or apoptosis ( ,C and data not shown).

Fig. 3A

Expression of Syk promotes senescence-like phenotypes

We observed that a major population of cells arrested by Syk displayed a flat enlarged morphology with increased granularity that is characteristic of senescent cells (

). This increase in cell size was also observed by flow cytometry-based assays where Fig. 4A Syk-expressing cells showed elevated side scatter that is characteristic of senescent cells. Senescence is accompanied by a series of genetic and metabolic changes that include accumulation of SA -gal. To confirm that the observed morphological changes were due to induction β of senescence, we assessed SA -gal activity 6 days post-infection. A blue staining indicative of acidic -gal activity was observed in the

β β
growth-arrested A375 infected with Ad-Syk ( ). Notably, about 40 of cells were -gal positive in Syk-expressing populations, Fig. 4B % β whereas less than 2 of A375 cells were positive in vector-infected cultures ( ). Lastly, we looked for the presence of % Fig. 4C senescence-associated heterochromatin foci (SAHF), another feature of senescent cells. Immunofluorescence analysis for HP1 , a marker β of SAHF revealed punctuated regions of DNA corresponding to heterochromatic foci in Syk-expressing cells but not in control Syk negative cells ( ). All together, these findings indicate that the cell-cycle arrest produced by Syk is mainly associated with induction Fig. 4D of senescence-like phenotype.

Expression of Syk up-regulates the p53-signaling pathway

Next, we sought to understand the molecular payhway(s) involved in senescence-like growth arrest after Syk reintroduction.

Immunoblot analysis was done to investigate expression and phosphorylation levels of specific proteins involved in cell cycle regulation.

Increased levels of activating phosphorylation of p53 at Ser15 and induction of the cdk inhibitor p21 were observed in Syk-expressing

A375 cells compared with controls (

). We also noticed that Syk expression resulted in modest, but consistent, increase in the levels Fig. 5A of total p53. In addition, Syk-expressing cells harbored dephosphorylation of pRb and lost cyclin A expression. Consistent with changes in p53 regulation, immunofluorescence of individual cells showed nuclear p53 accumulation in Syk-positive cells (

). The nuclear Fig. 5B accumulation of p53 was similar to the one observed after treatment with the radio-mimicking drug bleomycin. These data indicate that Syk expression results in up-regulation of p53/p21 and pRb pathways, which are known to mediate cell-cycle arrest and premature senescence.

To substantiate further these observations, we analyzed by quantitative RT-PCR expression level of more than 90 transcripts involved in p53-dependent cell cycle regulation and survival following Syk re-expression (see ). 29 genes supplemental information and Table S1 appeared to be reproducibly modulated by Syk and some of them have been validated at protein level ( and ). Among the Fig. 5A Fig. S5 14 genes up-regulated, 6 were known transcriptional targets of p53 Caspase 2 and survivin expression was confirmed at protein level ( ). Together, our results indicate that expression of Syk is linked Fig. S5 to altered expression of essential cell survival and cell cycle-regulatory genes that consequently results in G1 cell cycle arrest and inhibition of cell growth.

Inactivation of p53 prevents Syk-induced p21 expression

p21 is a well-characterized cdk inhibitor and a transcriptional target of p53 ( , ). The p53/p21 axis plays a crucial role in 33 34 mediating G1 arrest and senescence in normal and several tumor cells in response to multiple stress insults ( , ). Our studies show that 4 5

Syk-induced growth inhibition of melanoma cells is consistently associated with induction of this pathway. Therefore, we next investigated the requirement of p53 in Syk-induced p21 expression. To this end, A375 were incubated with the specific p53 inhibitor PFT-( ). The level of p21 normally seen after Syk expression was significantly lower in PFT-treated cells than in control cells ( ). α 35 α-Fig. 6A Similarly, we observed that induction of p21 by bleomycin was partially suppressed in PFT-treated cells. To rule out the possible αnonspecific effect of PFT-, we used siRNA to inhibit p53 expression. Cells were transfected with p53-specific siRNA (or unrelevant α control siRNA) and subsequently infected with adenovirus expressing Syk or empty vector as control.

shows that siRNA to p53 Fig. 6B led to an efficient knockdown of p53 protein as well as p21 basal expression. This experiment also revealed that induction of p21 by Syk expression or bleomycin treatment was totally prevented in p53-silenced cells. These data indicate that p53 plays a functional role in mediating Syk-induced p21 expression. According to this, no induction of p21 was seen when Syk is expressed in MeWo cells, which have no functional p53 ( ). Fig. 6C DISCUSSION There is accumulating evidence that the Syk tyrosine kinase is a novel tumor suppressor in melanoma. However, its mechanism of action with respect to tumor suppression is not known. Here, we show that re-expression of Syk in melanoma cells at levels similar to that seen in melanocytes, results in suppression of chemotaxis, sustained cell cycle arrest, and senescence accompanied by molecular events associated with activation of the p53 tumor suppressor pathway. Our observations on the antiproliferative effect of Syk support the hypothesis that inactivation of K gene confers a specific growth advantage in neoplastic melanocyte cells and promotes migration of SY tumor cells. Together, our findings agree with recent observations showing that Syk expression in xenografted melanoma cells results in dramatic reduction of tumor growth and metastatic abilities ( , ). 21 22 Consistent with a tumor suppressor function of Syk in melanoma, Syk levels are frequently low or undetectable in melanoma cells and primary tumors. The loss of expression occurs at transcriptional level, and, as a result of DNA hypermethylation ( ). Our study identified 22 a similar epigenetic regulation in the majority of melanoma cells analyzed. However, as shown in breast invasive tumors ( ), our results 18 suggested that mechanisms other than promoter methylation might account for loss of Syk expression in certain melanoma cells.

Nevertheless, our results underscored a potential role of cytosine methylation in gene inactivation in melanoma cells. It now remains SYK

to analyze the possible correlation between methylation rate and clinicopathological parameters in melanoma tumors. SYK Inhibition of colony-forming activity by Syk provides a model system to study the mechanism by which Syk exerts its growth suppressive effect. We found that kinase activation of Syk and further downstream signaling are required for Syk-mediated inhibition of colony formation. In contrast, the SH2 domains of Syk are dispensable for the colony growth suppressive effect. This observation is consistent with the view that function of Syk in melanoma cells does not require the interaction of the SH2 domains with phosphorylated ITAM tyrosines on putative receptors or adaptor proteins. However, it is possible that Syk activity in melanoma depends on its subcellular localization. Reports have showed that repression of breast tumor cell growth by Syk is associated with its nuclear localization and transcriptional repressor function ( , ). Also, loss of nuclear Syk expression was shown to be closely associated with the metastatic [START_REF] Wang | Transcription repressor activity of spleen tyrosine kinase mediates breast tumor suppression[END_REF] The growth suppression by Syk appears to be mediated through a G0/G1 cell cycle arrest and induction of senescence, and did not involve increased apoptosis or cell death. Interestingly, this activity seems specific to melanoma cells, as compared to breast carcinoma cells where expression of Syk triggered a non-apoptotic cell death through mitotic catastrophe ( , ). This is consistent with the idea 18 [START_REF] Zyss | The Syk tyrosine kinase localizes to the centrosomes and negatively affects mitotic progression[END_REF] that Syk mediates senescence or death depending on the genetic background of the tumor. At this stage, the mechanism of tumor type-specific activity of Syk remains unclear.

p53 is a major regulator of cell-cycle arrest, senescence or apoptosis ( ). Our findings provide the first evidence that tumor growth 40 inhibition associated with Syk involves activation of p53 as reflected by phosphorylation of p53, nuclear accumulation and regulation of various p53-responsive genes. The net effect of these changes tips the balance in favor of proliferative arrest and protects cells from p53-dependent apoptosis. In support of this, expression of Caspase 2, a protease implicated in p53-mediated apoptosis ( , ) was 41 32 strongly suppressed following Syk re-expression. One of the major contributors of p53-directed cell cycle arrest and senescence is p21 (also known as senescent cell-derived inhibitor) ( , , ), and we provide evidence for a functional requirement of p53 for p21 33 41 42 regulation by Syk. p21 functions by inhibiting cdk activity that results in hypophosphorylation of pRB and repression of E2F-targets genes, which blocks S phase entry and mediates cell cycle arrest. According to this, expression of Syk is consistently associated with hypophosphorylation of pRB and inhibition of E2F-targets genes such as cyclin A and cyclin B. Importantly, we also found a dramatic loss of E2F1 gene expression that can also account for the proliferative arrest and senescence experienced by melanoma cells expressing Syk.

A similar down-regulation of E2F1 has been recently reported in senescing melanoma cells treated by the anticancer agent diperpene ester ( ). [START_REF] Cozzi | Induction of senescence in diterpene ester-treated melanoma cells via protein kinase C-dependent hyperactivation of the mitogen-activated protein kinase pathway[END_REF] At present, we do not know the mechanism(s) through which Syk promotes phosphorylation and activation of p53. Phosphorylation of p53 may involve ATM/ATR DNA damage signaling pathways. However, it seems unlikely, as we did not observe activation of this pathway in cells expressing Syk (unpublished observations). The stress-activated c-jun N-terminal kinase might be a candidate for the kinase that phosphorylates p53 ( ). In this regard, it is interesting to note that Syk activates this pathway in melanoma cells (

). 44

Fig. S6 The results reported here might have important implications in our understanding of the physiological role of Syk in melanocytes. To date, the upstream signals involved in Syk activation and signaling pathways regulated by Syk in melanocytes are not known. However, because Syk modulates the p53 pathway in melanoma cells, we speculate that it may integrate p53-mediated stress responses in melanocytes. Accordingly, a link between Syk and oxidative stress signaling has been observed in B lymphocytes ( , ). Like 39 45 apoptosis, senescence is a stress response that prevents neoplastic transformation. Based on our observations, we propose that loss of Syk during melanoma development can lead to altered stress-dependent responses resulting in uncontrolled proliferation of abnormal cells.

Additional investigations using suppression of Syk in melanocytes will elucidate how inactivation of Syk contributes to melanoma pathogenesis.

To conclude, our results uncover a novel role for Syk in regulation of p53 and senescence program, and give a molecular explanation for its tumor suppressive function in melanoma. This provides an important insight for further understanding tumorigenic and invasive processes, particularly in the context that epigenetic inactivation of Syk is associated with numerous cancers.

Figure 1

Reintroduction of Syk in melanoma cells abrogates colony formation, growth in 3D culture and migration (A) The indicated melanoma cell lines were transfected with a puromycin resistance vector and pEF-V5-wt-Syk expression vector or control empty vector (pEF). Stable puromycin-resistant colonies were stained and counted. Colony growth of cells transfected with control vector is normalized to 100 for each cell lines.

, mean of triplicate of a representative experiment; , SD. Representative plates are

  15 days. Colonies were fixed with 3 paraformaldehyde and stained with 0.4 crystal violet. The number of colonies was scored % %

%Figure 2 Figure 3

 23 Figure 2 Syk suppresses colony growth independently of its two tandem SH2 domains (A) Functional domains of Syk and schematic of the mutants. (B) Control immunoblot shows expression of the V5-tagged Syk wt and mutants in transiently transfected A375 cells. (C) Cells were transfected with empty vector (pEF), Syk wt, the SH2 deleted ( SH2), the SH2 Δ mutated (SH2D) or the Kinase Dead (KD) Syk expression vector. Colony formation was analyzed as described in the legend of . Fig. 1 Representative plates are shown. , < 0.001, Student s tests. * P ' t

Figure 4

 4 Figure 4 Syk triggers senescence-like phenotypes (A) Phase-contrast images of A375 cells infected for 6 days with empty vector or with adenovirus encoding Syk. Syk-transduced cells show a large and flat morphology with increased cytoplasmic granularity. The figure shown is a typical representation of the experiment that was performed at least five times (B) Induction of SA--gal activity in A375 cells by Syk. Cells were fixed and stained for SA--gal (pH 6.0) after β β 6 days of infection. Upper panels are low-magnification images and lower panels are high-magnification images. (C) Percentage of SA-β -gal-positive cells 7 days post-infection. At least 200 cells were scored from multiple randomly selected fields. Values represent the mean ± SD of three independent measurements. (D) Syk induces senescence-associated heterochromatin foci (SAHF). A375 cells were stained by immunofluorescence with anti-Syk (red) and anti-HP1 (green) 6 days post-infection and viewed under a fluorescence microscope. The β percentage of SAHF positive cells is indicated in insert. Numbers given are representative of three independent experiments.

Figure 5

 5 Figure 5 Syk regulates expression and activity of senescence-associated markers (A) Lysates from A375 infected for 4 days with empty vector or with adenovirus encoding Syk were subjected to immunoblotting with the antibodies indicated on the left. (B) Immunofluorescent staining of vector-and Syk-expressing A375 cells. After 4 days of infection, cells were stained with anti-p53 antibody (green), anti-Syk antibody (red) or DAPI (blue). A treatment for 4 hr with bleomycin was used as positive control. Boxed regions correspond to larger magnifications of representative cells. The percentage of Syk-positive cells showing nuclear labeling of p53 is indicated in insert and is representative of three independent experiments.

Figure 6

 6 Figure 6Induction of the cdk inhibitor p21 by Syk expression is p53 dependent (A) A375 cells infected with empty vector or with adenovirus encoding Syk for 2 days were incubated with pifithrin (PFT ) (20 g/ml) or α α μ DMSO as a vehicle control for additional 2 days. Cell lysates were tested for Syk, p53, Phospho (Ser15) p53, p21 and ERK2 by immunoblotting. (B) A375 cells were transfected with p53-siRNA or luciferase-siRNA (control). 6 hr later, cells were infected with empty vector or with adenovirus encoding Syk for 4 days. A treatment with bleomycin was used as positive control. Total cell lysates were subjected to immunoblotting analysis as in A). (C) MeWo (p53 defective) human melanoma cells were infected with empty vector or with adenovirus encoding Syk for 4 days. Cell lysates were tested for Syk, p53, p21 and ERK2 by immunoblotting.

  It remains to be assessed whether such nuclear activity and localization of Syk occurs in melanoma cells and what are the structural element(s) of Syk involved in tumor suppression.

			37
	property of gastric cancer (	38	). Consistently, Zhou	et al	. (	39	) identified a shuttling sequence in Syk that accounts for its
	nucleocytoplasmic trafficking.

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

Egr-1 . Oncogene . 2005 ; 24 : 1423 -33

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

[START_REF] Turner | Tyrosine kinase SYK: essential functions for immunoreceptor signalling[END_REF]

Ackowledgements:

We thank H. Yamamura for Syk adenoviruses, M. Herlyn and F. Jotereau for melanoma cell lines. We also thank C. Gaggioli, G. Ponzio and R. Rottapel for helpful discussions. : INSERM, ARC grant N 3111 and INCa grant PL 2007. O. Bailet is a recipient of an INSERM-r gion Provence Alpes C te Grant support

Author Keywords Melanoma ; tumor suppressor ; senescence


Thomas Buffet

Semihchan Sermet

Olivier Marre

Bruno Cessac

Simone Bertoli

Dynamical Synapses in Retinal Surprise coding

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Gabriela Reyes-Castellanos

Alice Carrier
email: alice.carrier@inserm.fr

A novel anticancer pharmacological agent targeting mitochondrial complex I

Keywords: Mitochondria, oxidative phosphorylation system, complex I, anticancer therapies, cancer metabolism, tumor microenvironment

Targeting metabolic reprogramming has proven successful in oncology, but this field requires better identification of drugs that inhibit mitochondrial metabolism in cancer cells. Recent work from Dr. Wolf's group reveals that the primary target of the antitumor compound SMIP004-7 is mitochondrial complex I (NDUFS2 subunit), whose inhibition promotes anti-cancer immune surveillance.

Text

In order to develop new and efficient therapeutic strategies targeting cancer metabolism, determining the metabolic vulnerabilitie(s) of subsets of patients is required. In this context, the identification of dependencies on energetic metabolism (i.e. ATP production) has received considerable interest in recent years. Glycolysis and mitochondrial respiration are the two pathways responsible for the production of ATP. Cancer cells often show a high level of aerobic glycolysis (see Glossary), the so-called Warburg effect, i.e. high glycolytic activity in cancer cells even in the presence of oxygen. Notwithstanding, glycolysis is not the only provider of ATP in tumors, since mitochondria are fully functional in most of cancer cells, sustaining their aberrant proliferation.

Mitochondria play an instrumental role in normal cell homeostasis and cancer cell metabolic reprogramming, through their major functions in metabolism and cell death regulation [START_REF] Smith | Mitochondrial pharmacology[END_REF]. Mitochondria produce high levels of ATP by the oxidative phosphorylation (OXPHOS) system belonging to the mitochondrial respiration. The respiratory chain consists of five coupled protein complexes located in the mitochondrial inner membrane. The last complex (complex V or ATP-synthase) phosphorylates ADP into ATP by using the proton force generated by the flux of proton ions from the mitochondrial intermembrane space to the matrix. Proton ions accumulate in the intermembrane space via the transfer of electrons through complexes I, II, III, and IV (where oxygen is the last electron acceptor), collectively forming the electron transport chain (ETC). Electrons are provided by the reductive molecules NADH and FADH2, generated by the tricarboxylic acid (TCA) cycle in the mitochondrial matrix. The TCA cycle is fed by substrates from glycolysis, fatty acid oxidation (FAO), and amino acid catabolism.

There is now significant evidence that most cancers depend on mitochondria to meet their metabolic needs as they provide energy and key metabolites for the anabolic pathways of macromolecule (nucleic acids, lipids, proteins) synthesis [START_REF] Porporato | Mitochondrial metabolism and cancer[END_REF][START_REF] Vasan | Mitochondrial Metabolism as a Target for Cancer Therapy[END_REF]. Mitochondria not only promote tumor growth, but also tumor chemotherapy resistance, cancer cell invasiveness and metastasis [START_REF] Porporato | Mitochondrial metabolism and cancer[END_REF][START_REF] Vasan | Mitochondrial Metabolism as a Target for Cancer Therapy[END_REF]. A body of evidence demonstrates that different cancers are OXPHOS dependent, such as leukemia, melanoma, lung carcinoma, ovarian and pancreatic cancer (Figure 1A) [START_REF] Reyes-Castellanos | Mitochondrial Metabolism in PDAC: From Better Knowledge to New Targeting Strategies[END_REF][START_REF] Ashton | Oxidative Phosphorylation as an Emerging Target in Cancer Therapy[END_REF]. Therefore, cancers can be rationally targeted on the basis of metabolic dependencies in general and OXPHOS in particular, in order to develop new therapeutic strategies.

Reciprocally, a dysfunctional OXPHOS system is often accompanied by an increase in the partial reduction of oxygen into superoxide anion, a main reactive oxygen species (ROS). Even if superoxide anion can be eliminated by multiple antioxidant defenses, excessive production of ROS associated with dysfunctional OXPHOS often leads to oxidative stress [START_REF] Smith | Mitochondrial pharmacology[END_REF]. Importantly, drug-induced inhibition of OXPHOS leads to oxidative stress, which is one of the major pathways that triggers cell death. In this context, the identification of new cytotoxic molecules targeting OXPHOS and inducing tumor elimination is promising.

A paper published recently in Cell Chemical Biology by the group of Dieter Wolf uncovers the molecular mechanism of action of the chemical compound SMIP004-7 to inhibit the mitochondrial complex I (CI) [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF] (Figure 1B). SMIP004-7 is an analog of SMIP004 (N-[4butyl-2-methyl-phenyl] acetamide) which was identified by the same laboratory a decade ago, as a powerful antitumoral agent in prostate cancer, in both cellular and mouse xenograft models [START_REF] Rico-Bautista | Small molecule-induced mitochondrial disruption directs prostate cancer inhibition via UPR signaling[END_REF]. Cancer cell treatment with SMIP004 in vitro induced the disruption of mitochondrial respiration leading to oxidative stress, resulting in cell cycle arrest and apoptosis [START_REF] Rico-Bautista | Small molecule-induced mitochondrial disruption directs prostate cancer inhibition via UPR signaling[END_REF] (Figure 1C). The SMIP004-7 analog was tested in vivo since it had shown the highest cytotoxic activity in vitro, thus demonstrating its antitumor activity [START_REF] Rico-Bautista | Small molecule-induced mitochondrial disruption directs prostate cancer inhibition via UPR signaling[END_REF]. Now, Jain and collaborators report the identification of the molecular target of SMIP004-7 [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF].

By quantitative proteomic and metabolic analyses, the authors demonstrated that SMIP004-7 inhibits the NADH:ubiquinone oxidoreductase activity in mitochondrial CI [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF].

They also showed that promoting CI activity by genetic complementation allowed to rescue SMIP004-7-mediated cytotoxicity [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF]. These experiments point to the NDUFS2 subunit as the main target of SMIP004-7 in the CI matrix arm which contains seven catalytic subunits [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF]. Four functional modules and 45 subunits constitute the mammalian mitochondrial CI (Figure 1B) [START_REF] Smith | Mitochondrial pharmacology[END_REF][START_REF] Urra | The Mitochondrial Complex(I)ty of Cancer[END_REF]. Fourteen subunits are considered core subunits, including the NDUFS2, and the rest are called accessory or supernumerary subunits.

The correct assembly of CI is mandatory for its structure and function [START_REF] Smith | Mitochondrial pharmacology[END_REF][START_REF] Urra | The Mitochondrial Complex(I)ty of Cancer[END_REF]. Using native PAGE experiments, Jain and collaborators showed increased levels of cellular CI but not the other mitochondrial complexes in NDUFS2-overexpressing cells, suggesting the importance of NDUFS2 in de novo assembly of CI [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF]. SMIP004-7 treatment in vitro demonstrated a strong depletion of the assembled CI, suggesting a disassembly of the respirasome (i.e. respiratory supercomplex) and CI degradation (Figure 1B). Enzyme kinetics assays showed that SMIP004-7 is an uncompetitive inhibitor with regard to the ubiquinone, contrary to other compounds targeting CI such as piericidin A which interacts with the ubiquinone-10-binding site (Figure 1B) [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF].

Importantly, treatment with SMIP004-7 in immunocompetent preclinical models of breast cancer (orthotopic syngeneic allografts with murine 4T1 cells) confirmed that NDUFS2 is the target of SMIP004-7 in vivo as well, demonstrating its potential in the clinic [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF].

Moreover, SMIP004-7 treatment reversed hypoxia and increased infiltration with CD4 + and CD8 + T lymphocytes in the tumor microenvironment (TME) (Figure 1D) [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF]. It's possible that reduced oxygen consumption by the tumor cells upon SMIP004-7-driven mitochondrial disruption could explain decreased hypoxia, although this remains to be proven. In addition, reduced hypoxia by SMIP004-7 could promote the recruitment, proliferation, or survival of T cells. The exact nature of these T cells and of other immune cells that could be impacted by SMIP004-7 treatment remains to be investigated. In addition, the effect of SMIP004-7 on the metabolism and function of immune cells is worth further investigation. Nevertheless, the potential of SMIP004-7 in modulating the TME offers perspectives for the field of immunotherapy, including the use of immune checkpoint inhibitors such as anti-PD1 or PD-L1 antibodies, for which failures stem in part from hypoxic and immunosuppressive TME [START_REF] Anderson | Obstacles Posed by the Tumor Microenvironment to T cell Activity: A Case for Synergistic Therapies[END_REF].

In cancer, strategies are being developed to target mitochondrial metabolism to eliminate tumors [START_REF] Vasan | Mitochondrial Metabolism as a Target for Cancer Therapy[END_REF][START_REF] Reyes-Castellanos | Mitochondrial Metabolism in PDAC: From Better Knowledge to New Targeting Strategies[END_REF][START_REF] Ashton | Oxidative Phosphorylation as an Emerging Target in Cancer Therapy[END_REF]. The list of compounds targeting ETC and OXPHOS function is growing.

The most numerous are small molecules inhibiting mitochondrial CI, such as the biguanides Metformin and Phenformin, fenofibrate, and IACS-010759 [START_REF] Urra | The Mitochondrial Complex(I)ty of Cancer[END_REF][START_REF] Greene | Targeting OXPHOS and the electronic transport chain in cancer; Molecular and therapeutic implications[END_REF]. Some of these CI inhibitors induce cancer cell death through a mechanism that involves increased ROS production or interaction with AMP-activated protein kinase (AMPK) and hypoxia inducible factor 1-alpha (HIF1-α) signaling [START_REF] Urra | The Mitochondrial Complex(I)ty of Cancer[END_REF]. Importantly, targeting pharmacological molecules into mitochondria has proven to be a beneficial approach to increase their potency [START_REF] Urra | The Mitochondrial Complex(I)ty of Cancer[END_REF]. This can be done by conjugation to a lipophilic cation such as triphenylphosphonium (TTP), resulting in selective uptake of the attached bioactive moiety into the mitochondrial matrix [START_REF] Smith | Mitochondrial pharmacology[END_REF]. SMIP004-7 should now be added to the list of CI inhibitory pharmacological agents with clinical relevance in cancer [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF].

To conclude, multiple clinical trials are currently testing the efficacy of mitochondrial metabolism inhibition as a therapeutic treatment for cancer [START_REF] Vasan | Mitochondrial Metabolism as a Target for Cancer Therapy[END_REF]. Efforts to identify cancerspecific metabolic vulnerabilities, including mitochondrial dependencies, are essential to GLOSSARY Aerobic glycolysis or Warburg effect: In non tumoral cells, glucose is metabolized to pyruvate, which is either transferred to the mitochondria and used for respiration or metabolized via glycolysis to lactate under low oxygen conditions. The Warburg effect described in cancer cells stands for high levels of glycolysis and lactate production, even in the presence of normal oxygen concentrations.

Mitochondrial respiration:

The central process of energy metabolism. The respiratory chain (complexes I-IV) channels electrons derived from nutrients to oxygen, using the energy released to produce ATP.

OXPHOS system: It's composed of the four respiratory chain complexes that are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the fifth complex (ATP synthase) which uses this proton gradient to produce ATP.

Reduction of oxygen:

In the mitochondrial inner membrane, oxygen can be partially reduced to superoxide by the gain of one electron at the level of complexes I or III, or it can be fully reduced to water at the level of complex IV where it is the last acceptor of electron transfer. In the redox system, reduction is electron gain, and oxidation is electron loss. A reducing molecule is an electron donor (ex: NADH) and an oxidant is an electron acceptor (ex: NAD + ).

Figure legend Figure 1 .

 legend1 Figure legend

Figure 1 Cancer

 1 Figure 1

propose new effective therapeutic strategies against cancer. There is also a need to characterize the mechanism of action of pharmacological compounds that may affect metabolism, as has been done for SMIP004 compounds [START_REF] Jain | Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I[END_REF], and to chemically modify them to increase their efficacy and improve their pharmacology.

Declaration of interests

The authors declare no conflicts of interests.


Andrea Acri
email: andrea.acri@ephe.sorbonne.fr

Kesusastraan Jawa

Kuno ; Saskrit

; Śaivisme

; Buddhisme

Mpu Tanakuṅ

; Ḍaṅ

Hyaṅ Nirartha

ON MPU TANAKUṄ, DAṄ HYAṄ NIRARTHA, AND THE AUTHORSHIP OF THE BHUVANAKOŚA 1

Keywords: Bhuvanakośa, Bhuvanakośa, Old Javanese literature, Sanskrit, Śaivism, Buddhism, Mpu Tanakuṅ

This article takes as a point of departure the first Sanskrit verse of the Sanskrit-Old Javanese Śaiva scripture Bhuvanakośa, which has come down to us in Balinese palm-leaf manuscripts. It argues that this verse forms an introductory hymn (maṅgala or stuti) praising Śiva and, at the same time, revealing the identity of the compiler of the text-whether genuine or fictively attributed ex postthrough a "signature" expressed by means of a Sanskrit word hinting at the name Tanakuṅ, an author of Old Javanese literary and religious works who lived in the 15th century. It then elaborates on the socio-historical and religious background of that period, and links the figure of Tanakuṅ and the texts ascribed to him to Ḍaṅ Hyaṅ Nirartha, an important Balinese religious figure who is believed to have been active on the island between the late 15th and early 16th century.

INTRODUCTION

The Bhuvanakośa ("The Storehouse of Worlds") is a Sanskrit-Old-Javanese Śaiva tantric text of the tutur genre. 2 This textual source of anonymous authorship, probably compiled in various stages between the late first millennium CE and the early 16th century, 1 An early version of this article was presented at the International Symposium "Sañcaya Sari: Untaian Adikarya Stuart Robson" (Malang, East Java, 16-17 November 2019). I am grateful to Hadi Sidomulyo for his comments, and to IGA Darma Putra and Putu Eka Guna Yasa for having generously shared with me their knowledge about the Balinese literary tradition. Any mistakes are mine alone. I would like to acknowledge the support of the programme "Scripta-PSL. Histoire et pratiques de l'écrit", Investissements d'Avenir, launched by the French Government and implemented by ANR (ANR-10-IDEX-0001-02 PSL), and, from 2020 onwards, of the project DHARMA (ERC Synergy Grant 809994, "The Domestication of 'Hindu' Asceticism and the Religious Making of South and Southeast Asia").

2 On this class of texts, and the sub-genre of tattvas, see Acri 2006, 2017.

Andrea Acri

has come down to us through a handful of Balinese palm-leaf manuscripts (lontar), as well as typed Romanised transcriptions, kept in public or private collections in Indonesia and the Netherlands. Mainly arranged in a dyadic style (see Acri and Hunter 2020), the text is by far the bulkiest tutur known to us, comprising about five hundred Sanskrit stanzas (śloka) accompanied by translations, paraphrases, and/or commentaries in Old Javanese.

The Bhuvanakośa is still regarded in contemporary Bali as a "Classic" in the domain of Śaiva mysticism. The appreciation-or even "canonisation"-of this scripture is not a new phenomenon, but may date back to the pre-colonial period, for the Bhuvanakośa is included in a list of eight authoritative scriptures described as the "secret texts of the Brahmans" by the 19th-century observer Rudolph Friederich. 3 It is no wonder that this text has been known long since to both Western and Indonesian scholars. For instance, Roeloef Goris (1926) and Alexander Zieseniss (1939) described and studied parts of it in their pioneering works on Javano-Balinese Śaivism. The text was also used by Haryati Soebadio, who pinpointed several textual parallels with the Jñānasiddhānta, another Sanskrit-Old Javanese tutur preserved uniquely on Balinese manuscripts, which focuses on mysticism and yoga (Soebadio 1971). In spite of this state of affairs, the Bhuvanakośa has not yet been critically edited and fully translated into either Indonesian or European languages. 4 A common opinion among Balinese and Western scholars is that the Bhuvanakośa is an early text-probably one of the earliest of the tutur genre. While its formal organisation in Sanskrit-Old Javanese "translation dyads" instead of Old Javanese prose (whether interspersed with Sanskrit ślokas or not) would suggest that this is the case, one can never be totally sure: witness for instance the bulky Tutur Śivāgama, which is organised in just the same way, and yet was composed by Ida Pedanda Made Sidemen, probably in the first half of the 20th century. While the first section of the Bhuvanakośa may very well be early, what is important to stress here is that the text is a heterogeneous source, formed by at least two discrete textual units, structured as follows:

Ch. 1-5: Bhuvanakośa, Brahmarahasyaśāstra, prathamaḥ… pañcama paṭalaḥ. Ch. 6: Jñānasiddhāntaśāstraṁ, prathamaḥ paṭalaḥ. Ch. 7: Bhasmamantrasakalavidhiśāstraṁ, dvitīyaḥ paṭalaḥ. Ch. 8: Jñānasaṅkṣepaṁ, tr̥ tīyaḥ paṭalaḥ. Ch. 9: Bhuvanakośan, navamaḥ paṭalaḥ. Ch. 10: Siddhāntaśāstraṁ, jñānarahasyaṁ, daśamaḥ paṭalaḥ. Ch. 11 + explicit: Bhuvanakośa, paramarahasyaṁ, jñānasiddhāntaśāstraṁ, On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa śivopadeśaṁ samāptam.

The Bhuvanakośa deals with cosmography, geography, soteriology, ontology, yoga, subtle physiology, connections between microcosm and macrocosm, mantric mysticism (both sonic and graphic), and other aspects of Śaiva doctrine and practice. Although it documents a form of Śaivism that in terms of doctrine is related to that of texts of the tattva genre transmitted in Java and/or Bali, such as the Dharma Pātañjala, the Vr̥ haspatitattva, and the Tattvajñāna, the Bhuvanakośa does not deal extensively with metaphysics, and parts of it seem to betray a derivation from a different South Asian prototypical tradition. In this respect, some sections of the text present analogies that are found in the Mahābhārata and the Upaniṣads, early non-dualistic Śaiva Saiddhāntika texts, such as those belonging to the Kālottara (or Vātula/Āgneya) corpus, as well as Vedānta-influenced relatively late (post 10th-century) non-dualistic South Indian Śaiva Saiddhāntika texts. 5 While the Śaiva doctrine of the Bhuvanakośa displays several archaic elements, such as a list of 26 cosmic principles (tattva) encompassing the 25 principles of the Sāṅkhya plus Rudra at the top (vis-à-vis the 31 or 36 tattvas accepted by subsequent Śaiva schools), as in Pāśupata Śaivism (or Atimārga Śaivism), it espouses an Atimārgic gnostic approach to soteriology focusing on internalised yogic practices, including visualisations and the association of syllables to subtle centres of the body. Chapters 6 to 11 are characterized by a remarkable intertextuality with the Jñānasiddhānta, which shares with the Bhuvanakośa a similarly heterogeneous textual arrangement, and which may possibly be roughly contemporaneous to its eponymous second section, the Jñānasiddhāntaśāstra.

RESULTS AND DISCUSSION

Having described the general features of the Bhuvanakośa, I will now focus on the Sanskrit verse (maṅgala) that opens the text. While I have already dealt with this verse elsewhere (Acri 2015a), in the context of a parallel between the maṅgala verse found in the "colophon" of the Nītisārasamuccaya ("Compendium of the essence of policy"), a legal text written in Indic script and pre-Classical Malay language preserved in the Tanjung Tanah codex in the Kerinci highlands of Central Sumatra,6 in what follows I will analyse it to advance a tentative hypothesis about the authorship-or, more precisely, the identity of the compiler-of the text.7 This discussion will serve as a point of departure to elaborate on two emblematic literary and religious figures of the late Majapahit period in East Java and its aftermath in Bali, namely Mpu Tanakuṅ and Ḍaṅ Hyaṅ Nirartha (or Dvijendra).

Andrea Acri

The verse, together with its Old Javanese prelude and commentary, runs as follows: 8 The verse, as it is customary for maṅgalas or stutis in both Sanskrit and Old Javanese literature, is a hymn of praise directed to the paramount deity worshiped by the poet, uttered by the poet himself. In this case, the commentary explicitly tells us that the author or compiler of the text-who is the interlocutor of Śiva, seemingly receiving his revelation in the form of a vision-asked the deity about the state of extinction (nirvāṇa), thus triggering the Lord to impart to him the lessons constituting the text itself. The compiler seems to be hinted at by the expression saṅ saṅgrahakāri. I interpret saṅgrahakāri as a tadbhava (i.e., a Sanskrit loanword that has been morphologically altered) formed by the compound saṅgraha ("a compendium, summary, catalogue, list, epitome, abridgment, short statement", Monier-Williams 1899) plus kāri(n) ("doer, maker")-compare "[saṅgraha]kāra, m. the composer or author of the Saṃgraha" (Monier-Williams 1899). The label 8 Here I have used my own in-progress critical edition of the text; an edition of the first six Sanskrit verses plus Old Javanese glosses of the text, on the basis of the single manuscript (from Lombok) Leiden Cod.Or. 5022, may be found in Goris 1926, 78-81. 9 Em. All manuscripts read sasaṅgraha kāri sira. (The romanized version published by Pusat Dokumentasi Budaya Bali reads saṅ saṅgraha kāri (where kari is an "emphatic particle (often, but by no means exclusively, in interrogative sentences)", Zoetmulder 1982, 807), which is likely to be an editorial improvement rather than reflecting a genuine variant reading found in the manuscript prototype). 10 Em. All manuscripts read deva. 11 Conj. All manuscripts read vākya. See my discussion below. 12 Em. All manuscripts read amanmatha.

On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa saṅgraha used to define the text makes perfect sense in this context, as the Bhuvanakośa itself is a composite text, a compendium of material redacted together by a compiler. It may be noted here that in the maṅgala of the Old Malay Nītisārasamuccaya, which shares some similarities with the first verse of the Bhuvanakośa, the text expounded (vakti) by the poet is said to be "extracted from various authoritative sources" (nānāśāstroddhr̥ taṁ), which equals to say, it is a synthetic compilation or compendium (= saṅgraha). I should also like to point out that some early Śaiva Tantras from the Indian subcontinent are conceptualized as saṅgrahas, and bear that element in their titles-see, for instance, the Rauravasūtrasaṅgraha and the Svāyambhuvasūtrasaṅgraha; compare the Niśvāsatattvasaṁhitā (saṁhitā being a collection of texts).

The first quarter of the Sanskrit verse, praṇamya śirasā devam, is also attested, either in exactly the same form or with minor variants, in the Nītisārasamuccaya and in a number of Balinese and South Asian Sanskrit texts (Acri 2015a, 66-67). The second quarter, vakti munir amanmathaḥ, presents some grammatical and lexical problems, yet it is the most interesting, and constitutes the pivot of the argument presented in this article. Vakti is my emendation of vākya, from √vac, "to speak, declare, proclaim". Vākya may be either a nonstandard form of the optative participle vācyaṁ ("what is to be spoken/desired to be spoken/going to be spoken"), or a non-standard form equivalent to the substantive vākya(ṁ), "speech". Amanmathaḥ (nominative) is my emendation from amanmatha (vocative). If we accept either vākya = vācya(ṃ) or vākyaṁ (subst.),13 the verse-quarter could be the result of a contamination between a nominal sentence with genitive (vākyaṁ muner amanmathasya,14 which is, however, hypermetrical), and a verbal sentence, the correct form of which would be: vakti munir amanmathaḥ. Indeed, the shift from vakti to vākya(ṁ) is justifiable on palaeographic grounds, entailing the confusion between the subscript ya and ta, the drop of the hulu (or its transformation into an anusvāra), and the artificial lengthening of the first a (which is already metrically long as it occurs before a consonantal cluster). The form vakti, it should be noted, is not only reflected in the Old Javanese commentary (mojar ta sira), but, as I have mentioned above, also occurs in the maṅgala of the Nītisārasamuccaya. 15 As for amanmathaḥ, the context requires it to be a qualifier of muniḥ, the sage who is the saṅ saṅgrahakāri, and who, having bowed down to Śiva, utters (vakti) the invocation to him. The Sanskrit manmathaḥ means "agitating", "love or the god of love" (i.e., Kāma[deva]), "amorous passion or desire"; therefore, as an adjectival compound, amanmathaḥ could mean "he who is free from [erotic] desire". The form with an added alpha privans is, to the best of my knowledge, unattested in both the Sanskrit and Old

Andrea Acri

Javanese textual corpus. We are, therefore, dealing with a hapax,16 and a highly unusual form too, especially given the fact that it is used to qualify a person, for the same concept could have been conveyed through a number of well-attested synonyms (e.g. virāga, niṣkāma, niḥspr̥ ha, etc.). In view of this fact, I would like to tentatively advance the hypothesis that the Sanskrit bahuvrīhi compound amanmathaḥ could be a "signature", i.e., reveal (and hide at the same time)17 a reference to the name of the compiler of the text.

An element supporting the above-mentioned hypothesis is that one of the possible translations into Old Javanese of the compound amanmathaḥ would be tan akuṅ, "[he who is/one] without amorous passion/desire",18 and (Mpu) Tanakuṅ happens to be the (pen) name of an author who flourished in the second half of the 15th century in East Java. In their discussion about the name of this author, Teeuw et al. (1969, 13) translate kuṅ as "sexual love", "pangs of love", "amorous yearning", and the derivation akuṅ as "enamoured", "filled with amorous desire". They note that the former corresponds to the Sanskrit word rāga-tan akuṅ meaning "without amorousness", "without passion", "indifferent", being more or less the equivalent of the Sanskrit virakta; 19 and they conclude that "[c]learly it is a fitting pen-name for a poet who, in his own words, strives to attain a state of passionlessness or indifference" (Teeuw et al. 1969, 14). 20 Pen names (parab), either in Old Javanese or, less frequently, Sanskrit, were common in the literary world of kakavins, and poets often hid them in the introductory maṅgalas or final verses of their works as signatures (sometimes making playful and ironic allusions about themselves). 21 Clearly, premodern Javanese authors were keen to exploit the polysemy of Old Javanese and Sanskrit, and to use various synonyms to indicate their pen names. As noted by Supomo (1977, 4), since "the manipulation of synonyms is one of the most characteristic features of Old Javanese poetry […] it is not surprising to observe that in every kakawin a person, a god or a country is called by several proper names."

Now, we know that Mpu Tanakuṅ was the author of at least two Old Javanese kakavins: the Vr̥ ttasañcaya and the-strongly Śaiva in religious persuasion and theme-Śivarātrikalpa. Another didactic poem, the Udyalāka, explicitly states that its author is Tanakuṅ, but Teeuw et al. (1969, 13) cast doubt on the authenticity of this claim, and opine that it might have been either an early work of the poet (due to the presence of mistakes and other irregularities), or "it may be that it was written in Bali years later and for some reason traditionally ascribed to Tanakuṅ." Similarly, some short lyrical poems, like the Bhāṣa Tanakuṅ, are ascribed to this author; 22 but some among them are also ascribed to Nirartha (Teeuw et al. 1969, 13). In fact, in Bali, the collections of poems by Tanakuṅ and Nirartha are usually found in the same manuscript bundles. And some texts attributed to Nirartha, 23 like the Usana Bali Mayāntaka, do mention (and praise) Tanakuṅ. This association between the two figures, as we shall see below, is relevant. Teeuw et al. point out that at least two other Old Javanese poets had "negative" names, namely Tantular and, indeed, Nirartha. The latter is traditionally ascribed to the period between the late 15 th and early 16 th century, and plays a paramount role in the Balinese Śaiva tradition as the initiator of the lineage of the Pedanda Śiva, the highest Brahmanical ritual agents of Bali. The Śaiva didactic religio-philosophical poem Nirarthaprakr̥ ta, existing in only one manuscript from the Puri Cakranegara collection in Lombok, has a colophon declaring that the text was composed by Puputut Tanvriṅdeya (Student "Baffled") in Śaka year 1381 (AD 1459) in a location called Kañcana ("Gold"), a (predominantly Buddhist?) religious centre not far 1.3 runs: maṅgalyāni ṅhulun dhairya sahaja makәdә̄ mrākәtāṅ bhomakāvya, "May he (i.e., Kāma, and the poet's sponsor, whom he embodies) be a sure source of blessing for me too, as of my own accord I do my best to render the kāvya of Bhoma in Javanese" (trans. Teeuw 2005, 71). It seems that Teeuw translates dhairya + makәdә̄ (Zoetmulder 1982, 837: "feeling the urge to, feeling impelled to, set on; [wanting, seeking to obtain, etc.] at all costs [by any means]; keeping on, cannot but unremittingly, insistently, obstinately, unavoidably") as "I do my best"; however, dhairya here is more likely to represent a (Sanskrit or Sanskritic) epithet of the poet (who is writing in the first person, ṅhulun), which is the positive equivalent of the negative Old Javanese expression tan tular "immovable, unchanging, unperturbed". Some elements in the maṅgala evoke the East Javanese cultural milieu of Majapahit, in which Tantular was active. For instance, the expression dhyakṣeṅ kalaṅvan, which Teeuw translates "Chief Judge in Poetical Affairs", recalls the title dharmādhyakṣa, "Superintendent of Religion", a figure that is often mentioned in inscriptions of the Majapahit period (cf. below); and also the emphasis on Kāma, a divine figure whose significance becomes rather central in the religious and literary culture of the time. Teeuw (2005, 57) characterized the Bhomakāvya as "a well-thought out and well-composed literary creation by an intelligent, creative, and well-informed author", which Tantular undoubtedly was. Darma Putra (2020), in a short study on the maṅgala of the Bhomāntaka, has advanced a different hypothesis, connecting the text to an earlier historical period, namely the reign of Jayabhaya (AD 1135-1157), and interpreting dhairya sahaja as referring to the unwavering quality of the poet-viku who authored the text. This remains an open issue that needs to be explored further. On the hypothesis that Prapañca, the author of the Deśavarṇana/Nāgarakr̥ tāgama, is to be identified with (the young) Tantular (prapañca being an antonym of tan tular), or perhaps with an opponent, see Supomo 1977, 2-3. 22 Robson (1979, 304-305) considers this attribution verisimilar, on account of the author's seeming reliance on Sanskrit sources. 23 See Dvijendratattva 25a.

Andrea Acri from Majapahit. 24 Robson (1979, 305) correctly notes that "The use of a pen-name containing the word tan ("not") reminds us of the poets Tantular and Tanakung." 25 Both the Sutasoma by Tantular and the Nirarthaprakr̥ ta display signs of mixed Śiva-Buddha religiosity. 26 Nirartha himself was, according to the Balinese tradition, a Buddhist who converted to Śaivism in order to marry the daughter of a Śaiva priest. As it is often the case, Balinese chronicles and other folk-accounts, even though containing imaginific narratives elaborated to make sense of certain literary, historical, or religious realities, reflect a kernel of historical truth, or in any event preserve a memory of important historical personalities.

There is one more kakavin that needs to be mentioned in this context, namely the Dharma Śūnya, a Javanese manuscript of which contains a colophon dated Śaka 1382, or AD 1460. Commenting on the date of this colophon, Robson (1979, 305) notes that "Assuming that this reading is correct, we can see that the works of Tanakung were not entirely alone." Then, we have a major didactic-religious poem written at the same time in which Tanakuṅ flourished. Is it a coincidence, or could this text have been penned by Tanakuṅ himself (or by somebody in his "workshop")? An association going into that direction has already been made by the Balinese tradition, according to which the author of the Dharma Śūnya was not the Kamalanātha mentioned in the text, but Nirartha (Dharma Palguna 1999, 6). According to Dharma Palguna, Kamalanātha states that he is a man of religion (viku) without any possession (verse 153), which he links to the name Nirartha, nirartha meaning "without (nir) material possessions (artha)" in Sanskrit. 27 This attribution is difficult, but not impossible, to reconcile with the date of AD 1463 given in the above-mentioned colophon, for Nirartha's stay in Bali is traditionally ascribed by Babads to the period between AD 1488 and AD 1536. (On the other hand, the colophon of the Nirarthaprakr̥ ta gives the date of 1381 Śaka or AD 1459, but it must have been copied from a previous manuscript as the lontar in question is obviously Balinese, and the same bundle contains texts with later colophons). Now, some interesting connections between the Dharma Śūnya and Tanakuṅ have been advanced by Dharma Palguna. For instance, the late scholar has noted the presence 24 The Old Javanese inscription of Kañcana records the purchase of land for the establishment of a Buddhist foundation. According to Sidomulyo (2018, 231), this charter dates from the 9th century, and as such represents "the earliest documentary evidence of the central Javanese court's interest in the Brantas Delta region." More recently, however, Griffiths (2020, 133) has argued in favour of a 10th century dating, accepting the argument advanced by Krom in 1914. 25 This link was already pointed out by Poerbatjarka (1951, 204, note 9, along with the name or epithet Mapañji Tanutama (Tan Utama?), which is attributed to the dharmādhyakṣa of the Śaivas, Ḍaṅ Ācārya Śivanātha, in the Sarvadharma charter); cf. Supomo (1977, 3) and Teeuw et al. 1969, 14. Supomo (1977, 3) has noted how the words tan akuṅ and tamatan tular occur at the beginning of the first line of two stanzas of the Rāmāyaṇa kakavin (8.164a and 8.165a), which suggests that those pen-names could have denoted two authors living under the same ruler in Paṇḍan-salas, who must have had access to the same copy of the Old Javanese text. 26 I do not say "syncretic", because this "mixed" religiosity could actually be the outcome of an inclusivistic attitude (see Acri 2015b). 27 Another possible meaning is "without aim". But in the text Dharma Pitutur, attributed to Nirartha by the Dvijendratattva, the author uses the pen name "Braja Miskin", miskin meaning "poor" in high Balinese. Cf. Dharma Pitutur, 52b: kaputus dharma kavi tan pitutur makasadhananiṅ mpu braja miskin. I thank Putu Eka Guna Yasa of Udayana University in Denpasar for having pointed out to me this reference.

On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa of a praise to the Goddess Sarasvatī in both the Dharma Śūnya and the Vr̥ ttasañcaya (Dharma Palguna 1999, 227). Furthermore, he (1999, 307) pointed out that in Bali, some stanzas on Paramaśiva from the Dharma Śūnya and kakavin Bhārgavaśīkṣā, 28 as well as the Śivarātrikalpa-all works characterized by a similar religious content-are sung to introduce the devayajña, the worship to the highest Gods. I will discuss the implication of the name Bhārgava below; for the time being, let me note that the association between the Dharma Śūnya and the Śivarātrikalpa in Balinese worship seems to be relevant indeed. As a last point, I should mention that, as reported by Dharma Palguna (1999, 217), the Balinese scholar Ida Bagus Gede Agastia has linked the Kamalanātha mentioned in the Dharma Śūnya as its author to Kāmanātha, on account of the information found in Balinese babads that the Buddhist priest Daṅ Hyaṅ Smaranātha (son of Mpu Tantular) was the father of Daṅ Hyaṅ Nirartha (a Buddhist converted to Śaivism). 29 While the superficial resemblance between the kama-in Kamalanātha and the kāmain Kāmanātha seems to me not a strong piece of evidence to suppose an identity between the two (provided the former is, indeed, not the outcome of corruption), I believe that it is worthwhile to follow IBG Agastia's lead. Dharma Palguna (1999, 214) has noted that in the portions of the Dharma Śūnya and Dharma Putus 30 that attribute the authorship to Kamalanātha, the authors are faithful to the well-established convention of self-critique as being of modest understanding, of not mastering kakavin prosody properly, etc. This stands in contrast, and is not consistent with, the adoption of the self-aggrandizing name Kamalanātha ("The Lord [nātha] of/in the Lotus [kamala]") instead of such names as Tanakuṅ, Nirartha, etc. Indeed, Kamalanātha would virtually imply an equation of the poet with Paramaśiva, the supreme deity that is said to reside in the lotus (= heart) inside the human body in a number of tuturs. However, since kamala in Sanskrit may also have the (much less common) meaning of "desirous, lustful" (Monier-Williams 1899, on the basis of BRD, as per Atharvaveda VII, 6, 9; compare kāmala "libidinous, lustful", L.), then Kamalanātha could be a Javano-Sanskrit "neologism" intended to be a synonym of Kāmadeva, and equivalent to Smaranātha. 31 A version of the Babad Brāhmaṇa mentions Nirartha as the son of (A)smaranātha, himself the son of Tantular (Dharma Palguna 1999, 262), giving the following genealogy: Bhr̥ gu > Bajrasattva > Bharada > Bahula > Mpu Tantular (also Saṅ Hyaṅ Aśokanātha) 32 > 28 Another Bhārgavaśīkṣā is an Old Javanese prose version of the Bhuvanakośa without ślokas. The text is evidently named after the sage Bhārgava, mentioned at the beginning of the text. 29 See Rubinstein 2001, 83. 30 Kakavin Dharma Putus, Leiden Cod.Or. 19.526 (Dharma Palguna 1999, 207). 31 Intriguingly enough, kamala appears to be the antonym of tan akuṅ. 32 Tan tular in Old Javanese means "not moving, immovable, unchanging, unperturbed"; thus, Aśokanātha (sometimes spelled Aṅśokanātha) "The Lord (nātha) without (a) affliction/trouble (śoka)" would be an appropriate translation of tan tular.

Andrea Acri

(A)smaranātha 33 > Daṅ Hyaṅ Nirartha. 34 As noted by Rubinstein, Balinese babads narrating the genealogy and biography of Nirartha attribute to many of his Javanese ancestors (including his father Ḍaṅ Hyaṅ Asmaranātha) the status of royal purohitas and poets of Majapahit, identifying them as the authors of certain Old Javanese kakavins (Rubinstein 2001, 83-85). 35 Further, she points out that the list of Nirartha's ancestors given by these texts does not specify the existence of kinship ties, but rather depict a type of "intellectual genealogy" (Rubinstein 2001, 84).

All the above suggests that there was not only an association, but perhaps at some point even a "confusion", between the two figures of Tanakuṅ and Nirartha in Bali-or, at the very least, an attempt to link them together as belonging to the same spiritual, intellectual, and literary lineage. How is all this relevant to the issue of the authorship of the Bhuvanakośa? My tentative hypothesis is that the Sanskrit expression munir amanmatha in the maṅgala may be equivalent to the Old Javanese "Mpu Tanakuṅ"/"Asmaranātha", and that this could reflect a Balinese attempt to link the text to a (to some extent fictional, and to some extent historical) religious and intellectual tradition that orginated in Majapahit. 36 I will provide some further internal and external support to this idea below.

The Old Javanese commentary of the maṅgala of the Bhuvanakośa does not unpack this supposed "pen name", just passing over in silence the second quarter of the Sanskrit śloka. Yet, it does mention the name of the sage who is the interlocutor of Śiva, namely Bhārgava. Bhārgava means "son of Bhr̥ gu", or "descendant of Bhr̥ gu". As we have seen above, the Babad Brāhmaṇa traces the genealogy of both Tanakuṅ and Nirartha to Bhr̥ gu; the Dvijendratattva (2b) associates Nirartha to the "Bhr̥ guvaṅśa" of Griya Mās in Dāha by marriage. 37 Therefore, the mention of Bhārgava-who nota bene does not appear anywhere 33 Smaranātha seems to have been a fairly common name in East Java, for two Old Javanese charters from Majapahit, viz. the Decree Jaya Song of c. AD 1350 and the undated Charter of Batur (Pigeaud 1960, 104-114; cf. Sanderson 2009, 119, fn. 267), refer to the officials known as dharmopapatti ("Assessors of Religion"), among whom is a certain Smaranātha (the latter inscription actually refers to two different Smaranāthas); the Bendosari copper plates, issued under the reign of Hayam Vuruk (Śrī Rājasanāgara, r. 1350-1389), mention a Smaranātha (along with Śivanātha and Agreśvara) as belonging to the Bhairava denomination (Brandes 1913 I, 209). Compare the Ācāryas named Smaradahana and Smaradeva mentioned in the Sarvadharma charter of AD 1269 (issued under Kr̥ tanagara's reign: see Pigeaud 1960, 99-103). 34 The Dvijendratattva (2a-2b; compare the other versions of the same text, like the Babad Dvijendra) regards A(ṅ)śokanātha as the brother of Dvijendra, son of Asmaranātha, the purohita of Majapahit. Another version of the Babad Brāhmaṇa (Leiden Cod.Or. 13.733, 2a) gives the following genealogy: Mpu Śrīnātha > Mpu Aṅśoka and Mpu Nilartha. 35 For instance, to Tantular is attributed the authorship of the Arjunavijaya, the Sutasoma, and the Vr̥ ttasañcaya (contrast Supomo 1977, 1, reporting a passage from Friederich's account of a Balinese tradition assigning only the first two kakavins to "Mpu Tantular Boda" from Keḍiri). The last kakavin is by Tanakuṅ, but see Supomo (1977, 3) on the connection or relationship between Tantular and Tanakuṅ; cf. above, fn. 25. 36 Actually, the Balinese tradition rather consistently link Nirartha to Keḍiri rather than Majapahit. According to Hadi Sidomulyo (email dated 26/09/2020), this may suggest that he lived during the time when the Javanese royal family had abandoned the royal capital (after AD 1478) and fled southward. 37 Nirartha is said to have married the daughter of a Brahman priest, named Daṅ Hyaṅ Panavaran, whose ancestor was Bhr̥ gu. Rubinstein (2000, 76) notes that, according to one version of the Babad On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa else to my knowledge in Old Javanese or Balinese literature as the name of a historical person, nor it appears again as such in the Bhuvanakośa itself, except in the colophon of the Lombok manuscript Cod.Or. 5022 38 -may just be an appellative to denote the familial line of the author. Indeed, in 3.24, in the Sanskrit śloka the Lord addresses his interlocutor as bhr̥ gusattamaḥ ("the excellent Bhr̥ gu/the excellent among the Bhr̥ gus"), which the Old Javanese commentary glosses with the Sanskrit compound bhr̥ gutanaya, "the son of Bhr̥ gu". 39 To make things even more complicated, elsewhere in the first chapter of the Bhuvanakośa, the interlocutor of Śiva is addressed by the Lord as dvijarājendra "Supreme King of Brahmins" (Sanskrit verses, 1.15 and 1.16; Old Javanese commentary, 1.16) and dvijendra (Old Javanese commentary, 1.15). Another name of Nirartha was Mpu Dvijendra. Of course, dvijendra/dvijarājendra could just be a general appellative applied to Brahmins (like yogīśvara, or munisattamaḥ in 1.31), and perhaps it may have had some specific linkage with the Javanese and Balinese Brahmanical milieus of that time. But it could also reflect an attempt by the text to associate itself to both Tanakuṅ and Nirartha/Dvijendra, thereby confusing/coalescing them, or rather, it should be understood in the context of what was perceived by the tradition to be a "priestly family" or "intellectual genealogy". Or, could it be that Dvijendra was regarded as being present, along with Amanmatha/Bhārgava (alias Tanakuṅ/(A)smaranātha), on the occasion of the "hearing" of the text from the mouth of Śiva?

Let us now go back to Asmaranātha, whom, as we have seen above, some scholars have identified with Tanakuṅ. In regular Sanskrit, Asmaranātha could mean either "he who does not have Kāma as Lord", or "The Lord without [erotic] love". But, if this is indeed a Javanese Sanskritic compound intended to be a pen name, could it have been understood as a synonym of Amanmatha? Both Smaranātha ("God of Love") and Manmatha refer to Kāmadeva, and both are prefixed by an a-. My hypothesis is that Smaranātha (occurring in East Javanese inscriptions) and its synonym (?) Kamalanātha (occurring in the colophons of the Dharma Śūnya and Dharma Putus) denoted a different individual than Tanakuṅ, but they might have been coalesced with Asmaranātha, representing Tanakuṅ, in the Balinese babad tradition. 40 As for the identification of Kamalanātha (= Smaranātha?) with Nirartha, it is not impossible that it is the result of an ex post association between the sage, who obtained release disappeared from this world at Maśceti in Bali, and Kāma, who was regarded as being without a body (due to its burning by Śiva)-witness his epithets Atanu Brāhmaṇa (for instance, K. 237), "Nirartha acquired some ancestral writings from his father-in-law, a Brahmana Siwa priest from Daha, Java, around the period that Majapahit fell into decline". 38 Oṁ namaḥ śivāya | oṁ namo vāgīśvaraya | oṁ namo bhārggavāya || śrī gurubhyo namaḥ ||. 39 A recently discovered Merapi-Merbabu manuscript (dated Śaka 1493/AD 1571) of a Śaiva tutur entitled Mahāpadmaśāstra, belonging to a Griya in Buleleng, North Bali, appears to be arranged as a teaching imparted by Bhagavān Bhr̥ gu to his disciples. Although this Bhr̥ gu may represent the mythical sage rather than a historical figure, the possibility that it may refer to the Bhr̥ gu associated with Griya Mās of Dāha cannot be ruled out. (Although I have been unable to access this manuscript, a transcription of a few words at beginning and end of the text contained in it was kindly shared with me by Sugi Lanus via WhatsApp in July 2018. On this manuscript, see https://baliexpress.jawapos.com/read/2019/09/30/158436/lontar-merapi-merbabu-ditemukan-didesa-jinengdalem-usia-300-an-tahun). 40 Confusingly enough, the a-could be taken either as a Sanskrit alpha privans or as an Old Javanese adjectival prefix, resulting in the meaning of "he who has Kāma as Lord"/"he who is Kāma". See above, fn. 19, about the equivalence of tan akuṅ, virāga, and Kāma.

Andrea Acri

and Anaṅga, "the one without a body" (see above, fns. 19 and 40). This would evoke an important point of doctrine in the religious literature of the time, namely the achievement of release through a form of yoga causing the disappearance of the body into the nothingness/Supreme Reality (cf. e.g. Kiduṅ Dharma Pitutur 47-52a).

If Asmaranatha/Amanmatha was indeed Tanakuṅ, the legendary or pseudo-historical account of the Babad Brāhmaṇa does not seem to be too far off the mark, as the chronology of the two figures is not distant. As for Nirartha, Balinese oral accounts ascribe his arrival on the island to AD 1489, in the period of King Waturenggong of Gelgel. While Nirartha/Dvijendra was long deemed by Western scholars to be a fictitious figure, recently discovered manuscript evidence suggests that it might have been a historical figure after all. Worsley et al. (2013, 24) have described a colophon (actually, one of three) contained in a manuscript preserving the kakavin Sumanasāntaka (Leiden University Library Cod.Or. 5015), which states that "the copy was completed on 14 July 1537 in Bali, at 'the sima Kanaka by one whose parab was Nirartha' (tlas sinurat ing nūṣa Bali, ring sima Kanaka, de sang aparab Nirartha)." Worsley et al. note that all the elements of the elaborate dating system check, and conclude:

Taking the date to be genuine, we may further assume that Nirartha mentioned in this colophon must be none other than the Nirartha, the priest and poet, who migrated from Java to Bali to found the brahmana families during the turbulent period preceding the demise of the kingdom of Majapahit at the end of the fifteenth or beginning of the sixteenth century […]. As it happens, the date also conforms to the dates of Nirartha's other literary activities recorded in later Balinese sources. According to the Dwijendratattwa and the Babad Brāhmaṇa, in 1536-that is the year before he copied the Sumanasāntaka-he had just completed a work called Mahiṣa Mĕgat Kūng (Rubinstein 2000, 73).

Another element of interest is that the sima Kanaka mentioned in the colophon could be linked to the kañcana (both meaning "gold") mentioned in the colophon of the Nirarthaprakr̥ ta copied by Tan Vriṅdeya (allegedly in 1381 Śaka), identified by Poerbatjaraka as a Majapahit (Buddhist) religious site, mentioned as the place of origin of the author. This may correspond to the Griya Mas of Dāha (mās/mas meaning "gold" in Old Javanese and Balinese) of the Bhr̥ guvaṅśa mentioned in the Dvijendratattva. Could it be that Nirartha "replicated" in Bali this kañcana/Griya Mas in East Java, or that perhaps there existed kinship links between the priestly institutions in the two locations? 41 Further codicological evidence needs to be mentioned here. It is worth noting that the same manuscript bundle from the Lombok collection that preserves the Nirarthaprakr̥ ta (Leiden Cod.Or. 5023) also contains the Añaṅ Nirartha and Saṅu Sәkar by Nirartha, the Śivarātrikalpa by Tanakuṅ, and other late Majapahit works, some of which of Buddhist persuasion, such as the Deśavarṇana/Nāgarakr̥ tāgama, the Jinarthiprakr̥ ti, and the 41 I wonder whether the Buddhist dharmādhyakṣa Kanakamuni mentioned in the Decree Jaya Song (see above, fn. 33) could have been linked to this religious site, on account of the element kanaka-("gold") in his name, and his Buddhist affiliation.

On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa Kuñjarakarṇa. Here not only a work of Tanakuṅ is included, but in the whole, we come across the coexistence of Śaiva and Buddhist spirituality that was emblematic of late Majapahit, and that was attributed to Nirartha by the Balinese tradition (allegedly a "convert" from Buddhism to Śaivism). Furthermore, it may be not coincidental that one of the oldest known manuscripts of the Bhuvanakośa, Leiden Cod.Or. 5022, stems from the same collection.

One more intriguing piece of evidence is the Central Javanese Merapi-Merbabu codex Ms.Or.fol 410 preserved in the Berlin Staatsbibliothek, which contains the Dharma Śūnya and the Dharma Putus, plus other two unidentified mystical poems. Perhaps significantly, as I have mentioned above, the Dharma Putus, a text of mixed Śaiva-Buddhist religiosity, declares to have been composed by one whose name (parab) is Kamalanātha (Dharma Palguna 1999, 207). This text (along with the Dharma Śūnya Kәliṅ and the Dharma Pitutur, among many others) is attributed to Nirartha by the Balinese tradition, namely the Dvijendratattva (2a-2b; cf. pp. 116-117). The Merapi-Merbabu Dharma Putus bears a colophon dated 1535 Śaka or AD 1613. Dharma Palguna (1999, 278) notes that in Bali, too, several manuscripts of the Dharma Śūnya also contain the Dharma Putus.

There is much in common between Tanakuṅ and Nirartha as historical and literary figures. Both figures must have been remarkable indeed, and must have exerted an influence on the literary and religious culture of their time. Both Tanakuṅ and Nirartha were exponents of elite social milieus, and probably even held positions at the royal courts of Majapahit and Bali; Nirartha is depicted in Balinese texts as a poet, intellectual, wonderworker, and advisor to rulers. 42 Tanakuṅ is regarded as an innovator who had access to Sanskrit sources that were unavailable in Java at his time, such as the Padmapurāṇa and Sanskrit treatises on prosody, as well as Śaiva texts and practices that did not exist in Java before the 15th century, for instance the ritual of śivarātri (after which his chef d'eouvre, the Śivarātrikalpa, is named). 43 This has led to the speculation that he might have visited India-more precisely, South India, by then part of the Vijayanagara Kingdom-by the second half of the 15th century, and introduced the new doctrines and texts to Java after he returned back to his homeland (Teeuw et al. 1969, 19-20). The view that Tanakuṅ's "knowledge of the religious and cultural life of South Asia was gained first hand" was supported by Hunter (2001, 90) on the basis of the analysis of the Vr̥ ttasañcaya and its South Asian Sanskrit prototypes, which led him to conclude that the figures found in that text are not those "of a poet whose cultural horizons were bounded by the walls of an East Javanese court or monastery", his world being one in which the translocal "Sanskrit 42 Indeed, Balinese Pedandas-an institution founded by Nirartha, according to the Balinese-were regarded as religious and ritual advisers (purohita) to rulers (Rubinstein 1991, 64-66), as well as scholars and poets (Rubinstein 2000). 43 See Nihom 1997, 108, who suggests that the association between initiation (dīkṣā) and the five doctrinal and ritual items known as kalās found in South Indian Śaiva texts and, in Java, uniquely in the stuti of Tanakuṅ's Śivaratrikālpa, "is a reflection of the continued, or renewed (?), contacts between the East Javanese kingdom of Majapahit and the southern Indian kingdom of Vijayanagara." It is worthy of note that the only other locus where the five kalās are mentioned, both singularly and collectively as pañcakalās, is the Jñānasiddhānta (Chapter 12), which also contains circumstantial evidence of late southern Indian influences: cf. fn. 45.

Andrea Acri

Cosmopolis" was very much alive. As for Nirartha, he is depicted by the Balinese tradition as a well-travelled innovator or reformer, who transmitted to Bali the form of Śaivism that was in vogue in Majapahit during his time, which is believed to be practiced by the Pedanda Śiva to these days. 44 This could be interpreted as an attempt by the compiler (whether Tanakuṅ, or more likely, Nirartha, or a subsequent author linked to that Brahmanical family stemming from East Java) to reform the prevalent mode of Śaivism in Bali, and initiate a new line of priests. Nirartha must have adhered to the Śaivism of the cosmopolitan East Javanese court as opposed to the more archaic form of the religion that must have constituted the mainstream religiosity of priests in the more isolated and religiously conservative island of Bali.

Finally, as I have pointed out above (see also Acri 2021a, 2021b), the Bhuvanakośa contains illusionistic metaphors-e.g., the reflection of sun or moon on water, or the space in pot-that seem to derive from a form of Śaivism influenced by non-dualistic Vedānta that developed in South India after the 10th century, which we do not find in other Old Javanese Śaiva sources of the tattva genre. This suggests a possible influence from South India, and a relatively late date of compilation. 45 The Bhuvanakośa is also characterized by a somewhat higher degree of reconfiguration of Indic doctrinal elements, which may reflect a cultural context-either late Majapahit or Bali-in which religious ideology and practice had progressively moved away from their prototypical traditions. One also notes the appearance of Buddhist or Buddhist-flavoured terms such as nirvāṇa, śūnya, etc., although one never finds any traits of clearly and explicitly mixed or syncretic Śaiva-Buddhist religiosity. All these aspects are compatible with the biographies and chronologies of the figures of Tanakuṅ and Nirartha.

CONCLUSION

It would probably be a fruitless (not to say misleading) endeavour to try to establish the authorship and date of composition of such a multi-layered and heterogeneous source as the Bhuvanakośa. Yet, the hints at the author(s) or compilers of the text that are found in its maṅgala verse as well as in other parts of Chapter 1 of the text may give us some clues on the context in/for which it was produced and in which it circulated-namely, its reception. The proposed association with either Tanakuṅ or Nirartha, or both-whether genuine or fictitiously attributed ex post-suggests that the Bhuvanakośa may stem from a late Majapahit, "Javano-Balinese" context, and probably reflects the "horizon of expectation" of the Balinese religious specialists who were the prime vectors for its circulation.

44 Chapters 6-9 of the Bhuvanakośa give us some glimpses of what Nirartha's "reform" might have consisted in, namely a critique of a localized form Śaivism stemming from the Atimārga tradition from the perspective of a form of Mantramārga (Saiddhāntika) Śaivism, upholding a gnostic (niṣkala) stance against exterior (sakala) forms of worship. 45 The Jñānasiddhānta preserves a verse from a South Indian version of the Śaiva Saiddhāntika scripture Kiraṇatantra, which is not found in the earlier Nepalese recensions, thus suggesting a late South Indian line of transmission to Java (see Acri 2006).

saṅ saṅgrahakāri 9

 9 sira mavuvus | liṅnira | praṇamya śirasā devaṁ 10 | vakti 11 munir amanmathaḥ 12 | devadeva mahādeva | parameśvara śaṅkara || 1.1 śrī muni bhārgava | sira mahyun tumakvanakǝn ikaṅ pada nirbāṇa ri bhaṭāra | maṅkana pvābhiprāyanira | manambah ta sira ri bhaṭāra | śirasā | makakāraṇa hulunira sira | ri tәlasnira manambah | mojar ta sira || he devadeva | kita devaniṅ devata kabeh | he mahādeva | kita bhaṭāra mahādeva ṅaranta | he maheśvara | kita bhaṭāra maheśvara ṅaranta | he śaṅkara | kita bhaṭāra śaṅkara ṅaranta || The reverend author of the compendium spoke. His words were: Having bowed down with the head before God, the sage who is free from [erotic] desire spoke: 'O God of Gods, O Great God, O Supreme Lord, O Śaṅkara!' The sage Bhārgava desired to ask the Lord about the stage of extinction. Such was his intention. He worshipped the Lord with the head-he used his head. Having finished to worship, he spoke: O God of Gods-you are the God of all the Gods! O Mahādeva-You are Lord Mahādeva by name! O Maheśvara-you are Lord Maheśvara by name! O Śaṅkara-you are Lord Śaṅkara by name!

Friederich (1959, 28-29) listed the following titles [spelling as in the original 1849-50 edition in Dutch, pp. 22-23], 1) Boewana Sangksepa; 2) Boewana Kosa; 3) Wrĕhaspati Tatwa;

4) Sārasa Moestjaja; 5) Tatwa Djnjāna; 6) Kandampat; 7) Sadjotkranti; 8) Toetoer Kamoksa.4 A critical edition and English translation of the text by the present author is in preparation. The versions that have been published in Bali in recent years, which come with a loose Indonesian translation of the Old Javanese portions only (see, for example,[START_REF] Mirsha | Alih aksara dan alih bahasa (Brahma Rahasyam)[END_REF][START_REF] Gautama | Tutur Bhuwana Koṣa[END_REF], appear to be based on a single (unspecified) source, namely a rather faulty typewritten romanized transcription of a palm-leaf manuscript from the collection of the Pusat Dokumentasi Budaya Bali in Denpasar. Although these publications are of great importance for the "socialization" of the Hindu religion in the contemporary Balinese community, they are not very useful for scholarly purposes as they gloss over a number of crucial philological issues.

For a discussion of some of these analogies, see Acri 2021a and 2021b.

This may be the oldest Malay manuscript currently known (ca. 14th or early 15th century).

It is notoriously difficult to determine the authorship, dating, and region of compilation of tuturs, for they are esoteric texts mainly concerned with metaphysical realities. Just like the tantric scriptures in Sanskrit from the Indian subcontinent, these revealed scriptures were studiously freed from any references to worldly realia and human agents (including their authors) in order to keep them consistent with the mythical/atemporal plane in which they are set, where a dialogue between the supreme deity (usually Śiva) and his interlocutor (usually the Goddess, Kumāra, or a sage) takes place.

In Bhuvanakośa 1.3c we find the nominal construction vākyan te, which is (faithfully) glossed as nihan vuvusaniṅ hulun i kita ("my speech to you is as follows").

Being equivalent to amanmathinaḥ (manmathin meaning "enamoured/impassioned/in love").

Insofar that it obviously represents an introductory eulogy to (various manifestations of) Śiva, the verse mirrors the colophon of the Nītisārasamuccaya with respect to both context and function. The two passages contain a verbal praise (stuti) of, and represent a physical act of worship towards, a paramount deity-that is, Śiva-by either the text's author or, in the case of the Bhuvanakośa, the main interlocutor of Śiva and "revealer" of the text. Note the mention of Mahādeva in the line preceding the colophon of the Nītisārasamuccaya, and the epithets Mahādeva, Parameśvara, and Śaṅkara in the Bhuvanakośa.

One may suggest that the Old Javanese gloss mahyun ("desired") might represent the Sanskrit amanmathaḥ ("free from [sexual] desire") in the second verse-quarter, where the commentator would have taken a-as an Old Javanese prefix rather than as the Sanskrit alpha privans that it is. While this possibility cannot be discarded, it seems to me rather unlikely, for the Old Javanese gloss quite faithfully renders the other elements of the verse (cf. Acri 2015a), and it is only natural that the commentator would have wanted to add an explanation about what he defines as the abhiprāya of the sage for speaking to Śiva.

On the cryptic character of parabs or pen names, seeZoetmulder 1974, 151.

Cf. Zoetmulder

1982, 925-926 s.v. kuṅ: "love, amorous desire, lovesickness, languishing with love"; akuṅ "1. in love, infatuated, full of amorous desire, lovesick, languishing with love; 'ṅ akuṅ' may refer to a man or woman, but usually the former."19 Or virāga. If we go back to the word amanmathaḥ, we see that it may hide not only a pen name, but a playful pun: virāga (equivalent to virakta), if analyzed as Sanskrit vi "without" + Old Javanese and Balinese rāga "body", can also mean "the one without a body", an epithet of Kāma or Manmatha, the God of Love.20 Zoetmulder (1974, 365) noted that five out of the seven poems of the Bhāṣa Tanakuṅ, a collection of short lyrical poems attributed to Tanakuṅ, "are laments by the poet at his beloved being forced by her parents to marry another man, so that he (the poet) is now 'without love' (tan akung). Possibly it was such an event in his life which prompted him to adopt Tanakung as a nom-de-plume." I find this explanation rather unlikely, for Tanakuṅ's authorship of the Bhāṣa Tanakuṅ could have been spuriously attributed by the later Balinese tradition precisely on account of the contents of the poem rather than reflecting a genuine life event of the author (pace Robson: see below, fn. 22). Furthermore, one cannot even exclude the existence of two different Tanakuṅs. 21 This is the case of, for example, Mpu Tantular (tan tular), the author of the 14th-century Buddhist kakavin Sutasoma (cf. also below, fn. 27). I tentatively advance the hypothesis that the Sanskrit word dhairya occurring in the opening hymn of kakavin Bhomāntaka (1.3) could represent one such "signatures", alluding to the name Tantular. Dhairya in Sanskrit means "firmness" (OJ: "fortitude, courage", or as an adjective, "firm, constant, steady, self-controlled", etc.). The first line of verse

Jumantara: Jurnal Manuskrip Nusantara Vol. 13 No. 1 Tahun 2022 On Mputanakuṅ, Daṅhyaṅnirartha, And The authorship of The Bhuvanakośa