Inactivation of the Hippo tumor suppressor pathway promotes melanoma

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF-expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.


Introduction
Cutaneous melanoma arises from the malignant transformation of melanocytes, which are neural crest-derived cells mainly localized to the basal layer of the epidermis. When locally resected, melanoma is highly curable; however, melanoma is the most aggressive of all skin cancers and distant-stage disease is associated with significant lethality (1). Unraveling the molecular features underlying the pathogenesis of cutaneous melanoma is essential for the development of preventative and therapeutic treatment strategies.
The vast majority of melanocytic neoplasms are initiated by oncogenic mutations in the mitogen-activated protein kinase (MAPK) pathway, with activating mutations in BRAF and NRAS occurring in ~50% and ~20% of cutaneous melanomas, respectively (2). Within BRAF-mutant melanomas, the most common activating mutation results from a single amino acid substitution from a valine to a glutamic acid generating the constitutively active mutant BRAF V600E (3,4). Despite strongly inducing proliferative signaling, melanocyte-specific expression of BRAF V600E is insufficient to induce melanoma in multiple animal models; instead, BRAF V600E expression leads to the development of benign nevi (moles) comprised of growth-arrested melanocytes (5)(6)(7)(8)(9)(10). This is corroborated by clinical evidence as melanocytes within benign human nevi also frequently contain BRAF V600E mutations (11,12) and these melanocytic nevi rarely transform into melanoma (annual rate < 0.0005%) (13). Similarly, mutations within NRAS are commonly detected in congenital nevi and oncogenic NRAS expression in melanocytes in vivo does not rapidly yield melanoma (14)(15)(16). Although the risk of any single melanocytic nevus transforming into melanoma is minimal, understanding how such transformations occur is paramount as roughly one third of all melanomas co-exist with or arise from nevi (17).
These observations indicate that tumor suppression mechanisms restrain melanoma development following the acquisition of activating MAPK pathway mutations in melanocytes. A longstanding view is that strong oncogenic signals driven by mutations in MAPK pathway components leads to oncogene-induced cellular senescence (OIS), which safeguards against tumorigenesis (18)(19)(20). Supporting this view, it has been demonstrated that expression of BRAF V600E in primary melanocytes in vitro induces an immediate cell cycle arrest and that these arrested melanocytes exhibit all of the hallmarks of oncogene-induced senescence: they become large, flat, vacuolar, express p16 INK4A , display senescence-associated β-galactosidase (SA-β-gal) activity, and have increased heterochromatic foci and DNA damage (18,21).
However, while it is clear that oncogene-induced senescence occurs in vitro, the extent to which this mechanism operates to ward off tumorigenesis in vivo remains unclear (22)(23)(24). Several pieces of evidence argue against OIS as being the predominant mechanism restraining the proliferation of melanocytes harboring oncogenic mutations in vivo (extensively reviewed in (16)). Most notably, oncogene expression (e.g. BRAF V600E ) in melanocytes does not induce an immediate proliferative block in vivo. Rather, these oncogenes initially induce proliferation, as evidenced by the clonal outgrowth of melanocytes that ultimately form a nevus, which requires many rounds of cell division. Furthermore, melanocytes lacking proteins known to enforce senescence, such as p16 and p53, retain the capacity to enter a growth-arrested state, as melanocytes in Braf V600E /Cdkn2a -/and Braf V600E /Trp53 -/mouse models still primarily form nevi, with only a rare few melanocytes stochastically transforming into melanoma (7).
Collectively, these data suggest that additional tumor suppressive mechanisms have the capacity to restrain the proliferation of Braf V600E -positive mouse melanocytes, independent of inducing senescence. Recent modeling studies have led to the postulation that the growth arrest of nevus melanocytes is not solely due to oncogene activation and OIS in individual cells, but rather due to cells sensing and responding to their collective overgrowth, similar to what occurs in normal tissues (25). This cell growth arrest is very reminiscent of the arrest induced by activation of the Hippo tumor suppressor pathway, which is an evolutionarily conserved pathway known to regulate organ size. When the Hippo pathway is activated, the Hippo kinases LATS1/2 phosphorylate the transcriptional co-activators YAP (YAP1) and TAZ (WWTR1), resulting in their inactivation by nuclear exclusion and subsequent degradation (26,27). In contrast, when the Hippo pathway is inactivated, YAP and TAZ are active and form DNA-binding complexes with the TEAD family of transcription factors, which act synergistically with AP-1 complexes to stimulate the expression of genes mediating entry into S-phase and cell proliferation (28,29).
To examine if BRAF V600E activates the Hippo tumor suppressor pathway in vitro, we induced BRAF V600E expression and examined the relative levels of active LATS1/2 that is phosphorylated at the hydrophobic motif (T1079) (27). We found a significant increase in LATS phosphorylation following expression of oncogenic BRAF V600E ( Figure 1B). We then assessed total YAP phosphorylation (p-YAP) via phos-tag gel electrophoresis. We observed that BRAF V600E induction promoted phosphorylation of YAP at multiple sites ( Figure 1C). Consequently, expression of BRAF V600E led to nuclear exclusion of YAP and a corresponding decrease in the expression of the YAP target genes CYR61 and AMOTL2 ( Figures 1D and E). The observed effects on LATS and YAP activity were due to BRAF V600E , as overexpression of wild-type BRAF had no effect on LATS or YAP phosphorylation ( Figures S1A-D). We further confirmed these results in multiple cell lines, including nonimmortalized primary adult human melanocytes with an intact p53 pathway ( Figures 1F and S1E). Importantly, the observed effects of Hippo pathway activation were not limited to expression of BRAF V600E alone, as we also found that inducible expression of oncogenic NRAS Q61R similarly activates the Hippo pathway ( Figure S1F). Collectively, these data demonstrate that hyperstimulation of the MAPK signaling pathway through expression of oncogenic BRAF V600E or NRAS Q61R leads to activation of the Hippo tumor suppressor pathway in vitro.
We noted that expression of BRAF V600E reduced melanocyte cell number ~30-40% relative to uninduced controls over a 4-day period, despite the fact that these melanocytes were SV-40 immortalized ( Figure 1G). Live-cell imaging and population doubling assays revealed this was predominantly due to a proliferative arrest, rather than increased cell death ( Figures 1G, 2A-B). To test whether the observed Hippo pathway activation induced by BRAF V600E expression was responsible for this proliferative defect we used RNAi to knockdown the LATS1/2 kinases in the context of BRAF V600E expression. We found that loss of LATS1/2 prevented YAP phosphorylation following induction of BRAF V600E and fully rescued cell growth and viability ( Figure 1H-I). We then validated this result using soft agar growth assays. While parental Mel-ST cells and Mel-ST cells expressing BRAF V600E fail to efficiently grow under anchorage independent conditions, Mel-ST cells expressing BRAF V600E together with a constitutively active YAP mutant (YAP-5SA) demonstrated significant colony formation ( Figure 1J and data not shown). These data reveal that functional inactivation of the Hippo pathway, through either LATS1/2 depletion or constitutive YAP activation, is sufficient to restore proliferation to BRAF V600E -expressing immortalized melanocytes in vitro.   . ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 To test this possibility, we performed live-cell imaging of doxycycline-inducible BRAF V600E Mel-ST cells stably expressing the chromosome marker histone 2B-GFP (H2B-GFP). We observed that upon entering mitosis, cells expressing BRAF V600E often exhibited widely-oscillating chromosomes and were unable to maintain a tightlyaligned metaphase plate relative to uninduced controls ( Figure 2A). These chromosome alignment defects impaired the ability of many cells to satisfy the spindle assembly checkpoint, and consequently a portion of the BRAF V600E -expressing cells endured a significantly prolonged mitosis (Figure 2A-B). On average, cells expressing BRAF V600E experienced a mitosis that was double the length of uninduced control cells (control: ~74 min, induced: ~154 min).
Cells that cannot satisfy the spindle assembly checkpoint either undergo mitotic cell death, or exit from mitosis without undergoing cell division, a phenomenon termed mitotic slippage (38). Cells that undergo mitotic slippage often generate multinucleated tetraploid cells, and multinucleated melanocytes have been observed in human nevi (37,38). We observed that upon induction of BRAF V600E , the number of mitoses resulting in mitotic slippage increased significantly (control: ~1%, induced: ~20%) ( Figure 2B). We also noted cytokinesis failure following anaphase onset was increased following BRAF V600E expression (control: ~1%, induced: ~5%). Overall, melanocytes expressing BRAF V600E experienced a significantly increased occurrence of whole-genome doubling events (control: 2.47%, induced: 24.75%). We further confirmed the generality of these findings in another independent cell line ( Figures S2A-B). These data demonstrate that BRAF V600E , the most prevalent MAPK mutation in benign nevi, can impair mitosis leading to mitotic slippage and the formation of multinucleated tetraploid melanocytes. Indeed, this may be one mechanism by which multinucleated melanocytes form in human nevi. In support of these conclusions, it has recently been discovered that melanocyte-specific expression of BRAF V600E or NRAS Q61K in zebrafish leads to the formation of fish nevi largely comprised of binucleated tetraploid cells, suggesting oncogenic MAPK signaling can promote mitotic errors and tetraploidization in vivo as well (Darp et al, unpublished).
We examined whether BRAF V600E -induced tetraploidization was driving YAP phosphorylation, however our data suggested that mitotic errors leading to tetraploidization were not the major underlying cause of Hippo pathway activation in BRAF V600E -expressing melanocytes. First, BRAF V600E -expressing Mel-ST cells arrested in G1 (via thymidine) or G2 (via RO-3306 mediated CKD1 inhibition) still experienced Hippo activation despite their inability to become tetraploid ( Figure 2C). Second, our immunofluorescence experiments revealed mononucleated diploid cells also exhibit decreased nuclear YAP/TAZ, demonstrating tetraploidization was not necessary to observe Hippo pathway activation ( Figure 1D).

BRAF V600E -induced Hippo activation is ERK-dependent and partially mediated by changes in the actin cytoskeleton
We examined whether BRAF V600E specifically, or rather hyperactivation of the MAPK pathway generally, is responsible for Hippo pathway activation. We found that dampening of MAPK signaling via inhibition of the downstream kinases MEK1/2 or ERK1/2 fully prevented Hippo pathway activation, as measured by YAP/TAZ phosphorylation status, in BRAF V600E -expressing Mel-ST cells ( Figure 3A). These data demonstrated that Hippo pathway activation is entirely mediated by hyperactivation of MAPK signaling and requires factors downstream of ERK. We further confirmed this in another independent cell line ( Figure S3A). This result discounts the possibility that oncogenic BRAF V600E activates Hippo signaling via direct phosphorylation of key Hippo pathway components.
We noted that phosphorylation of YAP following BRAF V600E expression requires sustained MAPK stimulation over a period of 12-16 h, as transient MAPK activation only minimally affected YAP phosphorylation ( Figure 3B, S3B-C). Based on this result, we speculated that mounting oncogene-induced replication stress may be promoting Hippo pathway activation. However induction of replication stress by hydroxyurea treatment alone was not sufficient to activate the Hippo pathway ( Figure 3C).
Alternatively, it has been demonstrated that oncogenic activation of the MAPK pathway dramatically alters actomyosin cytoskeletal contractility and reduces RhoA activity in an ERK1/2-dependent manner (39)(40)(41). Reductions in active RhoA are known to promote Hippo pathway activation and, furthermore, ERK1/2-dependent cytoskeletal changes have previously been shown to modulate YAP/TAZ activity in melanoma cell lines (42). We   ! therefore posited that reduction of RhoA activity may represent one mechanism by which BRAF V600E -expressing cells activate the Hippo pathway in vitro. Indeed, we observed that there was a significant reduction in the number of actin stress fibers following activation of BRAF V600E , indicating reduced RhoA activity ( Figure 3D, S3D-E). We also found that inhibition of Rac1, which is known to antagonize RhoA, mitigated BRAF-induced Hippo pathway activation ( Figure 3E and S3F). These data suggest that BRAF V600E -induced Hippo pathway activation is at least partially mediated by prolonged MAPK hyperstimulation leading to ERK1/2-dependent cytoskeletal dysregulation. Supporting this view, endogenous Braf V600E expression in mouse embryonic fibroblasts has been shown to drastically reduce actin stress fibers, and a recent study has also demonstrated expression of BRAF V600E in RPE-1 cells leads to decreased RhoA activity (Darp et al. unpublished) (43).

Growth-arrested melanocytes in benign nevi show evidence of Hippo pathway activation
It is well established that melanocyte-specific expression of Braf V600E in animal models gives rise to benign nevi that harbor non-proliferating melanocytes. We hypothesized that these Braf V600E -positive melanocytes may also demonstrate evidence of Hippo pathway activation, similar to our observed results in vitro. To test this possibility, we analyzed a single-cell RNA sequencing dataset of whole-skin extracts collected at P30 and P50 from wildtype control mice and tamoxifen-painted Tyr::CreER T2 /Braf CA mice expressing active Braf V600E (25). We hypothesized any difference in Hippo activity may be only detectable at the transcriptomic level as we suspected Hippo activation in vivo to be subtle. We interrogated this dataset to examine if YAP/TAZ-dependent gene transcription is repressed in melanocytes expressing oncogenic Braf V600E . Following dimensionality reduction and initial clustering, melanocytes were identified utilizing the melanocyte-lineage marker Dct as previously reported ( Figure 4A) (25). This cluster was also found to have high expression of another melanocyte-lineage marker, Mlana ( Figure S4A). We then employed variance-adjusted Mahalanobis (VAM), a scRNA-seq optimized approach to obtain accurate signaling pathway scores, to examine if YAP/TAZ-mediated gene expression was decreased in Braf V600E -expressing mouse melanocytes relative to wild-type melanocytes utilizing previously published YAP/TAZ gene expression profiles (44)(45)(46). The most basic analysis, where all single-cells were binned by genotype, demonstrated that YAP/TAZ gene expression targets were significantly reduced in Braf V600Epositive melanocytes compared to wild-type ( Figure 4B).
We then performed unsupervised clustering, which generated five melanocyte subdivisions, to clarify which unique populations of oncogenic Braf V600E -expressing melanocytes exhibited the least YAP/TAZ activity ( Figure  4C). We theorized that clusters containing nevus melanocytes would be exclusively populated by cells isolated from Braf V600E mice, and be the primary melanocytic subtype isolated from 50 day old mice. We identified clusters 0 and 1 as Braf V600E -expressing melanocytes isolated from nevi ( Figure S4B). In support of this prediction, expression of Cdkn2a was found to be the highest in clusters 0 and 1, although Cdkn2a read-depth was limited throughout all clusters ( Figure S4C). Compared to all other melanocytes, irrespective of genotype, nevus melanocytes (clusters 0 and 1) exhibited the lowest YAP/TAZ activity scores of any cluster, demonstrating that YAP/TAZ-mediated gene expression is restrained following oncogenic Braf expression in mouse nevus melanocytes ( Figure 4D-F). Importantly, expression of Hippo pathway components remained unchanged regardless of genotype or cluster, suggesting decreased YAP/TAZ signaling was due to Hippo pathway activation, not increased expression of YAP/TAZ regulators ( Figure S4D-F).
Interestingly, not all melanocytes captured from Braf V600E mice exhibited low YAP/TAZ activity scores. Clusters 2 and 4, which contain an appreciable portion of melanocytes from both Braf +/+ and Braf V600E mice, demonstrated much higher YAP/TAZ activity relative to nevus melanocytes ( Figure S4G). However, within these clusters, YAP/TAZ activity was still decreased in Braf V600E melanocytes compared to wild-type cells. This suggests cell intrinsic mechanisms (e.g. RhoA inactivation) following Braf V600E expression are only partially leading to decreased YAP/TAZ activity and that other mechanisms, possibly cell extrinsic cues, may play additional roles in vivo. We suspect cluster 3, the only cluster that did not exhibit this trend, may be mainly comprised of proliferating, follicular melanocytes as cluster 3 mainly contains melanocytes isolated at P30 when most murine hair follicles are in anagen (25). Taken together, these data reveal Braf V600E -expressing mouse melanocytes largely exhibit decreased YAP/TAZ activity, with the most significant decreases found specifically within nevus melanocytes, strongly implying the Hippo pathway becomes activated in response to Braf V600E expression and nevus formation in vivo. In support of these conclusions, immunofluorescence staining of three human benign nevi revealed YAP localization to be predominantly cytoplasmic and thus presumably inactivated in human nevus melanocytes ( Figure 4G and S4H).

Lats1/2 -/-Deletion Promotes Melanomagenesis
Our data suggested that functional inactivation of the Hippo tumor suppressor pathway may enable Braf V600Eexpressing melanocytes to evade growth arrest and facilitate melanoma development. To test this, we generated mice carrying floxed alleles of both Lats1 and Lats2 (47) with Tyr::CreER T2 to allow for inducible, melanocytespecific inactivation of the Hippo pathway (Tyr::CreER T2 /Lats1 f/f /Lats2 f/f ). Deletion of LATS1/2 is well established to completely abrogate the Hippo pathway, and co-heterozygous loss of LATS1/2 is observed in ~15% of human melanomas, making deletion of Lats1/2 clinically relevant ( Figure 5A, S5A-B) (2,48). We also crossed Tyr:: Figure 5B). We confirmed the melanocytic specificity of our Tyr::CreER T2 expressing mice via incorporation of a fluorescent lineage trace (YFP LSL ), whose expression was only observed in cells that co-stained for melanocyte markers ( Figure S5C).
We observed that Braf V600E /Lats1/2 -/mice were highly prone to developing spontaneous dermal tumors within weeks after birth, even without topical 4-hydroxytamoxifen (4-HT) administration. A similar melanoma mouse model, Tyr:: , is prone to spontaneous melanoma formation in the absence of topical 4-HT, due to leakiness of the inducible Cre recombinase (49,50). This suggested that deletion of Lats1/2 was also playing a major role in promoting melanoma development, as Braf V600E expression alone in murine melanocytes does not generate tumors (5). In the few mice where spontaneous tumorigenesis was absent or delayed, 4-HT administration to Braf V600E /Lats1/2 -/flanks resulted in the potent formation of tumors which appeared similar to the spontaneously arising neoplasms ( Figure 5C). These tumors exhibited strong nuclear YAP/TAZ staining, indicating Hippo inactivation, and positively stained for the melanocytic markers SOX10 and S100 ( Figure 5D). SOX10 staining was nuclear and homogenous whereas S100 staining was weakly heterogeneous. Subsequent histopathologic analysis by a board certified dermatopathologist confirmed these infiltrative, spindle cell tumors to be mouse melanoma. Unlike other Braf V600E -driven mouse melanoma models (e.g. Braf V600E /Cdkn2a -/-, Braf V600E /Trp53 -/-), which still mainly induce nevus formation, we were unable to appreciate any obvious nevogenesis in Braf V600E /Lats1/2 -/mice. These data imply oncogenic Braf V600E -positive melanocytes may be incapable of entering an enduring growth arrest without a functional Hippo tumor suppressor pathway.
We also investigated the consequences of Lats1/2 loss in melanocytes in the absence of oncogenic Braf. Intriguingly, we found that following melanocyte-specific deletion of Lats1/2, mice exhibited no obvious hyperpigmentation, yet still rapidly developed cutaneous tumors with 100% penetrance after 4-5 weeks ( Figure  5E-F, S5D). Co-heterozygous deletion of Lats1/2 also promoted cutaneous tumorigenesis, albeit at prolonged time scales ( Figure S5E). Analysis of Lats1/2 -/tumor sections revealed non-pigmented neoplasms which were remarkably similar to invasive Braf V600E /Lats1/2 -/mouse tumors, exhibited a comparable staining profile, and were subsequently diagnosed as mouse melanoma ( Figure 5G). Previous studies have identified TEAD and AP-1 transcription factors as major regulators of the melanoma invasive state, which is marked by dedifferentiation and loss of pigmentation signatures (51,52). We hypothesized that YAP/TAZ-TEAD activation, driven by Lats1/2 deletion, may enable melanocytes to directly access this invasive gene program explaining our observed lack of pigmentation. In support of this hypothesis, both Braf V600E /Lats1/2 -/and Lats1/2 -/mouse melanomas exhibited markedly low staining for mature, differentiated melanocyte markers ( Figure S5F).
Given Lats1/2 -/tumors did not exhibit overt signs of pigmentation, we sought to generate additional data to validate that these neoplasms were melanocytic in origin. It has recently been demonstrated that initiation of the hair follicle cycle, via depilation, strongly promotes melanocyte transformation in Braf V600E /Pten -/mice (53, 54). We leveraged this unique characteristic of mouse melanomagenesis to test if Lats1/2 -/tumor formation was also promoted by depilation, suggesting a melanocytic origin. We induced loss of Lats1/2 on opposing mouse flanks and then depilated only one flank so as to compare the tumorigenic rate from depilated and non-depilated regions ( Figure 5H). We observed that skin regions depilated following 4-HT treatment demonstrated significantly faster tumorigenesis, with palpable tumors observed in 70% of depilated areas compared to 20% of non-depilated areas about one month following treatment ( Figure 5I). Not only did tumors appear faster in depilated areas, but these tumors also grew significantly larger ( Figure 5J). Collectively, our data demonstrate that melanocytespecific loss of Lats1/2 alone, or in conjunction with oncogenic Braf expression, promotes melanocyte transformation and melanoma formation in vivo.   Next, we investigated whether Hippo pathway inactivation was also occurring in other mouse models of melanoma. We surveyed publicly available datasets (7) and performed gene set enrichment analysis (GSEA) on microarrays gathered in an elegant series of experiments which isolated melanocytes that had either arrested or fully transformed into melanoma in either Braf V600E /Cdkn2a -/or Braf V600E /Cdkn2a -/-/Lkb1 -/mice. GSEA revealed that YAP/TAZ gene sets were significantly enriched in mouse melanoma as compared to arrested, nevus melanocytes or proliferating, non-tumorigenic melanocytes. These data thus indicate that as mouse melanocytes transform into melanoma they exhibit inactivation of the Hippo tumor suppressor pathway ( Figures  6A and S6A-D).

Melanoma Development Can Be Driven by YAP Activation
We sought to further define how deletion of Lats1/2 promotes melanoma development in vivo. While it is well described that Lats1/2 loss functionally inactivates the Hippo pathway and leads to the activation of YAP and TAZ, LATS1/2 can impinge upon additional signaling pathways that promote tumor development. For example, recent research has revealed inactivation of the Hippo pathway can promote mTOR signaling via multiple routes including YAP-driven expression of a micro-RNA, miR-29, which targets PTEN mRNA for silencing (55)(56)(57). However, we detected no observable changes in PTEN protein level following either RNAi-mediated knockdown of LATS1/2 or expression of constitutively active YAP (YAP-5SA) or TAZ (TAZ-4SA) in Mel-ST cells ( Figure 6B and C). We could also not appreciate any significant change in phosphorylated S6 levels following LATS1/2 silencing. Furthermore, examination of Lats1/2 -/tumors revealed PTEN remained strongly expressed in vivo ( Figure 6D and S6E). These data reveal that loss of Lats1/2 is not driving melanomagenesis by activating mTOR via miRNA-mediated depletion of PTEN.
It has also been demonstrated that active LATS2 can bind and inhibit MDM2 leading to increased p53 protein levels (58), raising the possibility that deletion of Lats1/2 leads to decreases in p53, which may facilitate Braf V600Edriven murine melanomagenesis (59). Discounting this, we found that p53 still accumulates in LATS1/2 depleted Mel-ST cells and Lats1/2 -/tumors ( Figures 6B and 6D).
To assess whether YAP activation alone is sufficient to broadly promote melanoma development, we generated a transgenic zebrafish model that expresses constitutively active YAP specifically in zebrafish melanocytes utilizing the miniCoopR system (60). While all EGFP-expressing controls experienced no tumor burden in this system, all Tg(mitfa:YAP-5SA) zebrafish rapidly developed pigmented fish melanoma ( Figure 6E). This result demonstrates that constitutively active YAP is sufficient to induce melanoma development, and highlights YAP1 as an important melanoma oncogene. Although deletion of Lats1/2 may still be promoting mTORC1 activity in our murine model, given activation of mTORC1 alone is insufficient to promote melanoma formation in vivo (7), whereas activation of YAP alone is sufficient, we believe activation of YAP/TAZ remains essential for the observed tumor formation in both Braf V600E /Lats1/2 -/and Lats1/2 -/mice.
As activation of YAP/TAZ signaling was observed to promote tumorigenesis across in vivo melanoma models, we investigated whether depletion of YAP/TAZ could inhibit melanoma cell growth. RNAi-mediated knockdown of YAP/TAZ in a Braf V600E /Pten -/mouse melanoma cell line (D4M.3A) resulted in significantly decreased viability as compared to immortalized melanocytes ( Figure 6F). This decrease in viability was predominantly driven by loss of YAP, as single knockdown of YAP was significantly detrimental to Braf V600E /Pten -/melanoma cells ( Figure  6F). To further confirm loss of YAP/TAZ signaling was driving decreases in viability, we treated either Mel-ST or D4M.3A cells with a recently discovered pan-TEAD inhibitor, MGH-CP1 (61). Strikingly, Braf V600E /Pten -/mouse melanoma cells were exquisitely sensitive to inhibition of TEAD by MGH-CP1 ( Figure 6G). Moreover, combined inhibition of TEAD and MAPK signaling further decreased D4M.3A cell viability ( Figure S6F).
Together, these data implicate YAP as a cutaneous melanoma oncogene and novel therapeutic target to further explore in the treatment of human melanoma. While YAP1 is not commonly mutated in human melanoma, YAP1 amplifications and mutations have been observed, and YAP staining in primary melanoma has been shown to significantly correlate with reduced patient survival (31,62,63).

Discussion
Discerning the molecular pathways that govern the growth arrest of nevus melanocytes, and how melanocytes ultimately overcome these barriers, is critical to fully understanding the mechanisms of melanomagenesis. A considerable body of work supports a role for OIS in preventing tumorigenic growth of melanocytes in vitro and in vivo; however, expanding lines of evidence demonstrate melanocytes within nevi retain proliferative capacity, in conflict with the absolute growth arrest implied by OIS (16). Indeed, up to 30% of melanomas are predicted to arise from pre-cursor nevi (17). These observations highlight nevi as being the product of "stable clonal expansion", rather than senescence (16).
Several genetic alterations that have the capacity to overcome the stable growth arrest of melanocytes expressing oncogenic MAPK mutations have been identified, including CDKN2A, PTEN, and TP53 (3,(5)(6)(7)59). While deletion of Cdkn2a and Trp53 with Braf V600E expression induces murine melanomagenesis, the vast majority of melanocytes still enter a stable growth arrest to form nevi (7). Further, recent murine single-cell sequencing analyses reveal Braf V600E -positive nevus melanocytes do not exhibit expression of senescence signatures (25). Together, these discoveries suggest additional unidentified tumor suppressive mechanisms exist to enforce the arrest of nevus melanocytes.
Our data demonstrate that the Hippo pathway represents one such tumor suppressive mechanism. We discovered that expression of BRAF V600E promotes activation of the Hippo tumor suppressor pathway across multiple cell lines and that Braf V600E -positive murine nevus melanocytes display significantly decreased YAP/TAZ signaling in vivo. We determined oncogenic BRAF expression induces Hippo pathway activation and cell cycle arrest in vitro via a cell-intrinsic mechanism, in which hyperactive MAPK signaling alters the cytoskeleton in part through decreased RhoA signaling. By contrast, our analysis of single-cell sequencing data indicated that while Braf V600E -expression leads to a general repression of YAP/TAZ signaling in most melanocytes in vivo, the effect is strongest in nevus melanocytes ( Figure 4D). This suggests cell-extrinsic cues, perhaps specific to the nevus microenvironment, may also be impinging upon YAP/TAZ signaling.
A recent modeling study has proposed that nevus melanocyte growth arrest is likely governed by collective cell behaviors (e.g. paracrine signaling (10)), rather than cell-autonomous mechanisms (25). Highly analogous to the collective cell processes mediating contact inhibition and organ growth, which are governed by the Hippo pathway, it is well known that a majority of Braf V600E -expressing melanocytes only enter a state of growth arrest following multiple rounds of division. It is therefore tempting to speculate that melanocyte overgrowth induced by oncogene expression significantly increases melanocyte density over multiple rounds of division ultimately promoting mounting activation of the Hippo tumor suppressor pathway and eventual growth arrest. If true, one would predict Hippo pathway activation would only engage after a nevus passes a critical size threshold and that nevus melanocyte growth may be restored by reducing local melanocyte density. Intriguingly, nevi that are partially resected have been observed to regain proliferative capacity and the vast majority of these recurrent nevi did not grow beyond the limits of the original surgical scar, suggesting these nevus melanocytes arrest once they attain a similar size (64,65).
We also found that Hippo pathway impairment alone is sufficient to induce melanoma formation in various in vivo models, even upon heterozygous loss of Lats1/2. This finding is clinically relevant, as co-heterozygous loss of LATS1/2 is frequently observed in human melanomas ( Figure 5A). However, it should be noted that Hippo pathway inactivation can also occur through mechanisms independent of LATS1/2 loss. For example, >80% of uveal melanomas and ~6% of cutaneous melanomas have mutations in GNAQ/GNA11 which can activate YAP/TAZ independent of LATS1/2 (66)(67)(68). Intriguingly, these activating mutations in GNAQ/GNA11 have been shown to stimulate RhoA activity (66). As YAP/TAZ-TEAD activation appears to be relatively common in melanoma, it will be important to define additional mechanisms by which Hippo inactivation occurs (63,69).
Our live-cell imaging revealed induction of oncogenic BRAF promotes chromosome alignment defects, prolonged mitosis, and a significant increase in cells experiencing a whole-genome doubling (WGD) event. We predominantly detected mitotic slippage events, in which cells expressing BRAF V600E exited mitosis without undergoing cell division, thus giving rise to multinucleated tetraploid cells which are commonly observed within benign human nevi (37). Typically caused by mitotic or cytokinetic failures, cells that undergo a WGD event (referred to as WGD + ) are genomically unstable cells known to drive tumorigenesis, and their contribution to human cancer is significant (70)(71)(72). In fact, the vast majority of metastatic melanomas and ~30-40% of human melanomas overall show evidence of having experienced a WGD event (71)(72)(73). WGD + cells are known to activate both the Hippo and p53 tumor suppressor pathways to restrain their ongoing proliferation (35,58,74,75). As such, Hippo pathway inactivation would restore proliferation not only to growth-arrested, diploid, nevus melanocytes, but also to the rarer, possibly WGD + , multinucleated melanocytes. Whether the ~40% of melanomas that have experienced a WGD enrich for alterations in the Hippo pathway and what specific defects cooperate with oncogenic MAPK mutations to promote WGD in human melanoma remains unknown.
Questions remain as to how Hippo pathway activation so strongly promotes melanoma development. For example, activating MAPK mutations appear to be critical for human melanocyte transformation. However, we establish that mouse melanocytes merely lacking Lats1/2 rapidly develop melanoma. Interestingly, while Lats1/2 loss does not stimulate the MAPK pathway in vitro, we found that Lats1/2 -/melanomas exhibit strong p-ERK staining ( Figure 5G). These data suggest that Lats1/2 -/melanocytes hyperactivate the MAPK pathway either through the acquisition of genetic alterations or, potentially, deletion of Lats1/2 in vivo is capable of stimulating MAPK activity through YAP/TAZ-dependent transcriptomic changes (67). Indeed, it has been observed that one common resistance mechanism to Vemurafenib, a BRAF V600E -specific inhibitor, is the amplification and/or activation of YAP (42,69,76). This suggests that YAP activity can compensate for loss of BRAF V600E signaling. Given MAPK inhibitor resistance remains a significant component of treatment failure, our data suggests that co-targeting MAPK and YAP-TEAD signaling could simultaneously prevent resistance (69) and decrease melanoma cell viability (32,77).
In summary, we demonstrate that activation of the Hippo tumor suppressor pathway promotes melanocyte growth arrest in response to the expression of oncogenic BRAF V600E . Further, disruption of the Hippo pathway, which promotes activation of YAP/TAZ, potently induces melanocyte growth in vitro and melanoma development in vivo in multiple model organisms. Collectively, our data implicate the Hippo pathway as an important melanoma tumor suppressor and highlight YAP/TAZ as promising therapeutic targets to investigate for the treatment of human melanoma.

Acknowledgments
We would like to thank Jackie King and Ross King for their unwavering resolve to promote melanoma awareness and research, as well Qi Sun, Shuyang Chen, and the entire Ganem Lab for wisdom and advice. We would also like to thank April Deng, Karen Dresser, Constance Brinckerhoff, Robert Weinberg, Craig Ceol, and Arthur Lander for sharing cell lines, reagents, and/or assistance. The results published herein are partly based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. We thank all the patients who . All FBS used in these studies was confirmed to either be naturally absent of tetracyclines or below 20 ng/mL by the manufacturer. All cells were maintained at 37°C with 5% CO2 atmosphere and maintained at sub-confluent levels for passaging and all experiments. Cultures were regularly checked for mycoplasma contamination utilizing a PCR detection kit (G238, ABM) or Hoechst staining. Bright-field images of tissue culture cells were captured on an Echo Revolve Hybrid Microscopy system at 10X or 20X (Echo Laboratories).

Cell Line Generation
To generate the BRAF V600E doxycycline inducible system Mel-ST, HEK293A, or hTERT BJ fibroblasts were first infected with lentivirus generated from pLenti CMV TetR Blast (Tet Repressor) and selected. Following selection, cells were infected with lentivirus generated from pLenti CMV/TO BRAF V600E Neo or pLenti CMV/TO BRAF Neo, selected, and single cell cloned to establish cell lines which demonstrated no basal expression at baseline and strong induction after doxycycline addition. Expression of BRAF V600E was confirmed using two different mutantspecific antibodies (VE1 and RM8 clones). To generate stably expressing H2B-GFP lines, cells were infected with lentivirus generated from pLenti H2B-GFP Blast. Stably expressing Mel-ST cells with empty vector (pLVX Puro), YAP-5SA (pBABE YAP-5SA Puro), and TAZ-4SA (pLVX Flag TAZ-4SA Puro) were generated via viral infection followed by selection. Tetraploid cells were generated by treating asynchronous cells with 4uM DCB for 16 h, followed by gentle washing to remove drug (5 x 5 min); completion of cytokinesis was confirmed by phasecontrast imaging.

Viral Infections and Transfections
Mel-ST, HEK293A, or BJ Fibroblasts were infected for 12-16 h with virus carrying genes of interest in the presence of 10 µg/mL polybrene, washed, and allowed to recover for 24 h before selection or single cell cloning. Short term viral infection of primary melanocytes was carried out similarly, but with 2 µg/mL of polybrene. All RNAi transfections were performed using 25-50 nM siRNA with Lipofectamine RNAi MAX according to the manufacturer's instructions. Briefly, cells were seeded in 6 or 12-well plates either by addition of reverse transfection mixture overnight, which was then washed and replaced with fresh media 18 h later, or forward transfection mixture for 4 h, which was then replaced with fresh media. Cells were then incubated for 48-72 h prior to lysis at sub-confluent levels.

Immunofluorescence and Confocal Microscopy
Cells were plated on glass coverslips, treated as indicated, washed in 1X phosphate-buffered saline (PBS) (Boston Bioproducts) and fixed in 4% paraformaldehyde for 10 min. Cells were then washed in PBS-0.01% Triton X-100, extracted in PBS-0.2% Triton X-100 for 10 min, blocked in Tris-buffered saline (TBS)-bovine serum albumin (BSA) (10 mM Tris, pH 7.5, 150 mM NaCl, 5% Bovine Serum Antigen, 0.2% sodium azide) for 1h, and incubated with primary antibodies diluted in TBS-BSA for 1 h at RT or overnight at 4°C in a humidified chamber. Primary antibodies were visualized using species-specific fluorescent secondary antibodies (Molecular Probes, Alexa Fluor secondaries, 488 nm, 568 nm, 1:500) and DNA was detected with 2.5 µg/mL Hoechst. F-actin was visualized using rhodamine-conjugated phalloidin (1:2000, Molecular Probes, R415). Immunofluorescence images for analysis were collected on a Nikon Ti-E inverted microscope equipped with a Zyla 4.2 PLUS (Andor) and X-Cite 120 LED light source at the same exposure. Confocal immunofluorescence images were collected on a Nikon Ti-E inverted microscope equipped with a C2+ laser scanning confocal head with 405 nm, 488 nm, 561 nm, 640 nm laser lines. Z-stacks were acquired with a series of 0.5-1 µm optical slices which were then converted into a single, max-intensity projected image. Images were analyzed using NIS-Elements Advanced Research (AR) and ImageJ. To assess YAP localization, two small square regions of interest were drawn at random in individual cells with one in the nucleus, and one in the cytoplasm. The background corrected, mean fluorescence intensity of YAP was subsequently measured in these regions of interest and a nuclear to cytoplasmic ratio was determined. To assess stress fiber quantity, images were background corrected, contrast normalized, and then fibers obvious to the naked eye were counted.

Live-Cell Imaging
Stably expressing H2B-GFP cells were grown on glass-bottom 12-well tissue culture treated dishes (Cellvis) and treated with drugs of interest. Immediately post-treatment imaging was performed on a Nikon Ti-E inverted microscope equipped with the Nikon Perfect Focus system. The microscope stage was enclosed within a temperature and atmosphere-controlled environment at 37°C and 5% humidified CO 2. Fluorescent or bright-field images were captured every 5-10 min with an 10X or 20X 0.5 NA Plan Fluor objective at multiple locations for 72-96 h. All captured images were analyzed using NIS-Elements AR software. Mitotic length was calculated by counting the duration from nuclear envelope breakdown to anaphase onset.

Tissue Staining
At the experimental endpoint mice were euthanized and mouse tumors or skin samples were dissected and immediately fixed in 4% paraformaldehyde (PFA) in PBS for 16h at 4°C. Tissues were then paraffin embedded for sectioning and mounting. PFA-fixed paraffin-embedded tissue sections were cut at 5 µm and mounted onto positively charged coverslips (Colorfrost Plus, Thermo). Mounted tissue samples were deparaffinized using xylenes and rehydrated via an ethanol:water gradient. For hematoxylin and eosin staining (H&E), the tissues were incubated in hematoxylin (#14166, Cell Signaling Technology), rinsed in water, differentiated with 1% acid ethanol, blued with 0.1% sodium bicarbonate solution, rinsed in water, dehydrated, cleared, and mounted with Cytoseal XYL (Thermo Scientific). For immunohistochemistry (IHC) or immunofluorescence (IF), antigen unmasking was performed on rehydrated tissue sections using either a citric-acid retrieval buffer (Vector Labs) or Tris-EDTA retrieval buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween-20). Heat-mediated antigen retrieval was performed using either a standard microwave (95°C, 20 min) or Decloaking Chamber NxGen (Biocare Medical) (110°C, 12 min), followed by cooling to room temperature (~30-60 min). Citric acid retrieval was used for most antibodies; Tris-EDTA retrieval was used for MelanA antibodies. For IHC, tissue sections were washed, endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS for 10 min, and then blocked for 1h in 10% goat serum in TBS (Sigma-Aldrich). For IF, tissue sections were blocked for 1h in 10% goat serum in TBS. Following serum block, if necessary, tissue was incubated with Rodent Block M (Biocare Medical) for 30 min to block endogenous mouse IgG prior to murine primary antibody addition. Primary antibodies were diluted in 10% goat serum in TBS and incubated overnight at 4°C in a humidified chamber. Following primary addition for IHC, slides were washed with TBS-0.01% Tween-20, incubated with anti-rabbit or mouse SignalStain Boost IHC detection reagent (Cell Signaling Technology) for 30 min and then developed with SignalStain DAB substrate kit (Cell Signaling Technology) according to manufacturer's instructions. Counterstaining was performed using hematoxylin, followed by dehydration, clearing, and mounting with Cytoseal XYL (Thermo Fisher). Images were captured at randomly selected points using a Nikon Ti-E inverted microscope equipped with a DS-Ri2 (Nikon). For IF, slides were incubated with species-specific fluorescent secondary antibodies (Molecular Probes) and 2.5 µg/mL Hoechst for 1h at room temperature in a dark humidified chamber. Autofluorescence was quenched using Vector TrueVIEW according to manufacturer's instructions, and slides were mounted using Prolong Gold Antifade (Invitrogen). Images were captured at randomly selected points using an Nikon Ti-E inverted microscope equipped with a Zyla 4.2 PLUS (Andor) and X-Cite 120 LED light source. For all staining experiments tissue-specific secondary controls were included in each staining experiment to ensure specificity and control for endogenous tissue pigmentation levels.

Protein Extraction and Immunoblotting
Cells were gently washed twice in ice-cold 1X PBS and lysed using ice-cold cell lysis buffer (50 mM Tris-HCl, 2% w/v SDS, 10% glycerol) containing 1X HALT (dual phosphatase and protease inhibitor, Thermo Fisher). Lysates were sonicated at 20% amplitude for 20 seconds, diluted in 4X Sample Buffer (Boston BioProducts), and resolved using SDS gel electrophoresis. For mouse tumor samples, 10 mg of tumor tissue was dissected and placed into RIPA lysis buffer (Boston BioProducts) supplemented with one Pierce protease inhibitor tablet and 1X HALT (Thermo Fisher). Tissue was mechanically dissociated initially using a 7cm pestle (Kimble) followed by further homogenization with a 20-guage needle and brief sonication for 20 s at 20 kHz. Tissue lysate was then centrifuged at 4°C and >15,000g for 10 min. Supernatant was collected, diluted in 4X Sample Buffer to 2X, and resolved using SDS gel electrophoresis. For phos-tag immunoblots, a phos-tag gel was prepared with diluted Phos-Tag™ Acrylamide reagent (Wako Chemicals, AAL-107) according to manufacturer's instructions utilizing manganese as the divalent cation. Proteins were then transferred onto PVDF membranes using a wet-tank transfer system (Bio-Rad), blocked for 1 h with TBS-0.1% Tween-20 containing 5% skim milk powder and then probed overnight at 4°C with primary antibodies diluted in TBS-0.1% Tween-20 containing 1% skim milk powder. Bound antibodies were detected by incubating membranes with horseradish peroxidaselinked, species-specific, secondary antibodies (1:5000, Cell Signaling Technology) for 1 h, followed by 3x10 min washing in TBS-0.1% Tween-20, and then addition of Clarity or Clarity Max ECL blotting substrate (Bio-Rad). Chemiluminescence acquisition was carried out using the Bio-Rad ChemiDox XRS+ system and quantitative densitometry was measured using Image Lab (Bio-Rad).

Human Nevus Sample Collection
Human skin tissues with melanocytic nevi and melanoma were retrieved from existing material in the pathology laboratory at UMass Medical Center. The tissue blocks are deidentified with new numbers before tissue sections (10 µm) were obtained for IHC stains. These studies were reviewed and determined to be IRB exempt by Boston University School of Medicine.

RNA isolation and qRT-PCR
Total RNA from cultured cells was isolated using a Quick-RNA kit (Zymo Research). cDNA libraries were generated from RNA using the Superscript III kit and random hexamer primers (Invitrogen). Quantitative real-time PCR was performed using SYBR Green reagents in a StepOnePlus system (Applied Biosystems) according to manufacturer protocol. For each individual experiment a technical triplicate was run which was then averaged to generate a single biological replicate. Primer sequences were as follows:

Soft Agar Assays
In 6-well dishes, sterile 2% noble agar stock solution in water was dissolved by heating to ~40-45°C, mixed with warm media, plated at final concentration of 0.6%, and allowed to cool and solidify at 4°C. Following solidification gels were warmed back to 37°C. Next, cells were trypsinized, counted, 1x10 4 cells were plated in 0.3% noble agar and allowed to solidify at room temperature or briefly at 4°C. Plates were maintained in a cell culture incubator for 2-4 weeks with feedings of 1.5 mL of 0.3% agarose solution weekly. All drugs were maintained at 2X concentration in underlays and independent experiments were done in technical triplicate. Total colonies per well were counted using phase-contrast imaging on an Echo Revolve Hybrid Microscopy system at 10X (Echo Laboratories). For imaging, gels were stained for 20 min with 0.1% Crystal Violet, gently washed multiple times overnight, and imaged on a Chemi-Doc XRS+ system under Fast Blast with 0.015-0.1 sec exposure.

Drug Treatments
The concentrations used for the MAPK pathway inhibitors (MEKi-1/2 and ERKi) were determined experimentally to be the doses at which phosphorylation of ERK1/2 and RSK1/2/3 returned to baseline despite the presence of oncogenic BRAF expression. The reagents used in these studies are as follows: MEKi ). For viability assays, 4x10 3 cells per well were plated into a white-bottom 96-well dish in technical duplicate to quintuplicate, dependent upon experiment. Cells were then treated with indicated siRNA and/or drugs for 4 days. The 96-well plate was then allowed to equilibrate at room temperature for 30 minutes followed by addition of Cell Titer Glo (Promega) to each well according to manufacturer instructions. Viability was then assessed via luminescence using a BMG microplate reader and analyzed using BMG Labtech Optima v2.0R2 software. All technical replicates were averaged to generate a single biologic replicate.

Copy Number Analysis
Log2 copy-number values and/or putative copy-number GISTIC 2.0 values for LATS1 and LATS2 were downloaded from cBioPortal for the TCGA skin cutaneous melanoma (SKCM) subset of the PanCancer Atlas and MSKCC, NEJM 2014 datasets. SKCM abbreviation represents skin cutaneous melanoma. Mutational status for BRAF and NRAS from the TCGA-SKCM dataset was also obtained from cBioPortal. Graphs were created in Prism 9.

Gene Expression Analysis
Publicly available microarray dataset GSE61750 was obtained from the Gene Expression Omnibus (GEO). For GSEA indicated groups were normalized via meandiv, probes collapsed to max probe, and a weighted enrichment metric (log2 Ratio of Classes) was utilized with 1000 permutations to determine enrichment in indicated gene sets. The YAP/TAZ target score gene set was derived from (45) and (46). Gene set can be found in Supplementary Table 1. The Hippo component gene set was derived from Reactome with additional genes added and can be found in Supplementary Table 2.

Single-Cell RNAseq Analysis
Count matrix of GSE154679 was downloaded from the GEO repository (25). Cells that had less than 200 expressed genes, more than 4000 expressed genes or > 17% of mitochondrial gene were removed from analysis. A total of 35214 cells were retained for downstream analysis. Normalization, dimensionality reduction, cell-clustering and data visualization were analyzed with Seurat package (78). PCA dimension reduction was performed using top 2000 highest variance genes. The top 15 principal components were utilized to calculate the k-nearest neighbors of each cells. Cell clusters were determined using Louvain algorithm at a resolution = 0.5, which was high enough to obtain clusters associated with cell lineage identity. We used UMAP to visualize and confirm cell clustering. Melanocyte cell population was identified based on the expression of canonical gene markers. To identify subclusters within melanocyte cell populations, we re-analyzed cells within melanocyte cell clusters using the same analysis pipeline described above. To quantify gene sets activity in each cell, gene set testing of scRNA-seq data was performed following VAM pipeline (44). Data Normalization and highest variable genes were analyzed using pipeline described above. VAM method was executed using vamForSeurat() function on melanocyte cell population for each gene set. Graphs were created in Seurat or data was imported into Prism 9 for graph creation.

Murine Studies
All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University (Protocol # PROTO201800236). All other mouse strains were sourced from Jackson Laboratory: Tyr::CreER T2 (Jax # 012328), Lats1 f/f (Jax # 024941), Lats2 f/f (Jax # 025428), BRAF V600E (Jax # 017837), R26-YFP LSL (Jax # 006148). 4-hydroxytamoxifen (4-HT [Sigma, H7904]) was dissolved in methanol to a concentration of 5 mg/ml. To induce knockout in Lats1/2 -/-, and Braf V600E /Lats1/2 -/-, mice aged 8-12 weeks had an approximately 2 cm square shaved on the right flank using surgical clippers and sufficient 4-HT solution to wet the skin was applied once daily to the shaved area for three consecutive days. For Braf V600E mice, a higher concentration of 4-HT was required; 4-HT was dissolved in dimethylsulphoxide (DMSO) to a concentration of 25 mg/ml, and topical administration was performed as above. For the depilation experiments using Lats1/2 -/mice, a 2 cm square was shaved on both the left and right flank of each mouse, and 5 mg/ml 4-HT was topically applied to each square for three consecutive days as above on the right flank, as well as on an approximately 2 cm length of the tail for three consecutive days. Two days after the final application, chemical depilation was performed by application of Nair hair remover onto the right flank for 15 seconds, followed immediately by wiping with a damp tissue to prevent irritation. For systemic knockout experiments, mice were injected with 20 mg/ml tamoxifen (Sigma, T5648) dissolved in corn oil to induce deletion in all Tyrosinase expressing cells. [8][9][10][11][12] week old mice were injected daily with 100 µl tamoxifen for 5 consecutive days. Tumor development was measured weekly using calipers.

Zebrafish Studies
Constructs Pmitfa:EGFP:pA and Pmitfa:YAP-5SA:pA were used in the miniCoopR assay as previously described (60). Briefly, mitfa(lf) mutant animals were bred, and single-cell stage embryos were injected with 25 pg of a single construct and 25 pg of Tol2 transposase mRNA. Successful Tol2-mediated integration of the construct into the genome rescued the mitfa(lf) phenotype, enabling melanocyte development. Melanocyte rescue was scored at 4-5 days of development, and rescued animals were grown to adulthood and monitored weekly for the presence of melanomas.

Hippo Component Gene Set
Supplemental Table 2