Inhibition of Metastatic Uveal Melanoma Cell Proliferation by the Histone Deacetylase 6 Inhibitor, Ricolinostat, is Linked to Altered Microphthalmia-associated Transcription Factor and Phospho-ERK Expression

Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase 6 inhibitor (HDAC6i), to attenuate MUM cell growth in vitro and in vivo, and elucidate the underlying molecular mechanisms. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo regression of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry analysis revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0006) following 24 hours of treatment and significant apoptosis was triggered in a time- and dose-dependent manner (p = <0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome-profiling, attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329, significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of MUM cells and are potential therapeutic options for MUM. Simple Summary Uveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body, or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting the cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM cancer that has spread. In our study, we identified that the drug ACY-1215 prevents the growth of UM cells from the liver. Our study has found a promising treatment approach for advanced UM.


ACY-1215 significantly attenuates long term proliferation of human uveal melanoma cell lines
Three commercially available HDAC6i (Tubastatin A, ACY-1215 and Tubacin) were selected to determine their efficacy in reducing long-term proliferation of human UM cell lines derived from primary (Mel285 and Mel270) and metastatic (OMM2.5) UM tumors [35]. Cells were treated for 96 hours at selected concentrations, treatment was stopped, cells were cultured for another 10 days in fresh complete media and colonies formed visualized with crystal violet staining and counted [36]. Initial screens at 10 -50 µM  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint showed a dose-dependent reduction in UM cell proliferation with all three HDAC6i tested ( Figure S1).
ACY-1215 was selected as the highest-ranked drug for subsequent studies based on its observed effects in all three UM cell lines tested and its existing approved use in clinical trials [27][28][29]. ACY-1215 was tested

Zebrafish OMM2.5 xenografts proved that ACY-1215 is efficacious in vivo
Our in vitro study provided preliminary evidence that ACY-1215 has anti-UM properties. Therefore, the efficacy of ACY-1215 in vivo was evaluated using a pre-clinical model of MUM, zebrafish OMM2.5 xenografts. A toxicity screen determined the maximum tolerated dose of ACY-1215 and Dacarbazine in zebrafish larvae, with both drugs well-tolerated at all tested concentrations ( Figure S2). OMM2.5 Dil labelled cells were transplanted into the perivitelline space of 2 days old larvae and xenografts were . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021.   We observed a significant increase in acetylated α-tubulin levels after 4 (3.56-fold increase, p = 0.001) and 24 (3.67-fold increase, p = 0.0002) hours post treatment (hpt) with 20 µM ACY-1215 compared to 0.5% DMSO treated OMM2.5 cells, confirming the inhibitory effects of ACY-1215 ( Figure 3B, 3B' and Figure   S7A). As a dose-dependent anti-cancer effect of ACY-1215 was observed in the clonogenic assays and zebrafish xenografts, correlations between expression level of HDAC6 and UM patient overall survival/progression free survival was analyzed. Extracting the gene expression data of 80 primary UM samples from The Cancer Genome Atlas (TCGA), Cox proportional-hazards models and Kaplan-Meier survival curves were generated. Kaplan-Meier survival curves were generated with a cut-off of 50% to demarcate as high or low HDAC6 expression, and Log-rank test was used to compare survival probability between groups. Interestingly, high HDAC6 expression was significantly associated with better overall survival but not with progression free survival (Cox OS, p = 0.007 and Cox PFS, p = 0.154) ( Figure 3C).
A known caveat of ACY-1215 is the non-selective inhibition of other HDAC isozymes at higher concentrations. The reported IC50 of ACY-1215 is 4.7 nM, at which ACY-1215 acts as a highly potent and . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  HDAC 2,3,1,8,7,5,4,9,11 and SIRT 1/2 ( Figure S4A and S4B) [37]. Thus, correlations between these HDAC isoforms and UM OS/PFS probability was analyzed ( Figure S4C)

Proteome profiling uncovers molecular signals altered in OMM2.5 UM cells by ACY-1215
Having observed beneficial effects against the growth and viability of UM cell lines in vitro and in vivo, proteome profiling of ACY-1215 treated OMM2.5 cells was performed to investigate the molecular mechanism of its anti-cancer action (Figure 4, Figure S5, Table S1 and S2). Changes in protein expression levels were analyzed after 4 and 24 hours of 20 µM ACY-1215 treatment ( Figure 4A). A total of 4,423 proteins were detected across all samples by mass spectrometry. At 4 hpt, 42 proteins were differentially . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint S5A and S5B). Using Cluego pathway analysis, the terms dendrite development and regulation of G protein-coupled receptor signaling pathways were identified as downregulated ( Figure S5C). A distinct pathway was not detected within the upregulated proteins. At 24 hpt, 150 proteins and 202 proteins were significantly down-and up-regulated, respectively ( Figure 4B). GO pathway enrichment analysis (fold change of > 1.2) for biological processes identified multiple pathways downregulated by ACY-1215, with pigment granule organization (11.24% of proteins) and pigment cell differentiation (7.87% of proteins) being prominently altered ( Figure 4C, Figure S6A and Table S1). Through enriched pathway analysis, biological processes such as regulation of microtubule polymerization or depolymerization (7.25% of proteins), DNA duplex unwinding (3.11% of proteins), regulation of chromatin silencing (3.11% of proteins), regulation of extrinsic apoptotic signaling pathway in absence of ligand (2.07 % of proteins), cellular senescence (1.55% of proteins), exit from mitosis (1.55% of proteins) and ERBB2 signaling pathway  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 5B). Furthermore, phospho-ERK and ERK expression levels were . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint analyzed in order to determine whether the MAPK/ERK signaling pathway played a role in the ACY-1215 mechanism of action. Through immunoblotting, a significant difference in p-ERK expression levels was not observed after 4 hours of 20 µM ACY-1215 treatment compared to vehicle control ( Figure 6B, 6B' and Figure S7C). Following 24 hpt with 20 µM ACY-1215, p-ERK expression levels were significantly downregulated (p = <0.0001) compared to vehicle control ( Figure 6B and Figure S7C). Overall, through proteomic analysis, MITF and p-ERK were identified as key players involved in the ACY-1215 mechanism of action in OMM2.5 cells.

ACY-1215 treatment arrests cell cycle progression in S phase
Outside of UM, previous studies have independently demonstrated that ACY-1215 and MITF regulate the cell cycle [38][39][40][41][42]. To determine whether ACY-1215 treatment altered cell cycle phases in MUM cells, to the DNA ploidy of UM cells [43,44]. Approximately 60% -70% of the cell population were diploid, in cell

MITF inhibitor treatment prevents MUM cell survival and proliferation in vitro
To further interrogate the requirement of MITF in MUM cell survival, the ability of OMM2.5 cells treated with the MITF pathway inhibitor ML329, to survive and proliferate was analyzed using colony formation assays. Cells were treated with increasing doses of ML329, ranging between 0.05 µM and 50 µM, given the reported IC50 value of 1.2 µM ( Figure 9A and 9B) [45]. The treatment regime was performed . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint aspreviously described , whereby OMM2.5 cells were treated with respective drug doses for 96 hours, and then maintained in culture, in fresh complete media for an additional 10 days ( Figure 9A).

Inhibition of MITF pathway hinder MUM cells survival in vivo in zebrafish OMM2.5 xenograft models
The efficacy of the MITF pathway inhibitor, ML329, on survival of MUM cells in vivo was determined using zebrafish xenograft models. ML329 was well tolerated by zebrafish in vivo, albeit with drug precipitation at higher concentrations (1 -100 µM) ( Figure S10). Although we observed effects in vitro at concentrations as low as at 0.25 µM ML329, we chose the concentration of 1.25 µM for our study to fit with the reported IC50 value [45]. As before, OMM2.5 Dil labelled cells were injected into the perivitelline space after which the larvae (2 dpf) were treated with either 0.5% DMSO or 1.25 µM ML329 for 3 days ( Figure 10A). There was no significant difference in the average number of disseminated cells to the caudal vein plexus of the OMM2.5 xenografted larvae at 0.5% DMSO (3.1 cells) or 1.25 µM ML329 (2.6 cells) treatment groups ( Figure 10B and 10D). However, on average, a 51% (p = 0.0006) reduction in OMM2.5 primary xenograft fluorescence was detected after normalization, following treatment with 1.25 µM ML329 compared to . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint vehicle controls ( Figure 10A and 10C). Experimentally, therefore we observe a beneficial effect of blocking the MITF pathway in MUM cell line in vitro and in vivo.

Figure 10
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Discussion
Metastatic UM (MUM) is a poor prognosis cancer lacking effective treatment options. Our study has provided evidence that inhibition of HDAC6 or MITF is efficacious in conferring anti-cancer effects in a MUM cell line, both in vitro and in vivo. To the best of our knowledge, this is the first study to provide evidence regarding the potential link between HDAC6 and MITF in MUM.
Three commercially available, first generation HDAC6i were screened in UM and MUM cell lines and ACY-1215 was selected for follow-up studies. ACY-1215 either as a monotherapy or in combination with other drugs, is presently in clinical trials for several cancers [27,46]. We observed strong anti-cancer effects elicited by ACY-1215 treatment in a dose-dependent manner in both UM and MUM derived cell lines, albeit weak HDAC6 expression is reported in UM tissues [47]. Notably, HDAC6 activity is significantly increased in inflammatory breast cancer even though HDAC6 is not overexpressed [48]. Hence, it is plausible that in MUM, there is increased HDAC6 activity but not HDAC6 expression. Our data indirectly supports the findings by Nencetti et al., whereby a novel synthetized quinoline derivative VS13, with high selectivity against HDAC6; led to a reduction in UM cell viability in vitro [26]. In addition, here, the anticancer effect of ACY-1215 was demonstrated in vivo in zebrafish OMM2.5 xenograft models, without any significant change to the number of disseminated cells. This is not surprising given the timeframe of the experiment, and a low burden in the average number of disseminated cells to the caudal vein plexus three days post transplantation in the vehicle controls. It would be worthwhile to perform follow-up studies to evaluate the efficacy of ACY-1215 on tumor metastasis, with long-term treatment regimens and in patientderived samples in vivo in larvae and/or in adult zebrafish [49][50][51][52].
However, pure HDAC6 inhibition mediated effects must be inferred with caution, as higher doses of ACY-1215, result in non-selective inhibition, and the observed beneficial effects are mediated by additional . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; targets [37,53]. In a study by Lin et al., CRISPR-induced HDAC6 knock-out lines (e.g. melanoma, triple negative breast cancer, colorectal cell lines) demonstrated that the cell viability/proliferation capability was comparable to wildtype controls; additionally ACY-1215 was able to mediate its anti-cancer effects at high concentrations (micromolar) even when HDAC6 was knocked-out [53]. Corroborating their findings, Depetter et al., revealed that treatment with 10 μM ACY-1215 in HAP1 cells with HDAC6 knock-out, led to a reduction in cell viability [37]. In another study, a distinct anti-proliferative effect was observed in highgrade serous ovarian cancer cells when a non-selective concentration of 10 μM ACY-1215 was used [54].
In both studies, the authors suggest that the true beneficial effects of HDAC6 inhibition might be reaped in combinatorial therapy rather than when administered as a single agent. Therefore, it has to be acknowledged that at our selected treatment concentration of 20 μM, we are likely to be non-selectively targeting other factors such as Class I HDAC isozymes, given the reported IC50 value for ACY-1215 is 4.7 nM. Importantly, HDAC6 was indeed inhibited by ACY-1215 at the concentration we used, as its substrate acetylated α-tubulin was significantly upregulated. Furthermore, from our proteomics data we also identified proteins involved in microtubule polymerization and regulation of microtubule polymerization or depolymerization to be significantly altered [55]. Irrespective of non-selective inhibition of HDAC isozymes, ACY-1215 still presents as a promising therapeutic for treatment of MUM, with its ability to prevent UM cell growth, that warrants further interrogation.
Proteome profiling of ACY-1215 treated OMM2.5 cells was key to deducing potential mechanisms of action. We discovered that the MITF signaling pathway and associated factors were significantly downregulated upon treatment with ACY-1215. Tying in with the concentration of ACY-1215 used, our findings are in line with another study, whereby it was reported that treatment of melanoma and clear cell sarcoma cells with different pan-HDAC inhibitors resulted in reduced MITF expression in vitro and in vivo in a mouse melanoma xenograft model [56].
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The role of MITF has been extensively studied in cutaneous melanoma [57][58][59]. MITF is a key transcription factor and a master regulator of melanogenesis and melanocyte differentiation. It also plays a multifaceted role regulating several cellular processes including cell cycle, DNA-damage repair, lysosome biogenesis, metabolism, autophagy, and oxidative stress [60][61][62][63]. MITF can be further distinguished into five different isoforms, MITF-A, MITF-B, MITF-C, MITF-H and MITF-M [64]. Particularly in cutaneous melanoma, MITF-M is involved in carcinogenesis events such as survival, proliferation, differentiation, invasion and migration [59]. Not surprising, certain types of mutations in MITF and MITF-associated members are linked to oncogenic functions in melanoma [60,65,66]. MITF plays a dual role in cutaneous melanoma, based on its expression levels and activity, however, there is controversy surrounding this matter [61]. For instance, some studies report that low MITF expression is necessary for proliferation and higher levels of MITF correlates to suppression of cell proliferation and promotes differentiation [59].
While others state that low levels of MITF expression is linked to invasiveness while high levels of MITF expression is required for cell proliferation/differentiation [40,58,67]. Nevertheless, targeting the MITF pathway shows promise as an anti-cancer approach. Aida et al., demonstrated that the growth of melanoma cells, SK-MEL-5 and SK-MEL-30 were inhibited by siRNA mediated knock-down of MITF [68].
Similarly, in another study, knock-down of MITF by shRNA, in MM649 cells resulted in reduced cell proliferation in vitro and tumor growth and dissemination in vivo in mouse xenografts [57]. Furthermore, pharmacological inhibition of the MITF signaling pathway using small molecule ML329 reduced cell viability in MITF-dependent melanoma (SK-MEL-5 and MALME-3M) cells without affecting the viability of A375 cells, a MITF-independent cell line [45]. Comparably, another compound, CH5552074, inhibited the growth of SK-MEL-5 cells via the suppression of MITF protein [68]. Interestingly, knock-down of MITF in B16F10 melanoma cells and overexpression of MITF in YUMM1.1 cells led to increased tumor growth in vivo in mice [69]. Apart from melanoma, studies have connected MITF with a role in multiple cancers including non-small cell lung cancer, pancreatic cancer, and hepatocellular carcinoma [70][71][72]. Most . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

cell proliferation and an increase in cells in S/G2 phases, suppressed cell migration and invasion
in vitro and tumor formation in vivo; an opposing effect was observed when MITF was overexpressed [41].
In the context of UM, MITF is upregulated in UM cells [73]. In our study, expression levels of MITF and several proteins involved in pathways associated with MITF, such as pigment cell differentiation and melanosome organization, were downregulated upon ACY-1215 treatment. This was consistent with the observed trend when MITF is downregulated. Taken together, there is ample evidence to suggest that targeting the MITF signaling pathway may be a novel therapeutic option for MUM.
Moreover, several studies have independently shown that ACY-1215 regulates cell cycle and cell death mechanisms in various cancers. In HCT-116 and HT29 colorectal cancer cells, a reduction in cell proliferation and viability was noted in a time-and dose-dependent manner; and apoptosis was observed as well at non-selective ACY-1215 concentrations [39,74]. Interestingly, ACY-1215 when used at HDAC6 selective concentrations (up to 2 μM) did not promote apoptosis, however, if used in combination with other anti-cancer drugs it proved to be more effective [74,75]. In esophageal squamous cell carcinoma cell lines (EC109 and TE-1), ACY-1215 treatment resulted in suppression of cell proliferation through the arrest of cell cycle in G2/M phase and an increase in apoptosis [76]. Similarly, 4 μM ACY-1215 treatment for 24 hours, prompted an increase in percentage of cells in G0/G1 phase; and a time/dose-dependent proapoptotic effects of ACY-1215 uncovered in lymphoma cell lines [38]. More recently, in gall bladder cancer cells, ACY-1215 inhibited cell proliferation and induced apoptosis as well as enhancing the chemotherapeutic effects of other anti-cancer agents upon co-treatment [77]. Collectively, in these studies it became evident that the PI3K/AKT and MAPK/ERK pathways played a central role in ACY-1215 mechanism of action. We postulated whether ACY-1215 treatment promoted cell cycle arrest and apoptosis in MUM cells. As expected, at the non-selective concentration, ACY-1215 treatment resulted in the halting of cell cycle progression in S phase and induced apoptosis. We observed a significant increase . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint in early apoptotic cells and a significant reduction in the number of viable cells at 20 and 50 μM ACY-1215 treatment by 24 hours. Additionally, expression of cleaved PARP, which is used as an indicator for apoptosis, was markedly upregulated in ACY-1215 treated MUM cells at 24 hours post treatment [78,79].
Further supporting evidence can be drawn from our proteomics data, whereby the pathways -regulation of extrinsic apoptotic signaling pathway in absence of ligand, exit from mitosis and cellular senescence were upregulated indicating an increase in expression levels of proteins associated with these biological is not surprising that the MAPK/ERK signaling pathway is involved in ACY-1215 mechanism of action. It was previously reported that ERK1/2 and HDAC6 are interacting partners involved in a positive feedforward loop [80][81][82]. In colon cancer cell lines, knock-down of HDAC6 resulted in reduced p-ERK expression but not total ERK expression levels [83]. Peng et al., showed that in A375 melanoma cells, inhibition with ACY-1215 alone and in combination with vemurafenib led to the disruption and inactivation of ERK [84]. Interestingly in prostate cancer cells (LNCaP), blocking of HDAC6 with . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Panobinostat led to increased ERK activity and as a consequence promoted apoptosis [85]. But this was not the case in PC-3 prostate cancer cells. While in another study, increased HDAC6 expression in lung cancer cell by Isoproterenol treatment led to the inhibition of the ERK signaling cascade [86]. Taken together, this indicates that there might be cell-specific context for HDAC6-ERK1 regulation and activity.
In UM, GNAQ/GNA11 mutations are associated with constitutive activation of the MAPK/ERK signaling pathway, although heterogeneity in MAPK/ERK signaling has been observed across UM samples with GNAQ/GNA11 mutations [87][88][89][90]. More specifically, the OMM2.5 cells used in this study carry a mutation in GNAQ, which is known to result in constitutively active MAPK/ERK signaling in UM [8,91]. Therefore, we postulate that inhibition of HDAC6 leads to reduced ERK activity that subsequently results in reduced p-ERK expression levels. In turn, reduced p-ERK expression levels decrease MITF expression and . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint consequently, associated biological processes downstream such as cell survival mechanisms are inhibited ( Figure 11). Collectively, our findings warrant an in-depth analysis into understanding the role of MITF in MUM and to consider targeting MITF and/or candidates in the MITF signaling pathway as additional potential therapeutic option(s).
Though promising, the role of HDAC6 and MITF still needs to be thoroughly investigated in UM and MUM patient samples. Currently, there is no clear evidence linking either HDAC6 or MITF in MUM prognosis.
Immunohistochemistry based expression analysis of 16 primary UM samples detected variable low levels of HDAC6 expression, with a clear correlation between HDAC6 expression levels and UM, unable to be drawn due to the limited sample size [47]. Based on TCGA data analysis comprising 80 UM patient samples, a significant correlation was found between HDAC6 expression and OS probability, highlighting a possible involvement of HDAC6 in UM prognosis. Moreover, HDAC2 and SIRT2 expression correlated to OS while HDAC4 expression showed correlations to PFS. HDAC 1 and 3 expression was not correlated to either OS or PFS. Although, HDAC 1, 2, 3, 4 and Sirtuin 2 (SIRT2) expression was detected in UM eye samples [47]. A limited number of studies have explored the expression of MITF in UM and MUM. MITF expression was found in 100% (15 out of 15) of 15) of UM samples in one study, however, in another study, MITF expression was detected in 65% (37 out of 57 samples) of choroidal UM patient samples, with levels of MITF expression not significantly associated with the survival of these patients [92,93]. Comparably, from our TCGA data analysis, there was no correlation between MITF expression levels and OS/PFS seen in UM patients. It has been previously suggested that MITF would be a useful marker for ocular malignant melanoma [94]. Taken together, it will be worthwhile to perform an extensive study with a larger cohort of UM and MUM patient samples to conclusively determine whether HDAC6 and MITF plays a part in MUM prognosis. Additionally, it needs to be determined whether targeting these pathways offer a broad treatment option for MUM irrespective of MUM causative mutation(s).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021. 10.28.466226 doi: bioRxiv preprint This study is pivotal in highlighting that MITF plays a critical role in the survival of OMM2.5 cells and provides evidence that the observed ACY-1215 mechanism of action in OMM2.5 cells is most likely through the regulation of MITF. Our data suggests that HDAC6 and/or pan-HDACs and the MITF signaling pathway offer novel options to identify therapeutic targets for treatment of MUM that needs to be considered and further evaluated. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021. 10.28.466226 doi: bioRxiv preprint Media were added to the plates and cells allowed to grow for an additional 10 days at 37 0 C with 5% CO2.
Clones were washed twice and fixed with 4% paraformaldehyde/formaldehyde for 10 mins at room temperature (RT). Clones were stained with 0.5% crystal violet solution (Pro-Lab diagnostics PL700; Richmond Hill, ON, Canada) for 10 mins -2 hours at RT, shaker, washed and dried (once desired staining is achieved). Plates were imaged using the GelCount™ system (Oxford Optronix; Oxford, UK) and analyzed using the ColonyCountJ Plugin in ImageJ v1.53e [95]. Statistical analysis was performed using One-way ANOVA with Dunnett's Test for Multiple Comparisons in GraphPad Prism v7.00 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). A p value of < 0.05 was considered as statistically significant. Experiments were performed in triplicates/quadruplicates.

OMM2.5 zebrafish xenografts
All animal work and husbandry were performed in accordance with ethical approval granted by Linköping Animal Research Ethics Committee. Zebrafish embryos/larvae from Tg(fli1a:EGFP) background were raised in embryo media containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM MgCl2, 0.33 mM CaCl2 and 0.003% phenylthiourea (PTU), in a petri dish at 28.5 0 C incubator. Adult Tg(fli1a:EGFP) zebrafish were maintained in a 14 hour light/10 hour dark cycle in a recirculating water system at 28 0 C. OMM2.5 cells were prepared for transplantation as described in previously published report [96] between 0 dpt and 3 dpt was measured, normalized and calculated using ImageJ. Before drug treatment, toxicity assays were performed with either 0.5% DMSO, ACY-1215, Dacarbazine or ML329 (ranges from 1 -100 µM). Eight larvae (4 larvae/well) per treatment group was exposed to the desired concentration of drug solutions for 3 days in 24/48-well plates at 35 0 C and imaged at 3 dpt. One-way ANOVA with Dunnett's Test for Multiple Comparisons or Student's T test statistical analysis was performed using GraphPad Prism.

Proteome profiling and mass spectrometry analysis
OMM2.5 cells were seeded at a density of 1 x 10 6 cells per well and drug treated for 4 or 24 hours with 0.5% DMSO or 20 µM ACY-1215, in duplicate (N = 4). Protein was isolated using PreOmics iST For protein/proteomics preparation kit (PreOmics GmbH; Martinsried, Germany) according to manufacturer's protocol. Mass Spectrometry and bioinformatic analysis of samples were performed as described previously [97]. Slight variations to methodology consist of raw data processing performed with MaxQuant v1.6.10.43, with MS/MS spectra and database search performed against Uniprot Homo sapiens database (2020_05) containing 75,074 entries [97]. Pathway analysis of enriched proteins (a fold change of (+/-) > 1.2 and a p value of < 0.05) was performed using ClueGo (v2.5.8) [98] and Cluepedia (v1.5.8) [99] plugins in Cytoscape (v3.8.2) [100] with the Homo sapiens (9606) marker set. GO: Biological Process functional pathway databases, consisting of 18058 genes, were used. GO tree levels (min = 3; max = 8) and GO term . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ; https://doi.org/10.1101/2021.10.28.466226 doi: bioRxiv preprint restriction (min#genes = 3, min% = 4%) were set and terms were grouped using a Kappa Score Threshold of 0.4. The classification was performed by two-sided hypergeometric enrichment test, and its probability value was corrected by the Benjamini-Hochberg method (Adjusted % Term p-value < 0.05).

Western blot analysis
Protein was isolated from Mel270, Mel285, OMM2.5 and ARPE19 cells at a cell density of 1 x 10 6 or 4 x 10 5 and immunoblotting was performed as described [36]. For validation of proteomics data, protein isolated for MS study was utilized. Protein concentrations were measured using BCA protein assay kit  Table S3. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Conclusion
This research provides evidence that HDAC6 inhibitors and MITF inhibitors should be considered and further investigated as a potential treatment option for MUM. Specifically, this study proves the efficacy of ACY-1215 as an anti-cancer agent for MUM in vitro and in vivo. We have additionally elucidated that ACY-1215 regulates MITF expression via p-ERK signaling when used at high concentrations.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ;  Enriched protein pathway analysis for GO term: biological processes, for down-and up-regulated proteins given a fold change cut off of +/-> 1.2, p < 0.05 displayed as pie charts. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 29, 2021. ;  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made