# BRN2 is a non-canonical melanoma tumor-suppressor

Michael Hamm, Pierre Sohier, Valérie Petit, Jérémy H Raymond, Véronique Delmas, Madeleine Le Coz, Franck Gesbert, Colin Kenny, Zackie Aktary, Marie Pouteaux, et al.

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## To cite this version:

Michael Hamm, Pierre Sohier, Valérie Petit, Jérémy H Raymond, Véronique Delmas, et al., BRN2 is a non-canonical melanoma tumor-suppressor. Nature Communications, 2021, 12 (1), pp.3707. 10.1038/s41467-021-23973-5. [hal-03437816v2](https://hal.science/hal-03437816v2)

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Submitted on 26 Nov 2021

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1 BRN2 is a non-canonical melanoma tumor-suppressor

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3 Michael Hamm¹⁻³$, Pierre Sohier¹⁻³$, Valérie Petit¹⁻³$, Jérémy H Raymond¹⁻³, Véronique

4 Delmas¹⁻³, Madeleine Le Coz¹⁻³, Franck Gesbert¹⁻³, Colin Kenny⁴, Zackie Aktary¹⁻³, Marie

5 Pouteaux¹⁻³, Florian Rambow¹⁻³, Alain Sarasin⁵, Nisamanee Charoenchon¹⁻³,⁶, Alfonso

6 Bellacosa⁷, Luis Sanchez-del-Campo⁸, Laura Mosteo⁸, Martin Lauss⁹, Dies Meijer¹⁰, Eirikur

7 Steingrimsson¹¹, Göran B Jönsson⁹, Robert A Cornell⁴, Irwin Davidson⁴,¹², Colin R Goding⁸#, and Lionel Larue¹⁻³##

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(1) Institut Curie, Université PSL, CNRS UMR3347, Inserm U1021, Normal and Pathological Development of Melanocytes, 91400 Orsay, France

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(2) Université Paris-Saclay, CNRS UMR3347, Inserm U1021, Signalisation radiobiologie et cancer, 91400 Orsay, France

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(3) Equipes Labellisées Ligue Contre le Cancer

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(4) Department of Anatomy and Cell biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

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(5) Laboratory of Genetic Instability and Oncogenesis, UMR8200 CNRS, Gustave Roussy, Université Paris-Sud, Villejuif, France.

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(6) Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

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(7) Cancer Epigenetics Program, Fox Chase Cancer Center, Philadelphia, PA, USA

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(8) Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford, OX3 7DQ, UK.

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(9) Department of Oncology, Clinical Sciences Lund, Lund University and Skåne University Hospital, Lund, Sweden.

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(10) Centre of neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom

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(11) Department of Biochemistry and Molecular Biology, and 5Department of Anatomy, BioMedical Center, Faculty of Medicine, University of Iceland, Sturlugata 8, 101

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30 Reykjavik, Iceland

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(12) Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UNISTRA, 1 Rue Laurent Fries, 67404 Illkirch Cedex, France. Department of Functional Genomics and Cancer.

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$ These authors contributed equally to this study.

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* Corresponding authors: Lionel Larue: lionel.larue@curie.fr

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# co last authors: Colin Goding & Lionel Larue

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The authors declare no potential conflicts of interest.

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Running title: BRN2 is a tumor-suppressor

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47 KEYWORDS

48 Melanoma; phenotypic switch; mouse molecular genetics; Haplo-insufficiency;

49 human; Mitf; Cre-LoxP; tamoxifen; POU3F2

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51 ABSTRACT (150 words)

52 While the major drivers of melanoma initiation, including activation of NRAS/BRAF

53 and loss of *PTEN* or *CDKN2A*, have been identified, the role of key transcription

54 factors that impose altered transcriptional states in response to deregulated signaling

55 is not well understood. The POU domain transcription factor BRN2 is a key regulator

56 of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in

57 a *Braf*^V600E *Pten*^F/+ context, we show that *BRN2* haplo-insufficiency promotes

58 melanoma initiation and metastasis. However, metastatic colonization is less efficient

59 in the absence of Brn2. Mechanistically, BRN2 directly induces *PTEN* expression and

60 in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target,

61 represses *PTEN* transcription. Collectively, our results suggest that on a *PTEN*

62 heterozygous background somatic deletion of one *BRN2* allele and temporal

63 regulation of the other allele elicits melanoma initiation and progression.

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65
66 INTRODUCTION

Cancer initiation is triggered by the activation of oncogenic signaling combined with senescence bypass. Yet while many typical oncogenes and tumor suppressors that affect cancer initiation have been identified, cancer initiation is likely to be modulated by additional genetic events. Understanding how non-classical driver mutations may impact cancer initiation is a key issue that has been relatively underexplored. Melanoma, a highly aggressive skin cancer, arises through the acquisition of well-defined genetic and epigenetic modifications in oncogenes and tumor suppressors and represents an excellent model system to address this key question.

As a highly genetically unstable cancer type, the initiation of melanoma requires the induction of melanocyte proliferation, which is mediated by several major founder mutations, the most common of which are *BRAF*<sup>V600E</sup> and *NRAS*<sup>Q61K/R 1,2</sup>. However, activation of BRAF or NRAS is insufficient to promote melanoma initiation without senescence bypass mediated by additional founder mutations or expression changes of several genes including *p16<sup>INK4A</sup>*, *CTNNB1*, *PTEN*, or *MDM4*<sup>3-7</sup>.

The transcription factor BRN2, also known as POU3F2 and N-OCT3, plays a critical role in neurogenesis and drives proliferation in a range of cancer types with neural or neuroendocrine origins, including glioblastoma, neuroblastoma, small cell lung cancer, and neuroendocrine prostate cancer <sup>8-10</sup>. In the melanocyte lineage, BRN2 is not detected in melanoblasts *in vivo* but is heterogeneously expressed in naevi and melanoma <sup>11-14</sup>. *In vitro* studies have shown that BRN2 expression is induced by a range of melanoma-associated signaling pathways including activation of the mitogen-activated protein kinase (MAPK) pathway downstream from BRAF, the PI3K pathway, the LEF-β-catenin axis, as well as FGF, TNF-α, EDN3 and SCF
signaling <sup>14-17</sup>. Consistent with BRN2 being expressed in a predominantly mutually exclusive pattern with the Microphthalmia-associated transcription factor (MITF) <sup>13</sup> that plays a crucial role in melanoma proliferation <sup>18</sup>, BRN2 is repressed by MITF via miR-211 <sup>19</sup>. However, the relationship between MITF and BRN2 is complex. For example, BRN2 was recently found to be regulated by E2F1, a cell cycle-regulated transcription factor that is also a target for MITF, and both BRN2 and MITF are regulated by PAX3 and WNT/β-catenin <sup>15,16,20-24</sup>. Indeed, both BRN2 and MITF can regulate expression of AXL <sup>21,25</sup>, with BRN2 repressing AXL expression, thus enabling some cells in human melanoma to adopt an AXL<sup>High</sup>, MITF<sup>Low</sup> and BRN2<sup>Low</sup> state <sup>25</sup>. The activation of BRN2 expression in a specific subset of melanoma cells in response to all three major signaling pathways (MAPK, PI3K/PTEN, and β-catenin) linked to melanoma initiation (early proliferation and bypass/escape senescence) and progression (including late proliferation and metastatic dissemination) suggests that Brn2 is likely to have a critical role in disease progression <sup>26</sup>. Most notably, BRN2 has been associated with MITF<sup>Low</sup> senescent or slow-cycling cells <sup>11</sup>, and identified as a key regulator of melanoma invasion and anoikis *in vitro* <sup>13,27,28</sup> and *in vivo* xenograft experiments <sup>20,29,30</sup>. Mechanistically, the ability of BRN2 to promote invasion has been linked to its ability to control expression of PDE5A-mediated cell contractility, phosphorylation of myosin light chain 2, repression of MITF and PAX3, and cooperation with bi-allelic loss of CDKN2A <sup>13,20,27,30</sup>. However, despite abundant information linking BRN2 to melanoma proliferation and invasiveness *in vitro* and *in vivo* xenograft experiments, the impact of BRN2 on melanoma initiation and progression *in vivo* has never been assessed.
113 In this work, we show that BRN2 acts as a tumor suppressor during melanoma

114 initiation and progression in a *BRAF-PTEN* context since BRN2 and MITF regulate

115 positively and negatively the transcription of *PTEN*, respectively.

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117 RESULTS

118

119 BRN2 loss or low expression correlates with reduced survival and worse prognosis.

120 Although BRN2 has been implicated in melanoma invasiveness, and its expression is
121 highly regulated, whether and how it may contribute to melanoma initiation or
122 incidence is not understood. To evaluate the prevalence of *BRN2* loss in human skin
123 *cutaneous melanoma* (SKCM), we retrieved copy-number alteration (CNA) data for
124 *BRN2* in SKCM metastases (stage IV) from the Cancer Genome Atlas (TCGA,
125 https://cancergenome.nih.gov/). The *BRN2* locus showed mono-allelic loss in 53%
126 and bi-allelic loss in 2.7% of all patient samples (n = 367, Figure 1A). Only a minority
127 (n = 29 of 367, corresponding to 7.9%) of SKCM samples showed a copy-number
128 gain (n = 27)/amplification (n = 2) for *BRN2*. We did not further analyze these
129 samples since the expression of BRN2 was slightly increased but not statistically
130 different (p=0.26, test of Kruskall-Wallis with a Dunn correction) between tumors that
131 gained and/or amplified this locus compared to the normal situation. We screened a
132 panel of human melanoma cell lines available in our laboratory (n = 23) for deletions
133 that affect the *BRN2* locus by comparative genomic hybridization. The *BRN2* locus
134 showed mono-allelic loss in 48% (11 out 23) of the human melanoma cell lines and
135 no bi-allelic loss, comparable to the TCGA-data (Supplementary Fig. 1A,
136 Supplementary Data 1). Notably, BRN2 mRNA levels were significantly lower in
137 SKCM metastases with bi-allelic BRN2 loss (Supplementary Fig. 1B). The mono- and
138 bi-allelic loss of *BRN2* was frequently associated with a large segmental deletion of
139 the long arm of chromosome 6 (Chr.6q) in SKCM metastases and in our cell line
140 panel (Figure 1B, Supplementary Fig. 1C, Supplementary Data 2). From the TCGA,
141 patients carrying the monoallelic loss of *BRN2* in metastases displayed a trend to
142 have a shorter overall survival than those with diploid status (Figure 1C). These
143 results were validated using an independent cohort of 118 regional metastases
144 previously described ³¹ (Figure 1D). Moreover, we evaluated the number of BRN2
145 alleles in nevi and melanoma that arose from these nevi using publically available
146 data ³². It appears that 28% (5 out of 18) or 22% (4 out of 18) of melanomas
147 presented either a mono-allelic loss or a gain of *BRN2* respectively compared to nevi
148 (Supplementary Fig. 1D). The situation is clearly complex, but we may conclude that
149 *BRN2* mono-allelic loss can occur during the early steps of melanomagenesis.

150 We next assessed the correlation between BRN2 mRNA levels and overall
151 patient survival to evaluate the effect of *BRN2* mono-allelic loss on melanoma
152 progression. We established “BRN2-high” and “BRN2-low” patient groups based on
153 RNA-seq data available from the TCGA (BRN2 subgroups defined as BRN2-low (≤ 1
154 transcript per million reads [TPM]) and BRN2 expressed/high [> 1 TPM]). Patients in
155 the “BRN2-low” group displayed significantly shorter overall survival than those of the
156 “BRN2-high” group (Figure 1E). Overall, the *BRN2* locus, frequently associated with
157 a large segmental deletion, is lost (mono- and bi-allelic) in ≈60% of human SKCM
158 metastases and correlates with significantly reduced overall survival. Since *CCNC*,
159 *ROS1* and *ARID1B* loci are distal to *BRN2* on chromosome 6, and are known to be
160 involved in melanomagenesis, we evaluated the overall survival of these patients
161 according to the presence and the level of mRNA expression of the corresponding
162 genes. We observed no significant difference between the presence or absence of
163 these three genes or their expression (Supplementary Fig. 1E-L). Finally, we
164 compared the loss of 6q with the mono-allelic loss (MAL) of *BRN2* (Supplementary
Fig. 1M) and found that 6q loss is associated with a worse prognostic than *BRN2* mono-allelic loss. This result indicates, as suspected, that other gene(s) located on 165 166 6q are of importance in melanomagenesis. Taken together, these data indicate that 167 in human melanoma BRN2 loss/low expression is associated with an adverse 168 outcome for the patient. 169

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Co-occurrence of *BRN2* loss with mono-allelic loss of *PTEN* 171

We next determined whether BRN2 loss co-occurs with melanoma driver mutations 172 by examining the TCGA CNA-data set and human melanoma cell-line panel. There 173 was no significant correlation between *BRAF* or *NRAS* mutation and *BRN2* loss 174 (mono- or bi-allelic), neither in human melanoma samples nor the human cell-line 175 panel (Supplementary Fig. 2A,B). We then searched for co-occurring CNAs of other 176 known melanoma-associated genes and found that mono-allelic loss of *BRN2* co- 177 occurred with mono-allelic loss of *PTEN* in approximately 40% of the human 178 melanoma samples in TCGA and in the human cell-line panel (Supplementary Fig. 179 2C,D). We next evaluated the concomitant *BRN2* locus alterations, *BRAF/NRAS* 180 mutations, and *CDKN2A/PTEN* alterations and found the most frequent genetic 181 constellation that co-occurs with *BRN2* loss in melanoma to be *BRAF*<sup>V600X</sup> mutation 182 together with mono-allelic *PTEN* loss (Supplementary Fig. 2E). Finally, we compared 183 the overall survival of human patients with a loss of one allele of *PTEN* who also had 184 a loss of *BRN2* (monoallelic loss = MAL) with a loss of one allele of *PTEN* and no 185 loss of *BRN2* (BRN2-normal). In this context, patients with loss of *BRN2* showed 186 significantly lower overall survival than *BRN2*-normal patients (Supplementary Fig. 187 1N). 188
189
190 In conclusion, in human melanoma, the loss of *BRN2* is preferentially associated with
191 *BRAF* mutation together with *PTEN* loss.

192
193 Loss of *BRN2* drives melanomagenesis *in vivo*.

194 These data suggesting that the loss of *BRN2* might be of importance in melanoma
195 prompted us to evaluate the potential causal role of BRN2 in melanomagenesis *in
196 vivo* by examining whether heterozygous (het) or homozygous (hom) loss of *Brn2*
197 affects melanoma initiation and/or progression in a mouse model. Note however, that
198 while genetic loss of BRN2 might be important, the complex regulation of BRN2
199 expression driven by melanoma-associated signaling pathways might also play a
200 major role, especially given that melanoma cells within a single tumor can exhibit
201 both high and very low BRN2 expression <sup>13,20</sup>. We therefore developed an inducible
202 genetically engineered mouse model system for generating Brn2-deficient melanoma
203 driven by the most common alterations in human SKCM (Braf<sup>V600E</sup> and *Pten* loss).
204 Specifically, we used *Tyr::Cre<sup>ERT2/°-Lar</sup>; Braf<sup>V600E/+</sup>* (called Braf from hereon) and
205 *Tyr::Cre<sup>ERT2/°-Lar</sup>; Braf<sup>V600E/+</sup>; *Pten<sup>F/+</sup>* (called Braf-Pten from hereon) mice carrying a
206 tamoxifen-inducible Cre-recombinase under the control of the tyrosinase promoter <sup>33</sup>-
207 <sup>35</sup>. This model system allows melanocyte lineage-specific induction of a *BRAF<sup>V600E</sup>*
208 mutation and mono-allelic deletion of *Pten* for Braf-Pten mice. Cre-mediated
209 defloxing leads to activation of the Braf<sup>V600E</sup> oncogene, inducing nevus and
210 spontaneous melanoma formation in Braf mice, reproducing many of the cardinal
211 histological and molecular features of human melanoma <sup>36</sup>. Bi-allelic and mono-allelic
212 loss of *PTEN* reduces tumor latency in *Braf*<sup>V600E</sup>- and *NRAS*<sup>Q61K</sup>-driven mouse melanoma models <sup>3,37</sup>

214 Using these models, we studied the effect of Brn2 insufficiency (het and hom) on *in vivo* melanomagenesis by introducing the floxed Brn2 locus into the genome by appropriate crossings (Supplementary Fig. 3A) <sup>38</sup>. Specifically, we generated the mouse lines *Tyr::Cre<sup>ERT2/°</sup>; Braf<sup>V600E/+</sup>; Brn2<sup>+/+</sup>* (Braf-Brn2-WT), *Tyr::Cre<sup>ERT2/°</sup>*, *Braf<sup>V600E/+</sup>; Brn2<sup>F/+</sup>* (Braf-Brn2-het), *Tyr::Cre<sup>ERT2/°</sup>;; Braf<sup>V600E/+</sup>; Brn2<sup>F/F</sup>* (Braf-Brn2-hom), *Tyr::Cre<sup>ERT2/°</sup>;; Braf<sup>V600E/+</sup>; Pten<sup>F/+</sup>;; Brn2<sup>+/+</sup>* (Braf-Pten-Brn2-WT), *Tyr::Cre<sup>ERT2/°</sup>*, *Braf<sup>V600E/+</sup>; Pten<sup>F/+</sup>; Brn2<sup>F/+</sup>* (Braf-Pten-Brn2-het) and *Tyr::Cre<sup>ERT2/°</sup>;; Braf<sup>V600E/+</sup>; Pten<sup>F/+</sup>; Brn2<sup>F/F</sup>* (Braf-Pten-Brn2-hom). Cre-mediated defloxing of *Braf*, *Pten*, and *Brn2* loci was induced by topical application of tamoxifen during the first three days after birth (Supplementary Fig. 3B). All mice were monitored for the appearance and growth rate of the first tumor, as well as for the number of tumors/mouse. Note that the ability to generate either homo- or heterozygous Brn2 KOs will mimic not only mono or biallelic loss in humans, but also reflect the variable levels of BRN2 observed within human tumors <sup>13,20</sup>. In the absence of PTEN (*Pten<sup>F/F</sup>*) on a *Braf<sup>V600E</sup>* background, the appearance of the tumors was too rapid to observe any difference between Brn2 +/+, Brn2 F/+ or Brn2 F/F mice.

230 Braf-Brn2-WT/het/hom mice showed no differences in the appearance of the first tumor, number of tumors/mouse or the tumor growth rate from those of Braf-WT mice (Figure 2, Supplementary Fig. 3C). However, compared to Braf-Pten-Brn2-WT, Braf-Pten-Brn2-het/hom mice significantly increased the number of tumors/mouse and the tumor growth rate, but not the timing of the appearance of the first tumor (Figure 2, Supplementary Fig. 3D). We verified that *Brn2*, *Braf*, and *Pten* were
correctly defloxed in the resulting melanomas (Supplementary Fig. 3E,F). In summary, our data show that Brn2 acts as a tumor suppressor *in vivo*. As it has been shown in humans and mice, induction of early proliferation induced by the presence of the BRAF<sup>V600E</sup> mutation leads to senescence, but it can be bypassed when the level of Pten is reduced <sup>3,37,39,40</sup>. It is important to note that in the absence of Pten in the physiological context, proliferation of melanoblasts and melanocytes is not induced as the reduction/lack of Pten promotes proliferation once the cells are transformed <sup>3,41</sup>. On a Braf-Pten context the loss/reduction of Brn2 appears to induce melanoma initiation after promoting proliferation and bypass/escape of senescence, and then allows the tumor growth as was previously showed in vivo <sup>3,37</sup>.

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Reduction of BRN2 levels increases proliferation *in vivo* and *in vitro*.

The effect of *Brn2* loss on tumor growth prompted us to investigate whether *Brn2* loss increases intra-tumor proliferation. Staining sections for Ki-67, a marker of cycling cells, revealed that melanomas from Braf-Pten-Brn2-het/hom mice displayed a significantly higher number of Ki-67+ cells than Braf-Pten-Brn2-WT melanomas (Figure 3A,B). To confirm this result, we injected Braf-Pten-Brn2 mice with bromodeoxyuridine (BrdU) two hours prior to euthanization, to determine whether melanoma cells were slow or fast-dividing. Braf-Pten-Brn2 melanomas had a significantly higher number of BrdU+ cells when Brn2 was heterozygous or homozygous (Figure 3C,D). These results indicated that heterozygous loss of *Pten* combined with heterozygous/homozygous loss of *Brn2* promotes melanoma proliferation *in vivo*.
We next assessed whether Brn2 knockdown favors proliferation in vitro and whether this mechanism is conserved (i) between human and mouse and (ii) between transformed and non-transformed cells using Dauv-1 human melanoma cell line, and the Melan-a mouse melanocyte cell line. These cell lines express both Pten and Brn2 mRNA and protein (Supplementary Table 1). Dauv-1 cells carry a BRAF<sup>V600E/+</sup> mutation identical to that used in the mouse melanoma model system, and Melan-a cells are WT for Braf. siRNA-mediated knockdown of Brn2 significantly increased cell number 72 h after transfection of both cell lines (Figure 3E-G). Brn2 knockdown, assessed by western blot, led to an increase in cyclin D1 protein levels in both cell lines, but did not alter cyclin D1 mRNA levels, suggesting a regulation of cyclin D1 at the protein level (Figure 3F-H). Overall, the reduction of the Brn2 protein induces cell proliferation of the melanocytic lineage *in vivo* and *in vitro*.

Colony formation assays *in vitro* are classically used to show the importance of a gene in tumorigenesis, and indicate that a single cell may survive *in vitro* and proliferate to form a colony. Previous work has demonstrated that a reduction in Brn2 levels in melanoma cells has no effect on colony formation<sup>42,43</sup>. However, to test this in our model, mouse melanoma cell lines were established and characterized from the different independent Braf-Pten-Brn2 C57BL/6J mouse melanoma; the m50 and m6 cell lines were Brn2-WT, m59 and m36 were Brn2-het, and m82 and m8 were Brn2-hom (Supplementary Fig. 4A-F). The presence or absence of Brn2 did not decrease the ability of these melanoma cell lines to grow in syngeneic mice (Supplementary Table 2). In other words, it appears that the absence of Brn2 in these melanoma cells does not affect the implantation of the cells on the body wall, the proliferation after their transformation or the induction of angiogenesis in an
283 immunocompetent environment. We evaluated the capacity of the m50 (Brn2 WT),
284 m59 (het), and m82 (hom) cells to generate colonies and observed, in agreement
285 with previous observations obtained using BRN2 depletion, that the three cell lines
286 have similar abilities to form colonies (Supplementary Fig. 4G). Moreover, re-
287 expression of Brn2 using lentivirus infection in two independent m8 and m82 Braf-
288 Pten-Brn2-hom mouse melanoma cell lines, also indicated that BRN2 does not affect
289 their capacity to form colonies under these conditions (Supplementary Fig. 4H,I). In
290 conclusion, as shown in human cell lines, the number of colony forming units is
291 independent of the presence or absence of Brn2 and consequently this assay is
292 uninformative regarding the role of Brn2 in melanomagenesis.

293 We also evaluated the activity of BRAF<sup>V600</sup> (PLX4720), MEK (Binimetinib), and
294 PI3K (LY294002) inhibitors on the capacities of m50, m59 and m82 mouse
295 melanoma cell lines to form CFU and determined the IC50 of these drugs
296 (Supplementary Fig. 4J-O). Consistent with observations suggesting that BRN2 may
297 suppress cell death <sup>43</sup>, Brn2-het/hom cells are more sensitive than WT cells to these
298 three drugs. We also evaluated the cooperativity of PLX4720 and LY294002 using
299 various concentrations of each drug, but we did not observe any cooperation/synergy
300 of these two drugs.

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302 Mono-allelic loss of Brn2 induces melanoma metastasis.

303 We next evaluated the effects of Brn2 on metastasis formation in vivo. Since human
304 SKCM is known to spread to proximal lymph nodes (LNs), we assessed the presence
305 of pigmented cells in the inguinal LNs of tumor-bearing Braf-Pten-Brn2 mice.
306 Specifically, we estimated the volume of the various metastases present in LNs and
307 the number of pigmented areas after haematoxylin & eosin (HE) staining of LN
308 sections (Figure 4A-C). All Braf-Pten-Brn2 mice, irrespective of BRN2 status, showed
309 the presence of pigmented cells in both inguinal LNs. However, the LN volume of
310 Braf-Pten-Brn2-het mice was significantly higher than that of Braf-Pten-Brn2-WT and
311 Braf-Pten-Brn2-hom (Figure 4A,B). Similarly, Braf-Pten-Brn2-het LNs showed a
312 higher number of pigmented areas per mm² than Braf-Pten-Brn2-WT/hom mice
313 (Figure 4C).

314 To verify that the pigmented cells in the lymph nodes did not arise from cells in
315 which the Cre recombinase had not worked efficiently, we tested whether these
316 pigmented cells were properly defloxed for Brn2. The targeted Brn2-flox allele, used
317 in our mouse model, has an eGFP-cassette inserted downstream of the floxed Brn2
318 locus (Figure 4D). Thus the production of eGFP occurs once the upstream Brn2 gene
319 is defloxed. Consistent with correct defloxing of Brn2, pigmented areas of LNs
320 expressed GFP in Braf-Pten-Brn2-het/hom mice, but not Braf-Pten-Brn2-WT mice
321 (Figure 4E). The pigmented cells present in Braf-Pten-Brn2-WT and Braf-Pten-Brn2-
322 het LNs also expressed Sox10, a melanocytic marker (Figure 4E), that co-localized
323 with eGFP-expression in Braf-Pten-Brn2-het mice confirming the melanocytic origin
324 of the pigmented cells observed.

325 To get a better understanding of melanomagenesis in the various Brn2-
326 WT/het/hom situations, we performed a transcriptomic analysis of the various Brn2
327 tumors and cell lines (Supplementary Data 3 and 4). The ontology enrichment
328 analysis indicated that the Braf-Pten-Brn2-het tumors were more inflamed than the
329 Braf-Pten-Brn2-WT tumors with increased neutrophil-associated gene expression
330 (Supplementary Fig. 5A). It also suggests that the extracellular-matrix was actively
331 remodeled and that these Braf-Pten-Brn2-het tumors were more subject to
332 angiogenesis (Supplementary Fig. 5A). Pathway enrichment analysis supported
333 these results as inflammatory gene expression signatures are enriched from both the
334 WikiPathways and KEGG databases (Supplementary Fig. 5B,C). Significantly, the
335 PI3K/AKT pathway was enriched in the Braf-Pten-Brn2-het tumors compared to the
336 Braf-Pten-Brn2-WT in both WikiPathways and KEGG databases (Supplementary Fig.
337 5B,C), suggesting that AKT could be more phosphorylated in the Braf-Pten-Brn2-het
338 tumors. This assumption was verified by Western-blot analysis where
339 phosphorylation of AKT on S473, a surrogate of the AKT activity, and the
340 phosphorylation of S6 on S235/236 were significantly increased in Braf-Pten-Brn2-
341 het/hom tumors compared to Braf-Pten-Brn2-WT tumors (Supplementary Fig. 5D).

342 Gene Set Enrichment Analysis (GSEA) using the melanoma invasive
343 signature from Verfaillie 44 indicated that Braf-Pten-Brn2-het tumors and cell lines
344 were more invasive than Braf-Pten-Brn2-hom tumors and cell lines (Figure 4F).
345 Moreover, from the GO, WikiPathways and KEGG 2019 analyses of the mouse
346 melanoma tumors, the immune system (cytokine, neutrophil, macrophage) and
347 angiogenesis are induced in Braf-Pten-Brn2-het tumors compared to Braf-Pten-Brn2-
348 WT tumors (Supplementary Fig. 5A-C). This information suggests that Braf-Pten-
349 Brn2-het cells have more potential to metastasize than Braf-Pten-Brn2-WT and Braf-
350 Pten-Brn2-hom cells. In this respect, we tested the capacity of Braf-Pten-Brn2-hom
351 (m82) mouse melanoma cell lines re-expressing or not Brn2 (m82 and m82+Brn2) to
352 invade matrigel in 3D. In the presence of ectopic Brn2, the m82 Brn2 KO melanoma
353 cell lines have more ability to invade (Figure 4G and S4I). Since AXL, a receptor
354 tyrosine kinase (RTK), is associated with melanoma metastasis 45, we also evaluated
the level of Axl in mouse melanoma tumors and cells. Braf-Pten-Brn2-het cells produce slightly more Axl mRNA than Brn2-WT/hom (Figure 4H). This upregulation does not affect genes that are in *Cis* of Axl suggesting a specific regulation of Axl in a Braf-Pten-Brn2-het context (Supplementary Fig. 5E-G). Moreover, when the levels of both BRN2 and MITF are reduced in human melanoma cell lines, the level of AXL mRNA is induced (Supplementary Fig. 6D,H,L,P,T). More precisely the level of AXL is slightly, but significantly, higher in Gerlach and DAUV-1 cell lines in which the level of AXL is already very high, and is higher in SK28 and 501Mel cell lines in which the level of AXL is much lower than in Gerlach and DAUV-1 cell lines.

In conclusion, compared with Braf-Pten-Brn2-WT mice, melanoma initiation is promoted in both Braf-Pten-Brn2-het/hom mice, but metastasis is promoted only in Braf-Pten-Brn2-het mice. These observations are consistent with the fact that on a Brn2-het/hom background melanoma initiation (proliferation and bypass/escape of senescence) is induced and melanoma invasion and/or survival is inhibited on a Brn2-hom background.

BRN2 binds to the *PTEN* promoter and BRN2 loss leads to the reduction of *PTEN* transcription.

The PI3K-AKT pathway is induced in melanoma and its induction abrogates BRAF<sup>V600E</sup>-induced senescence <sup>46,47</sup>. The loss of *Pten*, a suppressor of the PI3K pathway, induces melanoma initiation and proliferation *in vivo* <sup>3,37</sup>. Since our Braf-Pten mouse melanoma model retained one functional allele of *Pten*, we hypothesized that *Brn2* loss would induce less expression from the WT *Pten* allele, leading to the increased PI3K-AKT signaling observed (Supplementary Fig. 5D) and consequent
379 melanoma initiation and proliferation. We therefore evaluated the number of Pten
380 positive cells in the various Braf-Pten tumors by immunohistochemistry and found
381 that Braf-Pten-Brn2-het/hom tumors showed fewer Pten-pos cells than Braf-Pten-
382 Brn2-WT tumors (Figure 5A). This result was verified by western blotting of Braf-Pten
383 tumor samples. The reduction of Brn2 correlates with the reduction of Pten in Braf-
384 Pten-Brn2-het tumors compared to the Braf-Pten-Brn2-WT tumors (Figure 5B). In
385 accordance with the reduced protein levels, the mRNA levels of Brn2 and Pten were
386 significantly lower in Braf-Pten-Brn2-het/hom tumors than in Braf-Pten-Brn2-WT
387 tumors, suggesting regulation of Pten at the transcriptional level (Figure 5C).

388 Next, we evaluated the mechanism of Pten repression mediated by reduced
389 levels of Brn2 in the human Dauv-1, Gerlach, SK28, and 501Mel melanoma cell lines
390 and in the non-transformed mouse Melan-a cell line. siRNA-mediated BRN2
391 knockdown led to significantly reduced PTEN protein and mRNA levels in cell lines
392 having high BRN2 expression (Figure 5D,E and Supplementary Fig. 6). In cells with
393 lower level of BRN2 (501Mel), the reduction of PTEN was not observed.

394 We examined the *PTEN* promoter for BRN2 binding sites conserved between
395 humans and mice to determine whether BRN2 acts directly on *PTEN* and detected
396 two potential Brn2 binding sites (BS1 and BS2) at the positions - 2,049 and +761
397 (numbering relative to TSS). Chromatin-immunoprecipitation (ChIP) for BRN2,
398 performed on Dauv-1 melanoma cell extracts, followed by qPCR, revealed
399 quantitative BRN2 binding to both binding sites, comparable to BRN2 binding on the
400 PAX3 promoter (Figure 5F-H). IgG was used as a negative control.

401 To evaluate Pten promoter activity in response to BRN2, we cloned a 3.2-kb-
402 human Pten promoter fragment containing the two BRN2 binding sites upstream the
403 luciferase gene to generate the hsPten::Luciferase reporter construct (Figure 5H).

404 The analysis of human PTEN promoter activity showed an intrinsic activity of

405 hsPten::Luciferase in human melanoma cells (Supplementary Fig. 7A,B). A

406 consistent repression of PTEN transcription was observed when a smart-pool of

407 siBRN2 was added. In contrast, PTEN transcription was activated following co-

408 transfection of a BRN2 expression vector compared to empty vector in Dauv-1 and

409 SK28 cell lines in luciferase assay (Figure 5I-L). We performed similar experiments

410 with the mouse PTEN promoter, which was cloned upstream of the luciferase

411 construct (Supplementary Fig. 7C-F); Brn2 activated Pten transcription in three

412 mouse melanoma cell lines (m82, m59 and m50) (Figure 5M,N). In conclusion, BRN2

413 directly induces PTEN transcription.

414

415 MITF binds the *PTEN* locus and represses *PTEN* transcription.

416 The *MITF* gene encodes a key transcription factor that plays a major role in

417 melanocyte and melanoma biology ¹⁸. Several studies have reported that *MITF*

418 transcription is directly repressed by BRN2 whereas a reduction in BRN2 levels leads

419 to increased MITF levels ¹³,²⁷,⁴⁸, while another study showed that BRN2 induces

420 MITF ⁴⁹. Likely both results are correct due to the versatile role of BRN2 as a

421 transcription regulator whose activity may be dependent on context including genetic

422 status and/or the environment ²¹,²⁶,²⁷.

423 Knowing the importance of MITF in the melanocyte lineage, we analyzed the

424 consequences of modulation of MITF expression on PTEN. Using siRNA-mediated

425 *MITF* knockdown in three human melanoma cell lines expressing high levels of MITF

426 (501mel, SK28, HBL) led to a significant increase of PTEN protein and mRNA levels
(Figure 6A,B). In cells expressing lower levels of MITF, such as Gerlach and Dauv-1, we did not observe an increase of PTEN (Supplementary Fig. 6C,G). Next, to determine if MITF directly regulates PTEN expression through proximal enhancers we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with antibodies to MITF, as previously described,⁵⁰ and to H3K27Ac, revealing active chromatin, and against H3K4me3, revealing active and poised promoters, in SK28 cell lines that were wild type for *MITF* (MITF-WT) or had loss of function mutations in all alleles of it (ΔMITF) (Figure 6C) ⁵⁰. An MITF peak is present within the gene body of *PTEN* (intron 4), and a second one is 114kb downstream of the *PTEN* transcriptional start site (TSS) (i.e., +114kb). Both MITF peaks were centered on an M-box motif (5'-TCATGTG-3'). At both intron-4 and +114kb MITF peaks, the H3K27Ac signal was unchanged in ΔMITF mutant cell lines. However, six distal enhancers (painted light blue) exhibited a 2-fold greater H3K27ac signal in MITF mutant cell lines compared to wild-type cell lines (+140kb, +210kb, +230kb, +253kb, +301kb and +317kb), suggesting increased transcription of *PTEN* in the former. We confirmed the +140kb MITF peak after performing a quantitative ChIP experiments on 501Mel cells expressing MITF-HA (Figure 6D,E), using Tyrosinase (TYR) and PRM1 as positive and negative controls respectively. Overall, reduction of MITF results in an increase in PTEN mRNA expression. As such, it is plausible that Brn2 induces *Pten* transcription through two different, but concurrent mechanisms: (i) directly through BRN2 binding to the *PTEN* promoter to induce its transcription and (ii) indirectly via BRN2 modulating (repressing/inducing depending of the situation) MITF expression, with MITF binding to the 3' end of *PTEN* to inhibit its transcription. In the absence of BRN2, these mechanisms are disrupted and *PTEN* transcription is
451 downregulated in our conditions. In conclusion, MITF represses directly *PTEN*
452 transcription.

453 Overall, our results are consistent with a model in which reduction of BRN2
454 reduces PTEN transcription *in vitro* and *in vivo*, thus ramping up PI3K signaling and
455 inducing both the initiation of melanoma and the formation of metastases.

456

457 DISCUSSION

458 A well-established principle of cancer biology is that tumors are initiated by a
459 combination of oncogene activation together with loss of tumor suppressor
460 expression or activity. In melanoma, key oncogenic drivers, such as BRAF and
461 NRAS, have been well defined. Loss of P16 or PTEN tumor suppressor activity is
462 required to bypass oncogene-induced senescence and permit melanoma initiation.
463 However, while inactivation of tumor suppressors by mutation has been extensively
464 studied, it is less well understood how their activity may be modulated by changes in
465 their mRNA expression mediated by key melanoma-associated transcription factors.

466 At this point, it is important to note that factors involved in melanoma initiation will not
467 automatically reflect/translate into overall survival. Here, we identify BRN2, a key
468 transcription factor lying downstream of three melanoma-associated signaling
469 pathways (WNT/β-catenin, MAPK, and PI3K), as a tumor suppressor that functions
470 to regulate PTEN expression. Thus, monoallelic loss of *Brn2* promotes melanoma
471 initiation in a *Braf*<sup>V600E</sup>*/Pten*<sup>F/+</sup> background where mono-allelic loss of *Pten* sensitizes
472 cells to loss of *Brn2*.

473 Previous work has primarily linked BRN2 to melanoma migration and invasion
474 *in vitro* and *in xenograft experiments*<sup>13,20,42,51</sup>, but its role during melanoma initiation
and proliferation in vivo and in normal melanocytes had not been determined. We
report that, consistent with BRN2 playing a key role as a tumor suppressor in
melanomagenesis, its locus is frequently lost in human skin cutaneous melanoma
(SKCM) metastases, independently of their NRAS or BRAF status, and that BRN2
status contributes to overall patient survival. Significantly, the overall survival of
patients with a mono-allelic loss of PTEN is higher when the BRN2 locus is intact.

Although these observations are consistent with BRN2 affecting human
melanoma initiation and progression, loss of the BRN2 locus is frequently associated
with large segmental deletions that affect the long arm of chromosome 6 (6q) as it
was observed in 14 out of 53, and 9 out of 32 out of primary melanoma and in 17 out
of 21 melanoma cell lines <sup>52-54</sup>, and confirmed in this study on a total of 205 out of
338 melanomas, and 11 out of 25 melanoma cell lines. It has already been shown
that the loss of 6q was associated with a worse prognosis <sup>52</sup>. According to our
observations, the loss of 6q is more detrimental for the overall survival than the focal
loss of the BRN2 locus (Fig. 1 and Fig. S1). Moreover, in vitro studies have shown
that several genes are linked to melanomagenesis in the co-deleted region, including
ARID1B, CCNC, and ROS1, although none have yet been shown to be functionally
important in melanoma in vivo <sup>48,55-61</sup>. Similarly, we analysed the SKCM TCGA study
to evaluate the overall survival comparing the mono allelic loss and the diploid state
for CCNC, ROS1 and ARID1B (Fig. S1). There is no statistical significance for CCNC
(p = 0.086), ROS1 (p = 0.27) or ARID1B (p = 0.66).

Thus, it might be argued that any of the genes located in this frequently
deleted region may be acting to modulate melanoma initiation or progression.
However, our functional mouse molecular genetics models show conclusively that
499 the heterozygous or homozygous loss of Brn2 promotes melanoma initiation and
500 initial growth. Similarly, our transcriptomic analysis showed that the mRNA levels of
501 the seven genes in the region co-deleted in humans (*Arid1b*, *Mchr2*, *Ccnc*, *Cdk19*,
502 *Dll1*, *Ros1*, and *Crybg1/Aim1*) were not affected in the mouse tumors (Braf-Pten-
503 Brn2-WT, Braf-Pten-Brn2-het and Braf-Pten-Brn2-hom). Collectively, these results
504 strongly suggest that the reduction/loss of Brn2 levels is a critical event that
505 cooperates with heterozygous Pten to promote the initiation and growth of
506 melanoma, independently of the co-deleted genes in melanoma. Finally, independent
507 initiation events are promoted when the level of Brn2 is lower than normal -
508 heterozygous or homozygous- since the number of independent melanoma is higher
509 in Braf-Pten-Brn2-het/hom mice than in Braf-Pten-Brn2-WT mice (Figure 2B). Our
510 observations are therefore consistent with BRN2 acting as a tumor suppressor in
511 melanoma, and are in full agreement with the predominantly mutually exclusive
512 pattern of BRN2 and Ki-67 *in situ* staining of invasive melanoma <sup>20</sup>.

513 The presence of Braf<sup>V600E</sup> promotes proliferation prior to inducing senescence
514 and the loss of Pten results in senescence bypass <sup>3,34,37</sup>. As such, we believe that the
515 increased proliferation and tumor-initiation frequency observed in our
516 Braf<sup>V600E</sup>/Pten<sup>F/+</sup> model arising as a consequence of the reduction of Brn2, is likely to
517 occur as a consequence of the ability of Brn2 to activate Pten expression and
518 suppress PI3K signaling either directly or potentially indirectly via Mitf repression
519 during early melanomagenesis. In other words, inactivation of Brn2 or a reduction in
520 its expression would lead to low expression of the remaining Pten allele and as a
521 consequence increase the probability of senescence bypass. This explanation fits
522 with the fact that in the absence of Pten on a Braf<sup>V600E</sup> background, the appearance
523 of the tumors is too rapid to observe any difference between Brn2 +/+, Brn2 F/+ or
524 Brn2 F/F. According to our model the transcription regulation by Brn2 and Mitf would
525 not affect the level of Pten since it is already lost. Moreover, in a context in which
526 Pten is diploid (WT) and Brn2 is reduced (het or hom), the downregulation of Pten
527 would be limited and not sufficient to act efficiently as a tumor-suppressor.

528 Although it might be argued that the increase in visible tumor number in a
529 Braf-Pten-Brn2-het and -hom might be a consequence of the increased proliferation
530 caused by reduction/loss of Brn2, as evidenced by the increased proportion of Ki-67
531 positive cells within tumours, we feel this is unlikely. For welfare reasons the Braf-
532 Pten-Brn2-WT mice were euthanized with tumours (total volume 2 cm³) after 4 weeks
533 with about 8 tumours per mouse; by contrast the Braf-Pten-Brn2-het/hom mice were
534 euthanized after 1.3 weeks with about 17 tumours per mouse of a similar size to the
535 WT. Since tumour size is similar in the WT and Brn2 mutants, this indicates that the
536 total number of cells in the WT and mutant tumours is similar and have undergone a
537 similar number of cell divisions (though this occurred in a shorter time in the mutant).
538 Since the WT and mutants have undergone a similar number of cell divisions, if
539 proliferation were responsible for any increase in number of visible tumours then at 4
540 weeks the numbers of tumours in the WT should be the same as in the mutant at 1.3
541 weeks, whereas in fact, the tumour numbers in the WT were around 50% of those in
542 the Brn2 mutants. Although we do not want to rule out a contribution of proliferation,
543 the more likely explanation is that the reduction of BRN2 promotes the bypass of
544 senescence (by reducing the level of Pten) or/and promotes survival of proliferating
545 melanoma cells at the early stages of initiation.
Our *in vivo* data therefore reveal that melanocyte-specific Brn2 reduction in Braf-Pten mice promotes the initiation and progression of melanoma. Melanoma initiation is promoted after proliferation is induced through various proteins including Mitf and senescence bypassed in this case through reduction of the level of Pten. Melanoma progression is induced by promoting invasion when the level of Brn2 is intermediate after inducing Axl and modulating the immune system. In the future, we will have to evaluate the kinase activity of Axl in this context, and the consequences of its inhibition *in cellulo* and *in vivo* to understand the Mitf/Brn2/Axl *ménage à trois*. Altogether, we establish Brn2 as a central tumor suppressor, acting at different steps of melanomagenesis, and complement numerous other studies showing the effect of Brn2 on invasion<sup>13-15,20,29,51</sup>.

*Brn2* heterozygous mice were more prone to form LN metastases efficiently than mice that were *Brn2* WT when Pten was already heterozygous. The efficiency of the metastasis process depends on the status of *Brn2* as the number and size of each micro-metastasis was greater in Brn2 heterozygous than Brn2 wild-type melanoma. This is important as mono-allelic loss of *BRN2* in human, corresponding to *BRN2* heterozygous melanoma, occurs in 53% of human melanoma. In *Brn2* wild-type melanoma, the change of the level/activity of Brn2 is possible but the amount of protein found in Brn2 wild-type melanoma absorbs better transient Brn2 depletion than in *Brn2* heterozygous melanoma.

Some residual Brn2 activity might be required for efficient melanoma progression. On one hand, Braf-Pten-Brn2-het and Braf-Pten-Brn2-hom melanomas proliferate faster, and on the other hand, according to our results (Figure 4F,G) Braf-Pten-Brn2-hom melanomas have a reduced ability to invade compared to Braf-Pten-
570 Brn2-het melanomas. Moreover, the lack of BRN2 reduces migration and increases
571 the rate of apoptosis and/or anoikis<sup>20,28,43</sup>. In a more speculative way, one may think
572 that optimal melanoma progression may be associated with a series of proliferative
573 and invasive phases. In the absence of BRN2, cells are “fixed” in one stage and
574 cannot switch from “proliferative” to “invasive” states. These cells are mainly
575 proliferative and poorly invasive as we observed in Braf-Pten-Brn2-hom situation. In
576 the presence of Brn2 (as WT [two alleles] or heterozygous [one allele], the
577 corresponding mRNA and protein concentrations can be positively and negatively
578 modulated by external/internal factors. The modulation of the level of Brn2 is more
579 sensitive on a heterozygous background than on a WT background.

580 Altogether, one may understand that melanoma grows faster and forms
581 melanoma when Brn2 expression is not too high (proliferation handicap) but at the
582 same time not too low (migration/invasion/survival handicap). Indeed, on the one
583 hand, Brn2-het and Brn2-hom melanomas proliferate faster, and on the other hand,
584 Brn2-hom melanomas are handicapped to invade (Figure 4F,G), to migrate<sup>20</sup> and to
585 die by anoikis<sup>28</sup>.

586 The formation of metastases is a multistep process in which cells proliferating
587 in the primary tumor, and surviving the metastatic process, must undergo a switch to
588 an invasive phenotype prior to a switch to a proliferative phenotype on site. Since the
589 switch from proliferation to invasion, and back, has been associated with the activity
590 of MITF, it is possible that for efficient metastatic colonization cells must be able to
591 modulate MITF expression via BRN2. In this respect, it is especially important to note
592 that BRN2 has been identified as a key regulator of MITF<sup>13,49</sup>. Consistent with the
593 observation that MITF and BRN2 are frequently observed in mutually exclusive
populations in melanoma, and that BRN2 may act *in vivo* as an MITF repressor ¹³,
we have observed that in a non-tumoral context, the specific knock-out of Brn2 *in
vivo* in melanocytes increases the level of Mitf (publication in preparation).
Importantly, during progression of mouse Braf-Pten melanoma Mitf levels are
modulated. During the initial phase of growth, melanoma cells are pigmented,
indicative of Mitf activity, but later *in situ* they lose pigmentation and ability to produce
Mitf (⁶² and not shown). Although the reduction of Brn2 in Braf-Pten-Brn2 primary
melanoma is not sufficient to re-induce Mitf mRNA and pigmentation in all cells of
these primary melanomas, the Braf-Pten melanoma cells that formed LN metastases
were pigmented and re-expressed Sox10, a key transcription activator of Mitf. Thus,
during progression of Braf-Pten melanomas Mitf is produced during initial growth and
subsequently repressed during the second phase, before being re-expressed in LN
metastases. It seems therefore likely that one role of the residual BRN2 in the
heterozygotes may be to facilitate the modulation of MITF expression during
metastatic spread.

It has been shown that BRAF and PI3K induce BRN2 ¹⁵,¹⁶. In consequence, it
was expected that the level of BRN2 would decrease in the presence of such
inhibitors. In addition, in the presence or absence of BRN2, melanoma cells are
either more resistant or more sensitive to BRAF inhibitors, respectively ²⁸,⁴³. This
sensitivity would be associated with the function of BRN2 in DNA repair ⁴³.

Although here we have focused on the role of BRN2 in melanoma, BRN2 is
also expressed in a number of other cancer types including small cell lung cancer,
neuroblastoma, glioblastoma and neuroendocrine prostate cancer ⁸⁻¹⁰. While BRN2-
mediated regulation of MITF is not likely to be important for non-melanoma cancers
618 that do not express MITF, the ability of BRN2 to modulate PTEN expression
619 uncovered here may play an equally important role in promoting the initiation and
620 progression of these cancer types. In this respect the inducible knockout mice
621 described here may represent an important tool to examine the role of BRN2 in non-
622 melanoma cancers.

623 In conclusion, our results identify Brn2 as a key tumor suppressor through its
624 ability to modulate Pten expression that, given the high prevalence of monoallelic
625 mutations, is likely to play a key role in initiation of human melanoma and likely other
626 BRN2-expressing cancer types. Since BRN2 expression is activated by PI3K
627 signaling via PAX3 ¹⁶, its ability to suppress PI3K signaling by increasing PTEN
628 expression may also provide cells with a negative feedback loop to control the PI3K
629 pathway. Moreover activation of BRN2 by MAPK signaling downstream BRAF ¹⁵ as
630 well as WNT/β-catenin signaling ¹⁴, may also permit coordination between these
631 pathways and the PTEN/PI3K axis. Finally, given the importance of BRN2 in
632 melanomagenesis identified here as well as its frequent heterozygosity, it may be
633 important to further explore whether tumors with low BRN2 expression may be more
634 susceptible to PI3K pathway inhibition, as it was shown for MAPK inhibitors. In this
635 respect, this mouse model of BRN2-deficient melanoma could be useful for the pre-
636 clinical testing of inhibitors for clinical development especially since it has been
637 shown that BRN2 is involved in DNA repair ⁴³.

638
639 Figure legends

640

641 Figure 1. One *BRN2* allele is frequently lost in human melanoma and reduced BRN2
642 mRNA level correlates with reduced overall survival.

643 (A) Bar graph showing the status of the *BRN2* locus in human skin cutaneous
644 melanoma (SKCM) metastases (stage IV). Copy-number alterations (CNAs) were
645 estimated using the GISTIC algorithm. Two alleles (2a in black), one allele (1a in
646 red), no allele (0a in orange), and gain and/or amplification (G/Aa in blue) of the
647 *BRN2* locus are given.

648 (B) Pictogram showing the extent of segmental deletions (red or orange vertical lines)
649 that affect the *BRN2* locus on Chr.6q16 (dashed blue horizontal line) in SKCM
650 metastases.

651 (C) Kaplan-Meier curves comparing 10-year overall survival of SKCM patients diploid
652 for BRN2 (black line, n = 106) or those with mono-allelic (red n = 156). The TCGA
653 CNA-data set was analyzed (n = 309). Diploid vs. Mono-allelic loss: log-rank (Mantel-
654 Cox) test (p = 0.067). Data were retrieved from TCGA on August 8, 2019.

655 (D) Kaplan Meier curves comparing melanoma patients with diploid status or mono-
656 allelic loss of *BRN2* in 118 regional metastatic melanoma patients (p = 0.003, log-
657 rank test) ³¹ and unpublished data.

658 (E) Kaplan-Meier curves comparing 30-year overall survival of SKCM patients to
659 BRN2 mRNA levels. Log-rank (Mantel-Cox) test (p = 0.03). Data were retrieved from
660 TCGA on August 8, 2019. Significance was defined as * (p < 0.05) and *** (p≤0.001).

661

662 Figure 2. Brn2 loss potentiates melanomagenesis in Braf-Pten mice.
663 (A) Macroscopic pictures of the dorsal view of mice with cutaneous melanomas
664 carrying mutations in the melanocyte lineage for Braf, Pten, and Brn2 after tamoxifen
665 induction at birth (p1, p2, and p3 – see Supplementary Fig. 3). *Tyr::CreER<sup>T2/α</sup>;
666 *Braf<sup>V600E</sup>/*; Pten<sup>+/+</sup> (= Braf), Pten<sup>F/+</sup> (= Pten), Brn2<sup>+/+</sup> (= Brn2-WT), Brn2<sup>F/+</sup> (= Brn2-het),
667 and Brn2<sup>F/F</sup> (= Brn2-hom). Tumors are highlighted with arrows and the sizes of the
668 first growing tumors to appear are proportional to the diameters of the circles. F
669 means floxed allele.

670 (B) All Braf (n = 9), Braf-Brn2-het (n = 8), and Braf-Brn2-hom (n = 4) mice produced
671 cutaneous melanomas and their number was similar (1 to 2 tumors/mouse). All Braf-
672 Pten-Brn2-WT (n = 7), Braf-Pten-Brn2-het (n = 21), and Braf-Pten-Brn2-hom (n = 11)
673 mice produced cutaneous melanomas. Note that in the absence of Pten (*Pten<sup>F/F</sup>), the
674 appearance of the melanoma was too rapid to observe any difference between Brn2-
675 WT, Brn2-het, and Brn2-hom mice. Each dot corresponds to an individual mouse. As
676 control, mice of different genetic backgrounds were produced and not induced with
677 tamoxifen (Braf [n = 12], Braf-Brn2-het [n = 25], Braf-Brn2-hom [n = 11], Braf-Pten [n
678 = 7], Braf-Pten-Brn2-het [n = 13], and Braf-Pten-Brn2-hom [n = 6]; none of them
679 developed melanoma after 18 months, except one Braf-Pten-Brn2-het mouse that
680 developed one melanoma after 12 months. None of the mice that were wild-type for
681 *Braf* displayed any obvious phenotype, irrespective of the status of *Pten* or *Brn2*,
682 including melanomagenesis and hyperpigmentation.

683 (C) Growth rates of the first tumor appearing in each mouse for Braf-Brn2-WT, Braf-
684 Brn2-het, Braf-Brn2-hom, Braf-Pten-Brn2-WT, Braf-Pten-Brn2-het, and Braf-Pten-
685 het-Brn2-hom mice. The number of tumors is determined all along the life of the
686 mouse by checking the mice a minimum of twice a week. Statistical analysis was
performed using the two-tailed unpaired t-test. ns = non-significant, \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001. Data are presented as mean values +/- SEM. Braf-Pten-Brn2-het mice were euthanized in average 1.3 weeks after appearance of the first tumors with an average of 16 tumors/mouse. Similar results were obtained with Braf-Pten-Brn2-hom mice. Braf-Pten-Brn2-WT mice were euthanized at 4 weeks with an average of 8 tumors even though they did not reach a total volume of 2cm³ except for one mouse that was euthanized earlier (three weeks).

Figure 3. BRN2-het/hom induces proliferation *in vitro* and *in vivo*.

(A-D) Representative photomicrographs of Ki-67 (A) and BrdU (C) stainings of Braf-Pten-Brn2-WT/het/hom tumors. Ki-67+ cells are stained in red. Nuclei are stained in blue. Scale bar = 40 µm. Three independent tumors for each genotype were used for these experiments and three independent sections were used for each tumor. A 2-way ANOVA with Dunnett's multiple comparisons tests were performed. Ki-67+ (B) and BrdU (D) stainings of (A) and (C), respectively. Scale bar = 40 µm. Each dot represents the result for one tumor.

(E) Growth rate is induced in Dauv-1 and Melan-a cell lines after reduction of Brn2 using siBRN2 and siScr as control (Scr = scramble). Three independent biological and technical experiments were performed for each cell line and for each condition.

(F-H) Brn2 knock down induces Cyclin D1 protein but not its mRNA in melanocytes.

(F) Western blot analysis for Brn2, Cyclin D1, and actin after reduction of Brn2 in Dauv-1 and Melan-a cells. Experiments were performed independently three times.

One representative western blot is shown (raw data are presented in Supplementary Fig. 8). (G,H) Quantification of protein (G) and mRNA (H) levels for Dauv-1 cells after
711 siRNA-mediated knockdown (n = 3, independent experiments). For the proteins, all
712 values were normalized against the background and corresponding actin loading
713 control for each sample. Quantification was performed using *Image-J* software. For
714 mRNA, all values were normalized against those of TBP. au = arbitrary units.
715 Statistical analysis was performed using the two-tailed unpaired (B,D,E,H) and paired
716 (G) t-tests. ns = non-significant, \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001. Data are
717 presented as mean values +/- SEM.

718

719 Figure 4. Mono-allelic loss of *Brn2* induces melanoma metastasis.

720 (A) Upper panel: Representative photomicrographs of *in situ* inguinal lymph nodes
721 (LN) of Braf-Pten-Brn2-WT/het/hom mice. Scale bar = 1 mm. The pigmented volume
722 (mm³) was estimated for each LN. Lower panel: Representative photomicrographs of
723 haematoxylin & eosin (H&E) staining of LNs containing pigmented cells. Scale bar =
724 20 µm.

725 (B) Quantification of the pigmented volume of inguinal LNs in the upper panel of
726 Figure (A). n = 3, 5, and 5 for WT, het, and hom.

727 (C) Quantification of the pigmented areas per mm² of inguinal LNs in the lower panel
728 of Figure (A). Pigmented areas > 50 µm² were considered. n = 3, 5, and 3 for WT,
729 het, and hom.

730 (D) Scheme showing the defloxing strategy of Brn2 in melanocytes of the primary
731 tumor that releases eGFP expression upon the defloxing of *Brn2*.

732 (E) Representative photomicrographs, from four independent experiments, of serial
733 LN sections of Braf-Pten-Brn2-WT and Braf-Pten-Brn2-het mice stained with H&E
734 and the melanocyte marker Sox10. H&E staining was evaluated for one section and
735 GFP (green channel) and Sox10 staining (red channel) evaluated for an adjacent
736 section. Scale bar = 20 µm.

737 (F) A melanoma invasive signature was significantly enriched in the Braf-Pten-Brn2-het tumors (left) and in the Braf-Pten-Brn2-het melanoma cell lines (right) compared to their Braf-Pten-Brn2-hom counterparts.
738
739

740 (G) Left: photomicrographs of m82 and m82-BRN2 mouse melanoma cells
741 embedded as spheroids in 600 µg/mL matrigel at t0 (H0) and 18 hrs after (H18).
742
743 Right: Boxes and plots represent the area of invasion (red lines on
744 photomicrographs) quantified with ImageJ (n=54 for m82 cells and n=56 for m82-
745
746 BRN2 cells). P-value<0.0001. au = arbitrary unit.

745 (H) Axl mRNA is significantly overexpressed in Braf-Pten-Brn2-het melanoma and
746 melanoma cell lines (n = 10 and 2, respectively) compared to the Braf-Pten-Brn2-WT
747 (n = 5 and 4, respectively) and Braf-Pten-Brn2-hom (n = 10 and 3, respectively)
748 tumors.

749 Statistical analysis was performed using the two-tailed unpaired t-test for B, C, G and
750 H (tumors) and an Anova test for H (cell lines). Data are presented as mean values
751 +/- SEM for B and C, sd for H, and Box and whiskers (median, min to max) for G. ns
752 = non-significant, *p < 0.05, **p < 0.001, ***p < 0.0001.

753
754 Figure 5. Brn2 binds to the Pten promoter and Brn2 loss leads to Pten transcription
755 reduction.

756 (A) Representative photomicrographs of immunohistochemistry staining of Pten (red)
757 in Braf-Pten-Brn2-WT and Braf-Pten-Brn2 mouse melanomas are shown. Scale bar =
758 40 µm. Three independent tumors for each genotype were used for these
759 experiments and three independent sections were used for each tumor. A 2-way
760 ANOVA with Dunnett's multiple comparisons tests were performed. The percentage
761 of Pten⁺ cells in WT and mutant tumors is shown.

762 (B) Western blot analysis of Brn2, Pten and actin for Braf-Pten-WT and Braf-Pten-Brn2 from at least three tumors of each genotype. One representative example is
763 presented, raw data are presented in Supplementary Fig. 8. The relative intensities of
764 the band were estimated with ImageJ.
765

766 (C) RT-qPCR of Brn2 and Pten from Braf-Pten-WT and Braf-Pten-Brn2 melanomas.
767 Three independent mouse melanomas per genotype were analyzed. Data were
768 normalized against the values of Gapdh. au = arbitrary unit.

769 (D) Western blot analysis of BRN2, PTEN and ACTIN from Dauv-1 human melanoma
770 cells and Melan-a mouse melanocytes after siRNA mediated knockdown. A
771 representative western blot is shown, raw data are presented in Supplementary Fig.
772 8. Scr = Scramble.

773 (E) RT-qPCR of BRN2 and PTEN from human melanoma cells (Dauv-1) and mouse
774 melanocytes (Melan-a) after siRNA-mediated knockdown. Specific primers were
775 used for human and mouse samples. Dauv-1 (n = 6), Melan-a (n = 4), independent
776 experiments. Data were normalized against the values for TBP (Dauv-1) or Gapdh
777 (Melan-a).

778 (F) ChIP assays of BRN2 binding to the *PTEN* promoter in Dauv-1 melanoma cells.
779 All data shown are representative of at least three independent assays.

780 (G) Quantification of the ChIP-qPCR, plotted and normalized against IgG as the
781 reference. au = arbitrary unit. n=6, 3, 4 and 3 for BRN2 CDS, PAX3 prom, BS1 and
782 BS2, respectively.
783 (H) Scheme of the human *PTEN* promoter containing two BRN2 binding sites (BS)
784 represented as colored circles. Note that BS are conserved between humans and
785 mice. TSS = transcription start site. Exons (X) 1 and 2 are shown as horizontal
786 rectangles. The translation start site (ATG) and the end of exon 1 are indicated. All
787 numbering is relative to the TSS (+1). Representation of the reporter luciferase (luc)
788 construct with or without *PTEN* promoter.

789 (I-N) Human and mouse *PTEN* promoter activities were evaluated in human Dauv-1
790 (I,K), SK28 (J,L), and in mouse m82 [Brn2-hom], m59 [Brn2-het], and m50 [Brn2-WT]
791 (M,N) melanoma cell lines either in the presence of siScr (scramble) or siBrn2
792 (smart-pool) (I,J,M) or in the presence of expression vector of BRN2 (CMV::BRN2)
793 (L,N). The experiments were independently performed four (I,J,K) and three (L)
794 times. They were performed three independent times for m82 and m50, seven for
795 m59 (M), and seven times for m82, six for m59 and four for m50 (N).

796 Statistical analysis was performed using the two-tailed unpaired t-test for C, E, G and
797 paired t-test for I-N. Data are presented as mean values +/- SEM for C, E, G, I-M.
798 Box and whiskers (median, min to max) for N. ns = non-significant, *p < 0.05, **p <
799 0.001, and ****p < 0.0001.

800
801 Figure 6. MITF binds downstream of *PTEN* gene, MITF loss induces its transcription,
802 and enhancers flanking *PTEN* are activated in MITF-depleted cells.

803 (A) Western blot analysis of MITF and PTEN from human melanoma cells (501mel,
804 SK28, and HBL) after siRNA-mediated knockdown. Actin was used as a loading
805 control. A representative western blot out of three is shown, raw data are presented
806 in Supplementary Fig. 8. The molecular weight is indicated in kDa. Scr = Scrambled.
807 (B) RT-qPCR of MITF and PTEN from human melanoma cells (501mel and SK28)
808 after siRNA-MITF and Scr knockdown. All values were normalized against TBP. The
809 analysis was performed on three independent experiments with technical triplicates,
810 au = arbitrary unit.

811 (C) Screenshot of IGV genome browser (GRCH37/hg19) visualization of MITF,
812 H3K27Ac and H3K4me3 binding to the *PTEN* locus in SkMel28 cell lines that are
813 MITF-WT or mutant (i.e., MITF-ΔX6 = ΔMITF). Blue boxes below MITF and
814 H3K27Ac tracks: signal above IgG background (i.e., peaks) called by MACS2. PTEN
815 and downstream regions are shown, blue arrows indicate strand orientation and
816 horizontal rectangles the exons. Y-axes are scaled per antibody sample. Anti-MITF
817 CUT&RUN peaks present in WT cells that harbor an M-Box binding motif are painted
818 grey. Six distal enhancers (painted light blue) exhibited a 2-fold greater H3K27ac
819 signal in MITF mutant cell lines compared to wild-type cell lines. At least two
820 CUT&RUN biological replicates were performed for MITF, H3K27ac and H3K4me3.

821 (D) ChIP assays of MITF binding downstream of *PTEN* in 501mel human melanoma
822 cells stably expressing HA-Tagged MITF (location +140kb). ChIP assays are
823 performed using an antibody against HA and analyzed after a 30-cycle PCR
824 (exponential phase). The tyrosinase promoter (*TYR*) and *PRM1* were used as
825 positive and negative controls, respectively. Input represents approximately 3% of
826 the input used for the ChIP assay. H3 (histone H3) and IgG (Immunoglobulin G) were
827 used as positive and negative technical controls for each region of interest,
828 respectively. The oligonucleotides, their positions on the genome, and sizes of the
829 amplified fragments are shown in Supplementary Tables 3 and 4. All data shown are
830 representative of three independent assays. * corresponds to the remaining
831 oligonucleotides.

832 (E) Quantification of the ChIP-qPCR presented in (D) is plotted and normalized
833 against IgG as a reference. au = arbitrary unit. PRM1 (-): PRM1 (negative control),
834 TYR (+): tyrosinase promoter (positive control).

835 Statistical analysis was performed using the two-tailed unpaired t-test.

836 Data are presented as mean values +/- SD. ns = non-significant, *p < 0.05, **p <
837 0.01, and ***p < 0.001.

838
839 METHODS

840 Detailed materials and methods are given in the supplemental information.

841

842 *TCGA data*

843 All TCGA data for somatic mutations, copy number alterations (CNAs), RNA-seq
gene expression, and clinical data for skin cutaneous melanoma were retrieved from
the National Cancer Institute (NCI) Genomic Data Commons (GDC) thorough its
GDC Application Programming Interface (API) using the TCGAbiolinks package or
the CGDS-R package to query the Cancer Genomic Data Server (CGDS) from
cBioPortal.

849

850 *Copy number analysis (CNA)*

851 Preprocessed copy number data was obtained from cBioportal. Copy number data
sets generated by the GISTIC 2.0 algorithms, specifically the
allthresholded.bygenes.txt of the GISTIC output were used. Samples with GISTIC
copy-number values of "-1" were considered as mono-allelic loss (red) and samples
with GISTIC copy-number values of "-2" as bi-allelic loss (orange). TCGA dataset
CNA-data set (n = 367). BRN2 CNA classified as gain/amplification (GISTIC > "+1" or
"+2") are not shown (n = 29, 7.9%).

858

859 *Level of expression of BRN2 mRNA in melanoma patients*

860 BRN2 mRNA levels were obtained from RNA sequencing data analyzed using the
RNA Seq V2 RSEM pipeline as transcripts per million reads (TPM).

862
863 **Mouse Models**

864 Mice were bred and maintained in the specific pathogen-free mouse colony of the
865 Institut Curie, in accordance with the institute's regulations and French and European
866 Union laws. Experimental procedures were specifically approved by the ethics
867 committee of the Institut Curie CEEA-IC #118 (CEEA-IC 2016-001) in compliance
868 with the international guidelines. The transgenic *Tyr::Cre<sup>ERT2-Lar</sup>* (also called
869 *Tyr::Cre<sup>ERT2</sup>*, *Braf<sup>V600E</sup>*, *Pten*, and *Brn2* mice have been described, characterized,
870 and backcrossed onto a C57BL/6 background for more than ten generations <sup>33</sup>-
871 <sup>35,38,63</sup>. Genotyping were performed accordingly (Supplementary Tables 3 and 4).
872
873 In vivo gene activation/deletion and melanoma monitoring
874 Newborn mice were treated dorsally with 0.4 µg/day/ouse tamoxifen from P1 to P3.
875 Non-tamoxifen-induced mice of the same genotype were used as controls.
876 Developing skin excrescences > 3 mm diameter were considered to be melanomas,
877 and validated after growth. Mice were checked two-three times a week to estimate
878 the volume of the tumors. They were euthanized and autopsied four weeks after
879 tumor appearance or once the tumors reached 2 cm³. Mouse melanomas were
880 excised, rinsed in cold PBS snap-frozen in liquid nitrogen for subsequent
881 transcriptomic and western blot analysis and fixed in 4% PFA and embedded in
882 paraffin or OCT for histological analysis and immunostaining.
883
884 **Immunohistochemistry of mouse melanoma and inguinal lymph nodes**
885 Paraffin-embedded mouse melanomas were sectioned into 7-µm-thick transverse
886 sections and stained with hematoxylin/eosin (H&E), as previously described <sup>64</sup>.
887 Antibodies are from Nova-Costra for Ki-67, BD Biosciences for BrdU, from Abcam for
888 SOX10 and Cell signaling for PTEN. Images were captured using a ZEISS Axio
889 Imager 2 with Axiocam 506 color cameras. Image analysis was performed using
890 ZEISS ZEN, Adobe Photoshop, and ImageJ software. Quantifications of Ki-67 and
891 BrdU stainings were determined as a percentage. The percentage of Ki-67+ and
892 BrdU+ cells from three fields (1,000-2,000 cells/field) from three independent tumors
893 per genotype was determined and normalized.

894

895 Protein extraction from tumors

896 All steps were performed at 4°C. Tissues were transferred to a tube containing 2.8-
897 mm stainless steel beads and 1mL RIPA buffer supplemented with sodium
898 orthovanadate prior adding complete protease inhibitor and PhosSTOP (Roche).
899 Tissues were homogenized followed by centrifugation at 17,949 g for 1 min.
900 Supernatants were transferred and centrifuged for 20 min at 15,294 g. Supernatants
901 were collected and incubated with 200µL previously PBS-washed G-Sepharose
902 beads for 2h. Samples were centrifuged at 15,294 g for 5 min and quantified using
903 the Bradford assay.

904

905 Microarray analysis

906 Only one tumor per mouse was considered and corresponding to the biggest one.
907 Tumors had the same size since we harvested tumors for transcriptomic analyses
908 when they reached a size of 1cm³. RNA from mouse melanomas (5 Braf-Pten-Brn2-
909 WT, 10 Braf-Pten-Brn2-het, and 10 Braf-Pten-Brn2-hom) and mouse melanoma cell
910 lines (4 Braf-Pten-Brn2-WT, 2 Braf-Pten-Brn2-het, and 3 Braf-Pten-Brn2-hom)
established from the mouse melanoma tumors was extracted using the miRNeasy Kit (Qiagen, #217004). Complete protocol can be found in the supplementary information.

In brief, 11µg cRNA hybridized to mouse MOE430 gene expression Affymetrix microarrays (Affymetrix, #900443). The microarray data were normalized using the RMA (Robust Multichip Average) function of the edgeR package ⁶⁵. Differentially expressed gene analysis was performed with R, for the cell lines and the tumors using the limma package ⁶⁶ available from Bioconductor (http://www.bioconductor.org). An enriched gene-ontology (Gene-Ontology Biological-Process, 2018) and pathway (WikiPathways human, 2019 & KEGG human, 2019) analysis was performed on the 296 genes found overexpressed in the Braf-Pten-Brn2-het compared to the Braf-Pten-Brn2-WT using Enrichr ⁶⁷. Gene-set enrichment analysis (GSEA) was performed using the “Verfaillie” signature ⁴⁴.

### Cell lines

Melan-a and C57BL/6 9v cells were grown as previously described ⁴,⁶⁸. 501mel, 501mel-MITF-HA, HBL, SK-Mel-28 (SK28), Gerlach and Dauv-1 cells were grown in RPMI 1640 media supplemented with 10% FCS and 1% Penicillin-Streptomycin ⁶⁹⁻⁷¹. SK28Δex6 melanoma cells lacking Mitf was previously produced ⁵⁰. Cells are routinely tested for the absence of mycoplasmas using MycoAlert (Lonza). The origin and the main genetic characteristics of the cell lines are given in the supplemental information. Mouse melanoma cell lines were established as previously described ⁷².

### Genomic DNA extraction and genotyping from cell culture
935 Genomic DNA was extracted from melanoma cell lines and column-purified with the
936 QIAamp Kit (QIAGEN). See Supplementary Tables 3 and 4.

937
938 siRNA-mediated knock-down

939 siRNA targeting human BRN2 and MITF was purchased from Dharmacon as a
940 SMART-pool mix of four sequences. siRNA targeting PTEN from Santa Cruz Biotech.

941 Si Scramble (siSCR), with no known human or mouse targets, was purchased from
942 Eurofins Genomics (Supplementary Table 5). Briefly, cells were transfected with
943 Lipofectamine 2000 with 25-200 pmol siRNA or siSCR (according to the types of
944 experiments) and assayed for mRNA expression, protein content or luciferase activity
945 48 or 72 h post-transfection.

946
947 Clonogenic assays

948 For clonogenic assays, six-well tissue culture plates were seeded with 500 cells in
949 complete medium. The medium was changed 24 h after cell seeding and replaced
950 with complete medium containing the indicated concentrations of Binimetinib
951 (Selleck), MK-2206 2HCl (Selleck), LY294002 (Calbiochem) or PLX4720 (Axon
952 Medchem). After 9 days of incubation, colonies were fixed with 4% PFA
953 (paraformaldehyde), stained with Crystal violet in 10% ethanol, and counted on
954 images. IC50 were determined for each pharmacological agent and for each cell line
955 using the number of colonies in mock treated condition as the top response. Cell
956 lines have been treated with Binimetinib from 10⁻³ to 100 µM, with PLX4720 from 10⁻³
957 to 100 µM, with LY294002 from 5.10⁻³ to 50 µM and with MK-2206 from 10⁻³ to 10
958 µM. We used the resulted sigmoidal curves to calculate the IC50 with Graphpad
959 Prism. Experiments were performed at least in triplicate.
960

961 *Western blotting and detection*

962 Whole-cell lysate was prepared from cell lines using RIPA buffer supplemented with
963 sodium orthovanadate, complete inhibitor, and PhosSTOP. SDS-PAGE was carried
964 out on 10% polyacrylamide protein gels. Following the transfer of the proteins, the
965 nitrocellulose membranes were blocked in TBST with 5% non-fat dry milk for 1.5h at
966 RT and then probed with various antibodies. BRN2, Cyclin D1, PTEN, phospho-S6,
967 S6, phospho-AKT, and AKT antibodies were from Cell signaling, MITF from Abcam,
968 β-actin and vinculin from Sigma. Secondary antibodies were either HRP-conjugated
969 goat anti-rabbit or anti-mouse IgG. Blots were incubated in ECL (Pierce) and
970 revealed using ECL hyperfilm (GE Healthcare). All primary antibodies were used at a
971 dilution of 1/1,000, except β-actin and vinculin (1/5,000). All secondary antibodies
972 were used at a dilution of 1/20,000. Quantification of the western blots was
973 performed using ImageJ software. See table key ressources

974

975 *Chromatin immunoprecipitation*

976 ChIP experiments were performed as previously described ⁴, and see Supplementary
977 Table 6. ChIP assays of BRN2 binding to the *PTEN* promoter. ChIP assays were
978 performed using an antibody against BRN2 and analyzed after 30-cycle PCR in
979 exponentially growing phase of Dauv-1 melanoma cells. *PAX3* promoter (prom.) and
980 Brn2 coding sequences (CDS) were used as positive and negative controls,
981 respectively. Input represents approximately 0.4% of the input used for the ChIP
982 assay. H3 (histone H3) and IgG (Immunoglobulin G) were used as positive and
983 negative controls for each region of interest, respectively. The oligonucleotides, their
984 position on the genome, and the sizes of the amplified fragments are given in
985 Supplementary Tables 3 and 4.

986
987 RNA extraction and (ChIP) RT-qPCR

988 Tissues were crushed with a mortar and pestle, and stainless steel beads. Qiazol
989 was used to homogenize the samples prior extracting RNA using the miRNeasy Kit.

990 Purified RNA was reversed transcribed using M-MLV Reverse Transcriptase. Real-
991 time quantitative PCR (qPCR) was performed using iTaq™ Universal SYBR Green
992 Supermix. Each sample was run in technical triplicates and the quantified RNA
993 normalized against TBP (human) or Gapdh (mouse) as housekeeping transcripts
994 (Supplementary Tables 3 and 4).

995
996 Software

997 GraphPad PRISM, R version 3.6.3 (R Foundation for Statistical Computing, Vienna,
998 Austria), Adobe Illustrator, Adobe Photoshop, and Microsoft Power Point software
999 were used to analyze data and generate all graphs and figures.

1000
1001 Quantification and statistical analysis

1002 Cell culture-based experiments were performed in at least biological triplicate and
1003 validated three times as technical triplicates. P-values for the comparison of two
1004 groups were calculated using the unpaired Student t-test or Mann-Whitney test. P-
1005 values for the comparison of multiple groups were calculated using the analysis of
1006 variance (ANOVA) and Fisher's least significant difference tests. P-values for
1007 categorical data were calculated using the Chi-square test. P-values for the
1008 comparison of Kaplan-Meier curves were calculated using the log-rank (Mantel-Cox)
1009 or Gehan-Breslow-Wilcoxon test giving weight to the early events. P-values were
1010 reported as computed by Prism 6.

1011
1012 *Data availability*

1013 Microarray gene expression data that support the findings of this study have been
deposited in Gene Expression Omnibus (GEO) with the accession codes
1014
GSE126524 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126524) and
1015
GSE163085 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163085).

1017 The TCGA Skin Cutaneous Melanoma data referenced during the study are available
1018 in a public repository from the National Cancer Institute (NCI) Genomic Data
1019 Commons (GDC) website (https://portal.gdc.cancer.gov). Cut&Run assay data that
1020 support the findings of this study have been deposited in Gene Expression Omnibus
1021 (GEO) with the accession codes GSE153020
1022 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153020). All the other data
1023 supporting the findings of this study are available within the article and its
1024 supplementary information files and from the corresponding author upon reasonable
1025 request. A reporting summary for this article is available as a Supplementary
1026 Information file. The data that support the findings of this study are available from the
1027 corresponding author upon reasonable request.

1028
1029 Acknowledgements

1030 We are grateful to Dorothy C Bennett, Ghanem Ghanem, Florence Faure, and Meenhard
1031 Herlyn for providing cell lines. Hong Wu for providing Pten flox mice. We thank the Institut
1032 Curie staff responsible for the animal colony (especially P. Dubreuil), and the histology (S.
1033 Leboucher), FACS (C. Lasgi), and PICT-IBISA imaging (C. Lovo) facilities. This work was
1034 supported by the Ligue nationale contre le cancer, INCa, ITMO Cancer, Fondation ARC
1035 (PGA), and is under the program «Investissements d'Avenir» launched by the French
1036 Government and implemented by ANR Labex CelTisPhyBio (ANR-11-LBX-0038 and ANR-
1037 10-IDEX-0001-02 PSL). MH had a fellowship from PSL and FRM, PS had a fellowship from
1038 INSERM, and MLC had a fellowship from FRM. CRG was supported by the Ludwig Institute
1039 for Cancer Research. RC was supported in part by a grant from the National Institutes of
1040 Health (AR062547, RAC) and a postdoctoral fellowship from the American Association for
1041 Anatomy (CK). ES was supported from the Research Fund of Iceland (184861-053). LL and
1042 ES are supported by a Jules Verne grant.
1043
1044 Author contributions
1045
1046 M.H., P.S., V.P., J.R., V.D., M.L.C., C.P.K., M.P., F.R., N.C., A.S., L.S.C., L.M., M.L., and
1047 G.B.J. conducted the experiments
1048 M.H., P.S., R.A.C, C.R.G. and L.L. designed the experiments
1049 M.H., P.S., V.P., F.G., V.D., Z.A., A.S., A.B., I.D., C.R.G., and L.L. wrote and reviewed the
1050 manuscript
1051 C.R.G., and L.L. secured the funding
1052 D.M. and E.S. provided reagents
1053 I.D., A.S., C.R.G. provided expertise and feedback
1054
1055 Declaration of Interests
1056 The authors declare no competing interests.
1057
1058 Ethical rules
1059 Animal care, use, and experimental procedures were conducted in accordance with
1060 recommendations of the European Community (86/609/EEC) and Union (2010/63/UE) and
1061 the French National Committee (87/848). Animal care and use were approved by the ethics

1062 committee of the Curie Institute in compliance with the institutional guidelines.

1063

1064

1065
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<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
A

Braf (=Tyr::CreERt2/°; BRAF oncflox/+; PTEN +/+) Braf-Pten (=Tyr::CreERt2/°; BRAF oncflox/+; PTEN F/+)

Brn2-

WT

het

hom

<figure><img src="image_4.png" /></figure>

WT

het

hom

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>Figure 3</figcaption></figure>
A

Braf-Pten-Brn2

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /></figure>

D

Targeted allele

<figure><img src="image_20.png" /></figure>

E

Section 1

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

Braf
Pten
Brn2

F

<figure><img src="image_23.png" /></figure>

G

<figure><img src="image_24.png" /></figure>

H

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>Figure 5</figcaption></figure>
A

<figure><img src="image_43.png" /></figure>

B

<figure><img src="image_44.png" /></figure>

C

CUT&RUN-seq; anti-MITF, anti-H3K27Ac, anti-H3K4me3 (chr10:89,522,464-89,905,430)

<figure><img src="image_45.png" /></figure>

D

<figure><img src="image_46.png" /></figure>

E

<figure><img src="image_47.png" /><figcaption>Figure 6</figcaption></figure>