1. Author Response:

    Reviewer #1 (Public Review):

    In their manuscript, Urtatiz and colleagues propose that gain-of-function mutations affecting the G-alpha-q signaling pathway are not tolerated in melanocytes residing in the interfollicular epidermis because of paracrine signals from neighboring keratinocytes. This is an interesting and important hypothesis that would explain a mystery to the melanoma field - i.e. why are GNAQ/11 mutations common in uveal melanoma, among other rare subtypes, yet are exceedingly rare in cutaneous melanoma.

    Specific comments on experimental work:

    Previously, this group showed that forcible expression of oncogenic GNAQ during embryogenesis depletes melanocytes in the interfollicular epidermis. This paper offers an advance because TYR-cre mouse is inducible at later points in life, which also permits in vitro explant cultures to perform more in-depth studies. This is a major strength of the manuscript.

    Major concerns:

    The most significant issue with the experimental results is that in most of the explant cultures, the melanocytes are not proliferating. Instead, the authors are observing which melanocytes are dying slower than others. This seems a bit strange because there are countless examples of laboratories who have established healthy murine melanocytes in tissue culture, and it raises the question that there is something off with the culture conditions.

    Thank you to the reviewer for their thorough analysis and for raising interesting questions.

    Yes, it is true that the wildtype and GNAQ^Q209L expressing melanocytes in the survival curves (in Figure 2C, for example) did not increase above the number plated during the experiment. However, the Braf^V600E expressing cells did increase, which suggests that the culture conditions were not fundamentally off. It is important to note that the melanocytes in our study were sorted from mouse tail IFE. As far as we know, other labs have used mouse trunk skin or neonatal skin, which is populated by immature migrating melanocytes. These tissues have stem cells, for example in the hair follicle niche, and highly proliferative cell populations. We chose tail skin because it has a permanent population of IFE melanocytes.

    Our goal in these studies was a short observation with minimal interference. Encouraging the melanocytes to behave in a certain way in culture could change the results of the experiment or mask the differences between wildtype and GNAQ^Q209L melanocytes resulting from the microenvironment. We don't agree that studying the rate of dying off is not applicable, when the relevant in vivo phenotype is that GNAQ^Q209L melanocytes experience gradual attrition from the IFE.

    In addition, when sorting the GNAQ-mutant melanocytes, there is a selection for a subpopulation that did not die. This introduces a bias and seems, at minimum, worthy of discussion. One potential experiment to remove these doubts would be to isolate the GNAQ-mutant melanocytes prior to tamoxifen treatment and then induce the mutation formation in vitro.

    If we understand this correctly, the concern is that since some melanocytes have been lost in the GNAQ^Q209L IFE by the time we do the FACS, this approach may have selected for a subpopulation of melanocytes that are more resistant to the effects of GNAQ^Q209L. Maybe the GNAQ^Q209L cells survived better than wildtype in culture on fibronectin because they were naturally more robust. How then do we explain why the GNAQ^Q209L melanocytes survived less well than wildtype when cultured with IFE? If there is a subpopulation at play, it is behaving exactly as one would expect from the mice, so we don't see how it changes the conclusions.

    We have added this sentence to the manuscript: "Note, since some melanocytes have been lost in the GNAQ^Q209L IFE, this approach may have selected for melanocytes that are more resistant to the effects of GNAQ^Q209L.".

    The suggested experiment is difficult to do because Cre is required to turn on both Tomato and GNAQ^Q209L. To be able to do the suggested experiment, we would have to first obtain a different transgenic mouse line that constitutively expresses a fluorescent marker in melanocytes and cross it into our mouse model. Alternatively, we could try to sort melanocytes following antibody staining against a cell surface marker. In fact, neither of these approaches is free from the possibility of selecting subpopulations that vary in expression of the markers.

    Specific comments on bioinformatic work:

    Major issues:

    The evidence that there is selection against PLCB4 in cutaneous melanoma is weak. It is true that mutations frequently affect PLCB4, but this is equally true for a great number of genes in melanoma because of the high mutation burden in this cancer type. Following their lines of reasoning, the authors could make an equally compelling case that TTN, the gene encoding the muscle fiber titin which is the largest gene in the genome, is under selection. Unfortunately, the ratio of nonsynonymous to synonymous mutations is not supportive of the authors' argument that PLCB4 is under selection. It is somewhat bizarre that the authors' entirely disregarded synonymous mutations, but this reviewer looked them up in the TCGA study, and they are abundant. To identify genes under selection, there are much more sophisticated strategies that take into account the trinucleotide context of mutations, the ratio of nonsynonymous to synonymous mutations, and/or the ratio of exonic to intronic mutations. To be sure, the authors correctly point out these sophisticated algorithms have missed driver mutations in the past, but the missed mutations tend to be exceedingly rare, hotspot mutations. If the authors are going to make the case that loss-of-function PLCB4 mutations are under selection in melanoma, then the onus is on them to explain why the much more sophisticated strategies, previously invoked, have missed this finding, and they should employ even better methods to make their point. Unfortunately, the strategies that the authors do employ fall for the same traps that many older papers in the field stumbled upon. For example, they make the case that PLCB4 mutation are more frequent in melanomas with high mutation burdens. While this seems to denote a biological signal, it is exactly what one would expect to observe if a gene only harbored passenger mutations.

    We understand the concerns of the reviewer. To explain, we were led to the question of whether the frequent non-synonymous mutations in PLCB4 could play a role in cutaneous melanoma from a logical deduction based on biological insight, which we found quite convincing:

    1. We had found that oncogenic Gq inhibits melanocyte growth/survival when the cells are located in the epidermis.

    2. Phospholipase C beta (PLC-Beta) is the immediate effector of heterotrimeric G protein alpha subunits of the q class, GNAQ and GNA11. It is very likely to be involved in the pathway inhibiting melanocytes in the IFE.

    3. PLCB4 was already identified as a significant melanoma gene in uveal and CNS melanomas, through a recurrent hotspot mutation. Furthermore, the hotspot mutation in PLCB4 is mutually exclusive with the hotspot mutations in GNAQ or GNA11 in these cancers.

    We agree that more could have been done to describe the synonymous mutations and examine the ratio of non-synonymous (missense, nonsense) mutations to synonymous mutations. These studies can be found in the manuscript and in Supplementary File 1 Table 1f, 1g and Supplementary File 3. Using the approach described in Van den Eynden and Larsson (1), which takes into account the mutational signature of melanoma, we found that the synonymous mutations were significantly less frequent than expected based on the expected ratio (p = 7.35 x 10^-4; one-tailed binomial test). This indicates that there is positive selection for the non-synonymous mutations in PLCB4 in cutaneous melanoma.

    (1)Van den Eynden and Larsson (2017) Front Genet Jun 8;8:74 [https://doi.org/10.3389/fgene.2017.00074]

    In addition, not included in the above analysis were 13 more mutations having to do with splicing: 7 splice region, 3 splice donor site and 3 splice acceptor site mutations in the melanomas. While reviewing the literature, we noticed that PLCB4 was already identified in Chen et al. (2) as a gene subject to positive selection for splice-site mutations (p=7.86Ex10^-4; q=5.77Ex10^-2) in a genome wide analysis of melanoma.

    (2)Chen et al (2015) Mol Biol Evol. 2015 Aug;32(8):2181-5 (mentioned in the Supplement).

    These findings strengthen our hypothesis. We propose that loss-of-function mutations in PLCB4 simultaneously reduce Galpha_q and Galpha_11 signaling, releasing some of the inhibitory effects of the pathway caused by the epidermal microenvironment.

    As you know, due to a number of factors related to the process of mutagenesis, gene size and accessibility, different genes have different rates of mutation. PLCB4 might be a gene that is more likely to be mutated. It is the 99th most frequently mutated gene in the SKCM dataset. PLCB4 has more synonymous mutations (34 affected cases in total) than NF1 (14 cases), TP53 (0 cases) or PTEN (0 cases). Looking at the problem from our perspective, it seems that biological insight and hypothesis driven inquiry could help determine whether some of the genes with a higher inherent rate of mutation are playing a role in melanoma.

    We also would like to clarify the statement that "...PLCB4 mutations are more frequent in melanomas with high mutation burdens." What we saw was that the melanomas with 2000 to 3000 mutations overall had the highest rate of PLCB4 non-synonymous mutations (Figure 7E). Melanomas with more than 3000 mutations actually had a reduced rate of non-synonymous mutations in PLCB4. It was not simply that more overall mutations equaled more mutations in PLCB4.

    The Semaphorin mechanism is interesting but remains too speculative to warrant so much attention and space.

    We have reduced the emphasis on semaphorin signaling.

    In addition, there was too much speculation regarding signaling mechanisms in the apoptosis section of the manuscript. Generally speaking, RNA-sequencing is a powerful tool, but when there are >1000 differentially expressed genes, it is too easy to construct "in silico" mechanistic stories centered around a handful of genes. These would need be backed up with biological data, but in this case, additional mechanistic studies would go beyond the scope of the manuscript. Instead, this reviewer suggests removing these points.

    We have reduced the discussion of particular genes that supported increased apoptosis in GNAQ^Q209L melanocytes in our RNAseq analysis.

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  2. Evaluation Summary:

    In this manuscript, the authors study the discrepancy between the frequency of mutations in Gq alpha subunit (GNAQ and its paralogue GNA11) in uveal vs cutaneous melanoma. They hypothesize that the restriction of GNAQ and GNA11 mutations to non-epithelial melanomas is due to epidermal factors, which convert the impact of GNAQ Q209L mutation from being oncogenic to being inhibitory to melanocyte survival and proliferation, and reduce the maintenance, rather than the establishment of interfollicular epithelial melanocytes. This work provides new insights into the poorly understood difference in mutation frequency in different melanoma types.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    In their manuscript, Urtatiz and colleagues propose that gain-of-function mutations affecting the G-alpha-q signaling pathway are not tolerated in melanocytes residing in the interfollicular epidermis because of paracrine signals from neighboring keratinocytes. This is an interesting and important hypothesis that would explain a mystery to the melanoma field - i.e. why are GNAQ/11 mutations common in uveal melanoma, among other rare subtypes, yet are exceedingly rare in cutaneous melanoma.

    Specific comments on experimental work:
    Previously, this group showed that forcible expression of oncogenic GNAQ during embryogenesis depletes melanocytes in the interfollicular epidermis. This paper offers an advance because TYR-cre mouse is inducible at later points in life, which also permits in vitro explant cultures to perform more in-depth studies. This is a major strength of the manuscript.

    Major concerns:
    The most significant issue with the experimental results is that in most of the explant cultures, the melanocytes are not proliferating. Instead, the authors are observing which melanocytes are dying slower than others. This seems a bit strange because there are countless examples of laboratories who have established healthy murine melanocytes in tissue culture, and it raises the question that there is something off with the culture conditions.

    In addition, when sorting the GNAQ-mutant melanocytes, there is a selection for a subpopulation that did not die. This introduces a bias and seems, at minimum, worthy of discussion. One potential experiment to remove these doubts would be to isolate the GNAQ-mutant melanocytes prior to tamoxifen treatment and then induce the mutation formation in vitro.

    Specific comments on bioinformatic work:
    Major issues:
    The evidence that there is selection against PLCB4 in cutaneous melanoma is weak. It is true that mutations frequently affect PLCB4, but this is equally true for a great number of genes in melanoma because of the high mutation burden in this cancer type. Following their lines of reasoning, the authors could make an equally compelling case that TTN, the gene encoding the muscle fiber titin which is the largest gene in the genome, is under selection. Unfortunately, the ratio of nonsynonymous to synonymous mutations is not supportive of the authors' argument that PLCB4 is under selection. It is somewhat bizarre that the authors' entirely disregarded synonymous mutations, but this reviewer looked them up in the TCGA study, and they are abundant. To identify genes under selection, there are much more sophisticated strategies that take into account the trinucleotide context of mutations, the ratio of nonsynonymous to synonymous mutations, and/or the ratio of exonic to intronic mutations. To be sure, the authors correctly point out these sophisticated algorithms have missed driver mutations in the past, but the missed mutations tend to be exceedingly rare, hotspot mutations. If the authors are going to make the case that loss-of-function PLCB4 mutations are under selection in melanoma, then the onus is on them to explain why the much more sophisticated strategies, previously invoked, have missed this finding, and they should employ even better methods to make their point. Unfortunately, the strategies that the authors do employ fall for the same traps that many older papers in the field stumbled upon. For example, they make the case that PLCB4 mutation are more frequent in melanomas with high mutation burdens. While this seems to denote a biological signal, it is exactly what one would expect to observe if a gene only harbored passenger mutations.

    The Semaphorin mechanism is interesting but remains too speculative to warrant so much attention and space. In addition, there was too much speculation regarding signaling mechanisms in the apoptosis section of the manuscript. Generally speaking, RNA-sequencing is a powerful tool, but when there are >1000 differentially expressed genes, it is too easy to construct "in silico" mechanistic stories centered around a handful of genes. These would need be backed up with biological data, but in this case, additional mechanistic studies would go beyond the scope of the manuscript. Instead, this reviewer suggests removing these points.

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  4. Reviewer #2 (Public Review):

    The authors aimed at addressing the question of why constitutively activating somatic mutations in GNAQ and GNA11 that occur in almost 90% of uveal melanomas, and are common in blue nevi and melanomas of the central nervous system, are very rare in cutaneous melanoma tumors. They hypothesized that the restriction of GNAQ and GNA11 mutations to non-epithelial melanomas can be due to i) exposure of melanocytes in different anatomical locations to different mutagens that affect specific genes, ii) difference in the potential for melanomageneiss of melanocytes arising from different anterior-posterior positions during embryogenesis, and/or iii) differences in the microenvironment of melanocytes in different anatomical locations, that affect their regulation by paracrine factors. The authors arrived at the conclusion that epidermal factors convert the impact of GNAQQ209L mutation from being oncogenic to being inhibitory to melanocyte survival and proliferation, and reduce the maintenance, rather than the establishment of interfollicular epithelial (IFE) melanocytes.

    The strengths of the methods lie in the:

    1. In vivo comparison of the impact of GNAQQ209L mutation on melanocyte establishment and maintenance, by expressing the mutation in melanocytes, and comparing their number in the interfollicular epidermis (IFE) of the tail of wild type versus mutant mice.
    2. in vitro validation of the differences between wild type and GNAQQ209L mutant IFE melanocytes by sorting them from the tail IFE by FACS, and comparing their proliferation on fibronectin and their morphology. By removing the mutant melanocytes from their microenvironment (the tail IFE), they proliferated better than wild type melanocytes on a fibronectin matrix, demonstrating that the reduction in their number in vivo is due to paracrine regulation by their epithelial microenvironment.
    3. Demonstrating the impact of the microenvironment of GNAQQ209L mutant IFE melanocytes, melanocytes were co-cultured with IFE cells or tested in a transwell assay, with IFE cells in the bottom well, and the melanocytes plated on top of a permeable membrane. In both cases, IFE cells resulted in inhibition of proliferation and survival, and significant increase in the size and morphology of melanocytes.
    4. RNA sequencing to determine any change in gene expression that might account for the reduced survival, proliferation, and altered morphology of IFE melanocytes expressing GNAQQ209L , as compared to wild type melanocytes, using melanocytes immediately after sorting from the tails of mice. Bioinformatics analysis revealed altered expression of genes involved in pigmentation, cell adhesion, focal adhesion, extracellular matrix receptor interactions and structural constituents, axon guidance, nervous system development, stress and apoptosis, which account for the differences observed in the survival, proliferation, and morphology between mutant and wild type melanocytes.
    5. Analysis of TCGA-SKCM dataset to re-examine the data related to PLCB4, which encodes a protein that bins to Gαq and Gα11, and is the primary effector of signaling to downstream components of 1 class heterotrimeric G proteins, to confirm the previous report that PLCB4 is targeted by loss of function gene fusion events in some cutaneous melanomas. Demonstrating expression of loss of function of PLCB4 supports the authors' findings that activation of the q class heterotrimeric G proteins inhibits, while in contrast, inhibiting their signaling pathway is permissive to, epithelial melanoma tumor formation.
      The data presented are solid, and the results support the conclusions derived by the authors. The data were validated using the different experimental methods listed above. The results clarify the differences in the mutations observed in non-epithelial versus epithelial melanomas, and should improve the understanding of the various pathways that drive melanomagenesis under the influences of different microenvironments in different anatomical locations.
      The only weakness is in the detailed discussion of the various other mutations (pages 14-17). The data included could be summarized, since the detailed analysis detracts from the main take-home message of the manuscript that the epidermal microenvironment impairs the survival of GNAQQ209L mutant melanocytes, and deficiency of PLCB4 promotes cutaneous melanoma formation.
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