AKT1 E17K Activates Focal Adhesion Kinase and Promotes Melanoma

Published OnlineFirst May 28, 2019; DOI: 10.1158/1541-7786.MCR-18-1372

Cancer Genes and Networks Molecular Cancer Research AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis David A. Kircher1,2, Kirby A. Trombetti1, Mark R. Silvis1, Gennie L. Parkman1,2, Grant M. Fischer3, Stephanie N. Angel1, Christopher M. Stehn1, Sean C. Strain1, Allie H. Grossmann1,4,5, Keith L. Duffy6, Kenneth M. Boucher1,7, Martin McMahon1,2,6, Michael A. Davies3, Michelle C. Mendoza1,2, Matthew W. VanBrocklin1,2,8, and Sheri L. Holmen1,2,8

Abstract

Alterations in the PI3K/AKT pathway occur in up to 70% enhanced phosphorylation of focal adhesion kinase (FAK). of melanomas and are associated with disease progression. AKT1E17K expression in melanoma cells increased invasion The three AKT paralogs are highly conserved but data and this was reduced by pharmacologic inhibition of either suggest they have distinct functions. Activating mutations AKT or FAK. These data suggest that the different AKT of AKT1 and AKT3 occur in human melanoma but their role paralogs have distinct roles in melanoma brain metastasis in melanoma formation and metastasis remains unclear. and that AKT and FAK may be promising therapeutic Using an established melanoma mouse model, we evalua- targets. ted E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing Implications: This study suggests that AKT1E17K promotes AKT1E17K had the highest incidence of brain metastasis melanoma brain metastasis through activation of FAK and and lowest mean survival. Tumors expressing AKT1E17K provides a rationale for the therapeutic targeting of AKT displayed elevated levels of focal adhesion factors and and/or FAK to reduce melanoma metastasis.

Introduction Hyperactivation of the PI3K/AKT signaling pathway occurs in most metastatic melanomas and increased PI3K/AKT pathway Cutaneous melanoma is the most lethal form of skin cancer due activity correlates with disease progression (3, 4). Loss or inacti- to its propensity to metastasize. The disease exhibits organotrop- vation of the tumor suppressor PTEN is common in cutaneous ism for specific sites including the lung and brain (1). The melanoma and results in activation of the pathway leading to predilection of melanoma to preferentially seed and colonize elevated phospho-AKT (P-AKT) levels (5, 6). Interestingly, mel- select organs is particularly prominent in the form of brain anoma brain metastases exhibit higher P-AKT than those of the metastasis. Melanoma has the highest propensity to metastasize liver or lung (6). Melanomas with loss of PTEN also have a shorter to the brain among all adult malignancies (1) with up to 75% of time to brain metastasis (5) and AKT overexpression results in a stage IV patients with evidence of brain metastases at autopsy (2). shift from radial to vertical tumor growth (7). Taken together, However, the mechanisms that contribute to melanoma brain these data suggest that overstimulation of the PI3K/AKT pathway tropism are not well understood. plays a key role in melanoma progression and may be particularly important in brain homing and/or colonization. AKT1 is one of three closely related serine/threonine kinases 1Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, Utah. 2Department of Oncological Sciences, University of Utah Health (AKT1, AKT2, and AKT3). Although less common than PTEN Sciences Center, Salt Lake City, Utah. 3Department of Melanoma Medical loss, the "hotspot" mutation E17K in AKT1 and AKT3 (6, 8) Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas. also induces PI3K/AKT pathway hyperactivation (9) by disrupt- 4ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. ing the intramolecular interaction between the pleckstrin 5 Department of Pathology, University of Utah Health Sciences Center, Salt Lake homology domain and the kinase domain (10). AKT1Q79K has 6 City, Utah. Department of Dermatology, University of Utah Health Sciences also been detected in human melanoma and reported to not Center, Salt Lake City, Utah. 7Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah. 8Department of Surgery, University only result in constitutive activation of the enzyme but to also of Utah Health Sciences Center, Salt Lake City, Utah. mediate resistance to mutant BRAF inhibition (11). To date, mutations that result in constitutive activation of AKT2 have Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). not been observed in melanoma; however, one study reported that PTEN loss preferentially activates AKT2 over the other AKT Corresponding Author: Sheri L. Holmen, Huntsman Cancer Institute, University paralogs suggesting that aberrant AKT2 signaling may also of Utah, 2000 Circle of Hope Salt Lake City, UT 84112. Phone: 801-213-4237; fl fi Fax: 801-585-0900; E-mail: [email protected] in uence melanoma progression (12). This nding seems contradictory to a recent report demonstrating that AKT2 pref- Mol Cancer Res 2019;XX:XX–XX erentially binds phosphatidylinositol-3,4-biphosphate (PI(3,4)P2), doi: 10.1158/1541-7786.MCR-18-1372 which forms from phosphatidylinositol-3,4,5-triphosphate (PIP3) 2019 American Association for Cancer Research. by SHIP2 at the plasma membrane and delivered to early

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endosomes through clathrin-mediated endocytosis. Conversely, Cell culture AKT1 and AKT3 preferentially bind PIP3, a phosphatidylinositol The culture conditions for DF-1, YUMM 1.1, and primary sequestered to the plasma membrane (13). These distinct fates tumor cell lines are described in Supplementary Materials. All of AKT paralogs may contribute to differential exposure to sub- primary tumor cell lines were derived from Dct::TVA mice. strates. Studies aimed at defining the role of different AKT paralogs in cancer have provided some insight into paralog-specific func- Viral infections (in vivo and in vitro) tions in disease progression. Riggio and colleagues reported that Detailed experimental procedures describing the injection of while AKT1 overexpression promoted primary tumor growth in Dct::TVA mice, as well as the generation YUMM1.1 isogenic breast cancer–cell migration, invasion and metastasis were inhib- variants, and primary tumor cell line variants used in functional ited. Conversely, overexpression of AKT2 had the opposite experiments are described in Supplementary Materials. effect (14). AKT3 has been reported to inhibit migration and metastasis in breast cancer (15), whereas in melanoma, AKT3 has Histology and histochemical staining been implicated in tumorigenic potential (16). These studies Mice were euthanized at their experimental endpoints and imply that the different AKT paralogs may have specific and subjected to a full necropsy. Brain, lung, and primary tumor context-dependent functions in cancer, including tumor- tissues were fixed in formalin overnight, dehydrated in 70% suppressive properties, and therefore it is critical that these func- ethyl alcohol, and paraffin embedded. Sections were stained tions are accurately characterized so effective therapeutic strategies with H&E or left unstained for IHC. Expression of mutant AKT targeting AKT or its effectors can be developed. in each primary tumor of the mutant AKT cohorts was Using an established autochthonous mouse model of mela- confirmed by IHC for the HA epitope tag. Mice whose tumors noma, we previously demonstrated that activation of AKT1 were absent detectable HA were excluded from all analyses. through the addition of an N-terminal myristoylation sequence A separate portion of each primary tumor was frozen for use in (myrAKT1) reduced survival and increased the incidence immunoblot analyses, reverse phase protein array (RPPA), and of melanoma lung and brain metastasis in the context of RNA sequencing. BRAFV600E expression and Cdkn2a loss. Loss of Pten cooperated with myrAKT1 and further enhanced brain metastasis in this High-throughput arrays context (17). In this study, we evaluated the ability of constitu- Detailed experimental procedures and GEO accession numbers tively active E17K, E40K, or Q79K mutants of each AKT paralog to for the RPPA and RNA sequencing as well as TCGA data analysis promote tumor progression and metastasis in the context of are described in Supplementary Materials. BRAFV600E expression and loss of Cdkn2a and Pten. Expression of AKT1E17K promoted highly aggressive melanomas that metas- Spheroid formation, transwell invasion, and transwell tasized to the lungs and brain. This metastatic phenotype was not migration assays significantly observed in the case of other mutant AKT-positive All three-dimensional cell growth/motility assays were per- tumors, suggesting that the AKT paralogs have distinct, nonover- formed using YUMM 1.1 cells. Detailed experimental procedures, lapping roles in the development of melanoma brain metastases. including the pharmacologic inhibitors used in the transwell AKT1E17K-positive tumors showed AKT1E17K-dependent upregu- invasion study, are provided in Supplementary Materials. lation of multiple focal adhesion (FA) factors, which are key components of focal adhesions and established stimulators of Tail vein and intracranial injections cell motility, as well as phosphorylation of focal adhesion kinase NOD scid gamma (NSG) mice were injected via the tail vain or (FAK). Ectopic expression of AKT1E17K in nonmetastatic melano- intracranially with primary mouse melanoma cells. Additional ma cells increased cell invasion, a phenotype abrogated by phar- experimental details are provided in Supplementary Materials. macologic inhibition of AKT or FAK. These findings strongly suggest that one mechanism by which AKT1 promotes melanoma IHC, immunoblotting, and immunofluorescence lung and brain metastasis is through regulation and activation of Detailed experimental procedures for all antibody-based assays proteins involved in focal adhesions. This has important implica- are described in Supplementary Materials. tions for the development of therapeutic strategies aimed at preventing or treating disseminated disease. Statistical analysis Analytic methods used to determine significant differences between groups with respect to mouse survival, incidence of Materials and Methods metastasis, immunoblots, spheroid formation, RPPA, and RNA Mice and genotyping sequencing are described in Supplementary Materials. The aster- Dct::TVA;BrafCA;Cdkn2alox/lox;Ptenlox/lox mice have been described isks shown in figures correspond to P values as follows: ,P< 0.05; previously (17). The breeding scheme and genotype procedures are , P < 0.01; , P < 0.001. described in Supplementary Materials. Study approval Viral constructs and propagation All animal experimentation was performed in Association The avian retroviral vectors used in this study are replication- for Assessment and Accreditation of Laboratory Animal Care competent Avian Leukosis Virus splice acceptor and Bryan poly- International–approved facilities at the University of Utah merase-containing vectors of envelope subgroup A [designated (Salt Lake City, UT). All animal protocols were reviewed and RCASBP(A) and abbreviated RCAS]. Cloning details for RCAS approved prior to experimentation by the Institutional Animal AKT–mutant constructs and the method of viral propagation are Care and Use Committee at the University of Utah (Salt Lake described in Supplementary Materials. City, UT).

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Supplementary material AKT1 kinase activity is required for induction of melanoma For more details, refer to web version of PubMed Central for metastasis in vivo Supplementary Material. Propagation of AKT signaling is accomplished through kinase- dependent phosphorylation of downstream effectors that drive cell survival, growth, and motility. However, AKT1 has been Results reported to promote cell survival in a kinase-independent man- AKT1E17K promotes melanoma metastasis in vivo ner (18). To assess the requirement for kinase activity of AKT1E17K To evaluate the effect of distinct activating mutations in AKT for tumor growth and brain metastasis in our model, we on melanoma growth and metastasis, we used an established cloned a validated kinase-dead version of the E17K mutant autochthonous mouse model of melanoma based on the (AKT1E17K, K179M) into the RCAS vector (18, 19). Mice injected RCAS/TVA avian retroviral system (17). In this model, trans- with viruses encoding AKT1E17K, K179M and Cre showed no sig- genic mice express the TVA viral receptor under the control of nificant difference in survival (Supplementary Fig. S2) or inci- the dopachrome tautomerase (DCT) promoter, which allows dence of metastasis (Supplementary Table S1) compared with the targeting of the virus, and expression of the genes it contains, Cre-only control cohort. This was also true for the wild-type AKT1 specifically in melanocytes. Dct::TVA mice were previously and Cre cohort compared with the Cre-only control cohort. These crossed to BrafCA, Cdkn2alox/lox,andPtenlox/lox mice to model data demonstrate that the reduced survival and enhanced brain the most common alterations in cutaneous melanoma (17). metastases observed require AKT1 kinase activity, which is acti- Delivery of RCAS-Cre results in expression of BRAFV600E and vated by the E17K substitution. genetic loss of Cdkn2a and Pten. Activating mutants (E17K, E40K, or Q79K) of AKT1, AKT2, and AKT3 were created using Primary and metastatic melanomas are histologically similar to site-directed mutagenesis, engineered to contain an N-terminal the human disease HA epitope tag, and cloned into the RCAS retroviral vector. Primary tumors generated in Dct::TVA;BrafCA;Cdkn2alox/lox; Prior to in vivo studies, all mutant AKT viruses were shown to Ptenlox/lox mice were examined histologically following infect TVA-positive cells and express the mutant AKT protein in hematoxylin and eosin (H&E) staining by board-certified vitro (Supplementary Fig. S1). For in vivo analysis, newborn pathologists (A.H. Grossmann and K.L. Duffy; Fig. 2A). Tumors Dct::TVA;BrafCA;Cdkn2alox/lox;Ptenlox/lox mice were subcutane- displayed characteristics consistent with malignancy, including ously injected with viruses encoding mutant AKT1, AKT2, or mitotic figures and high nuclear grade with prominent nucleoli. AKT3 alone or in combination with RCAS-Cre. All mice injected They consisted primarily of short spindle cells but epithelioid cells with viruses containing mutant AKT alone, in the absence of were variably observed in addition to nonbrisk inflammation RCAS-Cre, remained tumor-free for the duration of the study (tumor-infiltrating lymphocytes), coagulative tumor necrosis, (150 days, n ¼ 59;Fig.1).Conversely,allmiceinjectedwith and intratumoral hemorrhage. Analysis of tumor sections by IHC viruses encoding Cre, alone or in combination with mutant revealed heterogeneous detection of mutant AKT (HA epitope AKT, developed primary tumors at the site of injection. Delivery tag; Fig. 2B), the presence of proliferation marker Ki67 (Fig. 2C), of RCAS-AKT1E17K or RCAS-AKT1E40K in combination P-AKT (Fig. 2D), P-ERK (Fig. 2E), and the melanoma marker S100 with RCAS-Cre significantly reduced survival compared with (Fig. 2F). delivery of RCAS-Cre alone (P ¼ 0.0063 and 0.0004, Microscopic examination of the lungs revealed that a subset of respectively; Fig. 1A; Supplementary Table S1). Likewise, mice had developed metastatic lesions; these tumors exhibited a delivery of viruses containing AKT2E40K, AKT3E17K,or malignant morphology consistent with those of the primary AKT3Q79K in combination with RCAS-Cre significantly reduced tumors (Fig. 2G and H). Lung lesions were evaluated by IHC survival compared with delivery of RCAS-Cre alone (P ¼ and exhibited loss of PTEN expression (Fig. 2I) as well as detect- 0.0025, 0.0026, and 0.0058, respectively; Fig. 1B and C; able P-AKT (Fig. 2J), and P-ERK (Fig. 2K). Likewise, and similar to Supplementary Table S1). Interestingly, delivery of viruses the primary tumors, lung metastases exhibited positivity for S100 containing AKT1Q79K, AKT2E17K, AKT2Q79K, or AKT3E40K in confirming the melanocytic and metastatic origin of these lesions combination with RCAS-Cre did not significantly reduce sur- (Fig. 2L). Similar analyses were performed on brain tissue from vival compared with delivery of RCAS-Cre alone (Fig. 1A–C; injected mice to identify those that had developed brain metas- Supplementary Table S1). tases and to characterize the histopathologic features of these To assess metastasis, full necropsies were performed and all lesions (Fig. 2M and N). These analyses confirmed the melano- major organs were examined histologically. Brain metastases were cytic and metastatic origin of these tumors as well as loss of PTEN detected in 38% (8/21) of mice injected with viruses encoding and positivity for P-AKT, and P-ERK (Fig. 2O–R). AKT1E17K and Cre (AKT1E17K cohort: BRAFV600E;Cdkn2a / ; Pten / ;AKT1E17K), whereas no brain metastases were detected Expression and activation of focal adhesion factors are altered in mice injected with viruses containing Cre alone (control cohort: in melanomas expressing AKT1E17K BRAFV600E;Cdkn2a / ;Pten / ; n ¼ 24; P ¼ 0.0009; Fig. 1D). Lung The AKT1E17K cohort exhibited the lowest mean survival and metastases were detected in 24% (5/21) of mice in the AKT1E17K highest incidence of brain metastasis of all mutant AKT cohorts cohort but this was not significantly different from the control tested. To identify key effectors essential for AKT1E17K-dependent cohort (P ¼ 0.225). No significant difference in the incidence of metastasis, we performed RPPA on five primary tumors samples brain or lung metastases was observed between all other exper- from each cohort. For RPPA, protein samples were probed for 245 imental cohorts and the controls (Supplementary Table S1). different epitopes and 15% (36/245) were significantly different These data suggest that AKT1E17K confers an enhanced capacity (P 0.05) between AKT1E17K and control tumors (Fig. 3A). to promote brain metastasis compared with the other AKT Several PI3K/AKT pathway–related proteins were differentially mutants tested. regulated in the AKT1E17K–positive tumors compared with

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Figure 1. AKTE17K reduces survival and promotes brain metastasis in mice with melanoma. A, Kaplan–Meier percent survival curves for Dct::TVA;BrafCA;Cdkn2alox/lox; Ptenlox/lox mice injected with viruses encoding either Cre (gray line) (control), Cre þ AKT1E17K (black line), Cre þ AKT1E40K (blue line), Cre þ AKT1Q79K (red line), or AKT1 (E17K, E40K or Q79K) alone (orange line). Cre þ AKT1E17K and Cre þ AKT1E40K reduced survival of mice compared with Cre alone (P ¼ 0.0063 and 0.0004 respectively). B, Kaplan–Meier percent survival curves with comparable color scheme for AKT2 mutant cohorts; Cre þ AKT2E40K reduced survival of mice compared with Cre alone (P ¼ 0.0025). C, Kaplan–Meier percent survival curves with comparable color scheme for AKT3-mutant cohorts; Cre þ AKT3E17K and Cre þ AKT3Q79K reduced survival of mice compared with Cre alone (P ¼ 0.0026 and 0.0058, respectively). A log-rank (Mantel–Cox) text was used to determine significant differences between cohorts. D, Mean survival SEM depicted on the y-axis; colored areas in circles represent the percentage of mice that developed no metastases (white), lung metastasis only (yellow), brain metastasis only (orange), or lung and brain metastasis (red). The number of mice (n) for each cohort is equal to those depicted in A–C. There was a significant increase in the incidence of brain metastasis (asterisk) in AKT1E17K mice compared with controls (38% vs. 0%, respectively; P ¼ 0.0009). A Fisher exact test (two-tailed) was used to determine significant differences in the incidence of metastasis. P values are as follows: P < 0.05 (), P < 0.01 (), P < 0.001 (); ns, not significant.

controls (Supplementary Fig. S3A). Of these proteins, c-KIT effector that contributes to brain metastasis. Likewise, a second (downregulated; P ¼ 0.0255), and p70-S6K1 (downregulated; FA factor, phospho-focal adhesion kinase (Y397; P-FAK) was also P ¼ 0.0346) were found to exhibit consistent patterns of dysre- upregulated in AKT1E17K mouse tumors compared with controls gulation in patient brain-metastatic melanomas compared with (P ¼ 0.0016); however, P-FAK was not an included epitope in the non-brain metastatic melanomas (TCGA data; ref. 20). However, TCGA RPPA panel. The Y397 phosphorylation event in FAK acts as downregulation of c-KIT and p70-S6K1 would not be predicted to a molecular switch that promotes focal adhesion turnover and enhance tumor progression. Interestingly, we found that paxillin, ultimately drives cell motility (21). Paxillin is one of several FA a FA factor known for the ability to stimulate cell motility, was scaffolding proteins recruited to nascent FAs by P-FAK and is a upregulated in both AKT1E17K mouse tumors compared with necessary component of the FA turnover process (22). RPPA controls (P ¼ 0.0060), as well as patient brain-metastatic mela- analysis of tumors from AKT2E17K or AKT3E17K cohorts revealed nomas compared with non-brain metastatic melanomas (P ¼ no differential regulation of P-FAK or paxillin compared with 0.0138). This ﬁnding revealed a potential AKT1 downstream controls (Supplementary Fig. S3B and S3C, respectively).

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Figure 2. Histologic analysis of the primary tumor and metastatic tumor tissue show similarities with human melanoma. Representative images of serial sections from a primary tumor, lung lesion, and brain lesion in BRAFV600E;Cdkn2a/; Pten/;AKT1E17K mice are shown. A, Primary tumor stained with H&E. B, IHC for HA performed on the primary tumor tissue revealed AKT1E17K expression as well as the presence of Ki67 (C)P-AKT (S473; D) P-ERK (T202/Y204; E), and S100 (F). G and H, H&E staining was used to identify lung metastases. I, IHC for PTEN revealed expression in normal tissue but absence of expression in lung metastases and the presence of P-AKT (S473; J), P-ERK (T202/Y204; K), and S100 (L). M and N, H&E staining was used to identify brain metastases. These tumors were subject to identical IHC analyses as the lung metastases. Analyses revealed the absence of PTEN (O)and presence of P-AKT (S473; P), P-ERK (T202/Y204; Q), and S100 (R). Scale bars, 100 mm.

To validate these findings, protein levels of P-FAK, total FAK, tent with the immunoblot results (Supplementary Fig. S4). These phospho-paxillin (P-paxillin), and total paxillin were measured data suggest that the AKT1E17K mutant potentiates AKT signaling in five AKT1E17K and five control primary tumors via immunoblot beyond that of Pten loss alone. (Fig. 3B). Consistent with the RPPA data, a significant increase in FAK and paxillin have been widely reported to promote cancer the relative levels of P-FAK and total paxillin was observed in the cell motility and have been implicated in tumor progression and AKT1E17K tumors compared with control tumors (P ¼ 0.0004 and metastasis (23), including in melanoma where FAK has been 0.0030, respectively). P-paxillin (Y31), which was not included in shown to have a role in proliferation, motility, and inva- the RPPA analysis, similarly exhibited an increase in AKT1E17K sion (24, 25). FAK and paxillin are critical for FA disassembly tumors compared with controls (P ¼ 0.0130; Fig. 3C). This is (FA turnover), which involves a FAK–Src–paxillin complex, consistent with P-FAK–mediated phosphorylation of paxillin on although the precise mechanism(s) are unclear (26). Consistent Y31. A significant increase in the relative levels of P-AKT (T308; with these reports, our data suggest that the increase in P-FAK, P ¼ 0.0227) and (S473; P < 0.0001) was also observed in the paxillin, and P-paxillin by AKT1E17K may contribute to tumor cell AKT1E17K tumors compared with controls (Fig. 3C). IHC analysis spreading. Because only a small fraction of the RPPA platform was of P-FAK, paxillin, and P-paxillin in primary tumors was consis- dedicated to FA factors, we set out to determine whether

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Figure 3. Phospho-FAK (P-FAK; Y397), paxillin, and phospho-paxillin (P-Paxillin; Y31) are upregulated in AKT1E17K-positive primary tumors. A, Reverse-phase protein array (RPPA) was performed on BRAFV600E;Cdkn2a/;Pten/ (control) and BRAFV600E;Cdkn2a/; Pten/;AKT1E17K (AKT1E17K-positive) primary tumors to identify differentially regulated proteins and phospho-proteins between the two groups. Signiﬁcant results (P 0.05) are presented as a heatmap based on

log2 fold change. B, Immunoblot analysis was performed on control and AKT1E17K-positive tumors followed by densitometry analysis from three independent blots to quantify total protein levels in each group (C). A significant increase in P-FAK expression (P ¼ 0.0004), paxillin (P ¼ 0.0030), P-paxillin (P ¼ 0.0130), P-AKT (T308; P ¼ 0.0227), and P-AKT (S473; P < 0.0001) was detected in AKT1E17K-positive tumors compared with controls. The numbers listed at the top of each column for A and B represent individual mouse numbers; 9869.1 and 9869.2 denote two separate primary tumors from mouse 9869. Significant differences between groups were determined using a two-tailed unpaired t test. P values are as follows: , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

additional FA proteins were differentially expressed between forming transmembrane heterodimers. The proteins not only AKT1E17K and control tumors. RNA was isolated from the same provide an anchor to the ECM required for cell motility during primary tumors used in the RPPA analysis and subjected to RNA myosin II contraction of actin filaments, but channel mechanical sequencing. Of the 10 primary tumors sequenced, one AKT1E17K information from the ECM through intracellular FAs to modulate tumor did not pass quality control procedures and thus was cell responses to the external environment. Each cohort retained excluded from further examination. Assessment of global differ- similar integrin gene expression profiles within their respective ential gene expression by principal component analysis and groups (Supplementary Fig. S6A), but 14 of 27 integrins were unsupervised hierarchical clustering of gene expression profiles differentially expressed between the AKT1E17K cohort compared revealed that samples within each cohort clustered together with the controls with 6 genes significantly upregulated (Itgav, appropriately (Supplementary Fig. S5A). The majority of the most Itga5, Itga8, Itgbl1, Itgb1, and Itgb5) and 8 genes significantly highly differentially expressed genes were downregulated in downregulated (Itgad, Itgae, Itgal, Itgax, Itga2b, Itga10, Itga11, and AKT1E17K tumors compared with controls (Supplementary Itgb6) in the AKT1E17K tumors (Supplementary Fig. S6B). These Fig. S5B). In contrast, out of 18 different FA genes that have data indicate that AKTE17K also influences the expression of reported roles in FA assembly and/or disassembly (21), 16 of integrins. these genes were upregulated in AKT1E17K tumors compared with controls (Fig. 4A and B). These data strongly suggest that AKTE17K AKT1E17K promotes melanoma cell migration and invasion has a critical role in upregulating numerous FA factors. To test whether expression of AKT1E17K promotes cell motility and/or proliferation in vitro, we utilized Yale University Mouse Expression of AKT1E17K alters integrin expression Melanoma (YUMM) cell line 1.1, which expresses BRAFV600E and Expression patterns of alpha and beta integrin genes were also is deficient for Cdkn2a and Pten (27). These molecular alterations assessed between AKT1E17K and control tumors. Integrin proteins mirror the tumors within our control cohort. The YUMM 1.1 cells link the actin cytoskeleton to the extracellular matrix (ECM) by were used to generate isogenic cell lines that stably express either

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Figure 4. Expression of AKT1E17K promotes the upregulation of focal adhesion factors. The mRNA levels of 18 focal adhesion factors were compared between BRAFV600E;Cdkn2a/; Pten/ (control, gray) and BRAFV600E;Cdkn2a/;Pten/; AKT1E17K (AKT1E17K-positive, black) primary tumors. A, Dendrogram with heatmap using unsupervised hierarchical clustering of focal adhesion factors illustrates similarities in gene expression profiles within groups. B, Expression of AKT1E17K promoted the upregulation of FAK (Ptk2; P ¼ 0.0173), Paxillin (Pxn; P ¼ 0.0116), Src (P ¼ 0.0026), Vcl (P ¼ 0.0005), Tln1 (P ¼ 0.0001), Actn1 (P ¼ 0.0098), Actn4 (P ¼ 0.0002), Zfyve21 (P ¼ 0.0299), Dnm2 (P ¼ 3.2 105), Grb2 (P ¼ 0.0051), Capn2 (P ¼ 2.2 105), Ptpn12 (P ¼ 0.0003), Ptpn1 (P ¼ 0.0007), Pin1 (P ¼ 0.0083), Bcar1 (P ¼ 0.0017), and Mapk1 (P ¼ 0.0018). An adjusted P value generated by a Benjamini– Hochberg correction was used to determine significant differences between groups. P values are as follows: , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

wild-type AKT1 or AKT1E17K (Fig. 5A). Immunofluorescence with the parental cells was observed (P ¼ 0.0041 and 0.0038, followed by total internal reflection fluorescence (TIRF) and respectively; Fig. 5C). However, because wild-type AKT1 expres- confocal microscopy was used to further visualize AKT1E17K and sion does not confer a metastatic phenotype in vivo (Supplemen- FAs in these cells. Distinct paxillin puncta, indicative of FA tary Table S1), the increased capacity of AKT1-expressing cells to formation, were located primarily at the cell periphery in both form spheroids in this context does not translate to the prometa- the parental and AKT1E17K cells; in some cases, AKT1E17K colo- static phenotype seen in vivo by AKT1E17K tumors. This distinction calized with paxillin (Fig. 5B). could potentially be explained by differences in invasive capacity. Cell proliferation assays were performed at multiple time Indeed, we observed almost a 5-fold increase in the number of points over 5 days in the presence or absence of growth factors. AKT1E17K-expressing spheroids that invaded the ECM compared With the exception of an increase in proliferation for cells expres- with the parental cells (P ¼ 8 10 15), but less than a 3-fold sing wild-type AKT1 compared with the parental cells on day 5 increase in invasion with cells expressing wild-type AKT1 (Supplementary Fig. S7A), no differences were observed between compared with the parental cells (P ¼ 3 10 5; Fig. 5D and conditions (Supplementary Fig. S7B). These data suggest that E). These data suggest that AKT1 signaling increases the ability of AKT1E17K does not confer a proliferative advantage compared melanoma cells to invade the ECM and that the oncogenic with controls in this context. When the same cells were plated on mutation of AKT1 (E17K) enhances this effect. Matrigel, an increase in the ability of AKT1E17K and wild-type To determine whether AKT1E17K promotes cell migration, we AKT1-expressing cells to form melanoma spheroids compared performed a transwell migration assay using the isogenic YUMM

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Figure 5. Expression of AKT1E17K activates FAK and promotes invasion of melanoma spheroids. A, Yale University Mouse Melanoma (YUMM 1.1; BRAFV600E;Cdkn2a/; Pten/) cells engineered to express wild-type AKT1 or AKT1E17K. Expression of either AKT1 or AKT1E17K resulted in upregulation of P-AKT (T308 and S473) compared with the control parental cell line, but P-FAK (Y397 and Y925) was only elevated in cells expressing AKT1E17K. B, Immunofluorescence used to detect paxillin (green) and AKT1E17K (red) in YUMM 1.1 cells. Top (TIRF microscopy), a single YUMM 1.1 control (left) and AKT1E17K-expressing cell (right). Red arrows specify paxillin puncta, demonstrating the formation of focal adhesions (FAs). Yellow arrows denote the colocalization of paxillin and AKT1E17K in FAs. Black scale bar, 20 mm; white scale bar, 2 mm. Bottom (confocal microscopy), multiple YUMM 1.1 cells. AKT1E17K expression was abundant in YUMM 1.1 cells engineered to express AKT1E17K (red). DAPI stain (blue) was used to label nuclei. Scale bar, 400 mm. C, YUMM 1.1 Control, AKT1, or AKT1E17K cells were plated on Matrigel and the number of melanoma spheroids formed were counted on day 5. Expression of AKT1 promoted spheroid formation compared with controls (P ¼ 0.0038), as did AKT1E17K compared with controls (P ¼ 0.0041). There was no difference in spheroid formation between AKT1 and AKT1E17K conditions. The mean number of spheroids formed for each condition were compared using an unpaired t test (n > 15 for each condition). D, The number of spheroids in C that were found to invade the ECM was increased in both the AKT1 condition and the AKT1E17K condition compared with controls (P ¼ 5 106 and 8 1015, respectively). AKT1E17K-induced invasion was also significantly elevated compared with AKT1 (P ¼ 3 105). The percentage of spheroids that invaded the ECM for each condition was compared using an unpaired t test. E, Representative images of YUMM 1.1 control, AKT1, and AKT1E17K conditions are shown. Scale bar, 1 mm. P values are as follows: , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

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1.1 cell lines. AKT1E17K-expressing cells demonstrated a substan- AKT1E17K in the 5610 cell line (5610E17K; Fig. 7A and B) and tial increase in their ability to migrate through transwell pores intracranial injection of this cell line resulted in illness and early toward a chemoattractant compared with both the parental cells euthanasia for all mice. In addition, all mice injected with (P ¼ 0.0006) and cells expressing wild-type AKT1 (P ¼ 5610E17K were found to have detectable brain lesions (Fig. 7D 0.0046; Fig. 6A and B). These data demonstrate that unlike and E) and this was statistically significant when compared with wild-type AKT1, AKT1E17K promotes migration in this context. mice injected with the 5610 parental line (P ¼ 0.0048). These data indicate that while aberrant PI3K/AKT pathway signaling does not Inhibition of AKT or FAK reduces AKT1E17K-mediated invasion appear to impact the ability of tumor cells to promote lung We next sought to determine whether pharmacologic inhibi- colonization, it does promote melanoma brain colonization tion of AKT or FAK interferes with cell invasion. To address this either through Pten loss, expression of AKT1E17K, or both. question, parental, AKT1, and AKT1E17K-expressing isogenic YUMM 1.1 cells were plated into Matrigel-coated transwell cham- bers and treated with vehicle alone, 1,000 nmol/L GSK-2141795 Discussion (AKT inhibitor), 500 nmol/L PF-573228 (FAK inhibitor), or We performed an objective and comprehensive in vivo analysis 50 nmol/L dasatinib (Src inhibitor). Src acts at the intersection of three separate AKT-activating mutations (E17K, E40K, or of the FAK-initiated FA assembly and disassembly by forming a Q79K) in each of the three AKT paralogs on melanoma progres- complex with FAK and phosphorylating several key FAK tyrosine sion using an established autochthonous mouse model of mel- residues, one of which (Y925) stimulates FA turnover (21). Each anoma. Each of the AKT mutants used in this study engender compound was found to reduce levels of P-FAK (Y397 and Y925) constitutive activation of the protein (9, 10, 19, 28). The mech- in culture (Fig. 6C). Expression of AKT1E17K significantly enhanc- anism of AKT constitutive activation was elegantly described by ed the ability of cells to invade compared with both the parental Carpten and colleagues in 2007 using the E17K mutant, whereby (P ¼ 0.0010) and AKT1 isogenic cells (P ¼ 0.0186; Fig. 6D and the lysine substitution of glutamic acid at amino acid 17 was E), consistent with our observations of spheroid invasion and shown through X-ray crystallography to disrupt the intramolec- transwell migration. Inhibition of either AKT or FAK signifi- ular interactions that occur between the PH domain and kinase cantly reduced the invasion of AKT1E17K-expressing cells domain. This perturbation forces a conformational change in the (P ¼ 0.0200 and 0.0205, respectively) compared with vehi- protein resulting in increased affinity of the PH domain for cle-only–treated AKT1E17K-expressing cells (Fig. 6D and E). phosphatidylinositols at the plasma membrane where the protein Although inhibition of Src trended toward decreased invasive is phosphorylated (10). Since this time, dozens of amino acid capacity of AKT1E17K-expressing cells, this difference was not substitutions in the PH domain of AKT that are predicted to significant compared with vehicle-only–treated AKT1E17K- interfere with the PH-Kinase domain interface have been shown expressing cells (P ¼ 0.0847). Collectively, these data indicate to promote kinase hyperactivity, including those found in that AKT1E17K promotes invasion of mouse melanoma cells in a cancers (9). FAK-dependent manner. Importantly, the invasive phenotype To measure the impact of AKT-activating mutations on mela- of oncogenic AKT1 can be impaired by pharmacologic inhibi- noma, we used overall survival and the incidence of metastasis as tion of either AKT or FAK. readouts of disease progression. We discovered a mutant- dependent effect on survival for all three AKT paralogs. Although Either PTEN loss or AKT1E17K expression is sufficient for brain AKT2 and AKT3-mutant tumors reduced the survival of mice colonization compared with BRAFV600E;Cdkn2a / ;Pten / control tumors in While enhanced invasive properties allow the melanoma cells some cases, they did not significantly promote lung and brain to exit the primary tumor site, it is unclear whether activation of metastasis. However, an analysis of AKT1 mutants in the same the PI3K/AKT pathway is sufficient to allow circulating tumor cells context revealed that AKT1E17K-positive tumors not only reduced to colonize distal organs, including the brain, and promote tumor the survival of mice compared with controls, but also increased growth in these distant sites. To test this, we generated three the incidence of brain metastasis to nearly 40% (Fig. 1D). This primary tumor cell lines with variations in PI3K/AKT activity from discovery is particularly noteworthy as AKT1 was reported in one Dct::TVA mice: 5610 (BRAFV600E;Cdkn2a / ), 9678 (BRAFV600E; study to be "the most important hub gene" as determined by the Cdkn2a / ;Pten / ), and 7788 (BRAFV600E;Cdkn2a / ;Pten / ; PageRank algorithm used to identify top hub genes in melano- AKT1E17K; Fig. 7A and B). Each of the three cell lines were injected ma (29), and to exhibit the "second highest centrality score" in into the tail vein of NOD scid gamma (NSG) mice to assess the melanoma as determined by an algorithm that combines PageR- ability of each cell line to colonize lungs. All mice, regardless of the ank with closeness and betweenness (30). Furthermore, the E17K cell line injected, developed detectable lung lesions (Fig. 7C). To substitution in AKT1 is the most common mutation found among assess the ability to colonize the brain, each of the three cell lines all AKT paralogs in human cancer, representing over one-third of were injected intracranially into NSG mice. Mice injected with AKT1 mutations in cBioPortal (31). Specific to melanoma, cBio- 5610 remained healthy for the duration of the study, whereas all Portal indicates that approximately 3% of these tumors have mice injected with either 9678 or 7788 demonstrated signs of missense substitutions in AKT1. Of those, the most common is illness and were euthanized prior to the experimental endpoint. the hotspot mutation AKT1E17K, which represents 17% of these The brains of all mice were examined post-necropsy, sectioned, mutations. Recent evidence also suggests that this alteration may and stained with H&E in an effort to detect melanoma growth. All be a more common occurrence in advanced melanomas that are mice injected with 9678 and 7788 were found to have detectable resistant to MAPK inhibition. Shi and colleagues detected brain lesions, whereas no mice injected with 5610 had detectable AKT1E17K in a melanoma that had acquired resistance to BRAFi lesions (Fig. 7D); these differences were statistically significant (vemurafenib), but this mutation was not detected in the (P ¼ 0.0119 and 0.0048, respectively; Fig. 7E). Expression of pre-treated tumor (11).

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Figure 6. Expression of AKT1E17K promotes transwell migration and invasion. A, YUMM 1.1 control, wild-type AKT1, or AKT1E17K-expressing cells were plated on the apical surface of transwells. The ability of cells to migrate through pores towards a chemoattractant was measured on day 3. Cells on the basolateral side of the transwells were fixed and stained with crystal violet. The relative absorbance of solubilized crystal violet was compared between conditions using an unpaired t test (n ¼ 6 for each condition). AKT1E17K-expressing cells demonstrated enhanced migration compared with control (P ¼ 0.0006) and AKT1 cells (P ¼ 0.0046). B, Representative images of crystal violet staining for each condition in the migration assay are shown. Scale bar, 1 mm. C, YUMM 1.1 AKT1E17K–expressing cells were treated with either DMSO vehicle control, 500 or 1,000 nmol/L GSK-2141795 (AKTi), 100 or 500 nmol/L PF-573228 (FAKi), or 10 or 50 nmol/L dasatinib (Srci) daily for 5 days. Immunoblot analysis demonstrated targeted inhibition of P-FAK (Y397 and Y925) for drug-treated cells, as well as P-PRAS40 (T246) for AKTi-treated cells. D, YUMM 1.1 control, wild-type AKT1, or AKT1E17K-expressing cells were plated on the apical surface of transwells above a layer of Matrigel in media supplemented with DMSO vehicle control (n ¼ 10 transwells per condition); AKT1E17K-expressing cells were additionally plated in 1,000 nmol/L AKTi, 500 nmol/L FAKi, or 50 nmol/L Srci (n ¼ 4 transwells per condition). The ability of cells to invade through Matrigel and pores toward a chemoattractant was measured on day 5. Cells on the basolateral side of the transwells were processed as per the migration assay in A. AKT1E17K-expressing cells (black bar) demonstrated enhanced invasion compared with control cells (gray bar; P ¼ 0.0010) and AKT1-expressing cells (white bar; P ¼ 0.0186). Inhibition of AKT (light blue) and FAK (blue) significantly reduced invasion of AKT1E17K-expressing cells (P ¼ 0.0200 and 0.0205, respectively). The difference between AKT1E17K- expressing cells (DMSO) and those treated with the Src inhibitor (dark blue) was not significant (P ¼ 0.0847). E, Representative images of crystal violet staining for each condition in the invasion assay are shown. Scale bar, 1 mm. P values are as follows: , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

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Figure 7. Aberrant PI3K/AKT pathway activity promotes melanoma brain colonization. A, The following primary tumor cell lines sourced from Dct::TVA mice were used: 5610 (BRAFV600E;Cdkn2a/; green box), 5610E17K (BRAFV600E;Cdkn2a/;AKT1E17K; green dashed box), 9678 (BRAFV600E;Cdkn2a/;Pten/; gray box), and 7788 (BRAFV600E;Cdkn2a/;Pten/;AKT1E17K; black box). Alterations within each cell line are denoted by check marks. B, The four cell lines listed in A were subject to immunoblot analysis; results demonstrate hyperactivity of canonical PI3K/AKT pathway signaling in cells deficient in Pten and/or those that express AKT1E17K as evidenced by an increase in P-AKT, P-PRAS40, and P-GSK3b. C, H&E staining was used to detect lesions in the lung tissue sections of all mice. Mean survival SEM are reported for each cohort and all mice injected with 5610 (31.3 1.7, n ¼ 6), 9678 (29.2 1.9, n ¼ 6), or 7788 (35.5 0.3, n ¼ 6) developed lesions. Representative images are shown. Scale bar, 1 mm. D, H&E staining was used to detect lesions in brain tissue sections of all mice. Representative images of a normal brain from the 5610 cohort, and brains with detectable lesions from the 5610E17K, 9678, and 7788 cohorts are shown. Scale bar, 200 mm.. E, Mean survival SEM are reported for each cohort and no mice intracranially injected with cell line 5610 (150 day endpoint, n ¼ 6) developed detectable lesions, whereas all mice injected with cell lines 5610E17K (64.3 8.6 days, n ¼ 4; green dashed box), 9678 (28.7 0.7 days, n ¼ 3; gray box), or 7788 (32.8 0.8 days, n ¼ 4; black box) were found to have tumor growth in the brain and these differences were statistically significant (P ¼ 0.0048, 0.0119, and 0.0048, respectively). A Fisher exact test (two-tailed) was used to determine significant differences in incidence. P values are as follows: , P < 0.05; , P < 0.01; , P < 0.001.

The differences in tumorigenic potential among E17K, E40K, functional modification between variants of a single AKT paralog and Q79K variants of each AKT paralog is likely due to the unique would likely translate to corresponding paralogs, for which molecular features imparted by each amino acid substitution. unique target substrates have already been identified. Indeed, Parikh and colleagues reported that AKT1 wild-type, To develop mechanistic insight into the molecular and cell E17K, and Q79K variants possessed varying magnitudes of intra- biological features that cause AKT1E17K-positive tumors to metas- molecular interactions between the PH and kinase domains as tasize to the brain, we used RPPA to screen for differentially well as contrasting levels of phospho-AKT (S473; ref. 9). These regulated proteins and phospho-proteins in control versus differences may, in turn, produce a range of conformational AKT1E17K-positive primary tumors. FA factors P-FAK and paxillin modifications that could ultimately influence activity, including were found to be among the most highly upregulated factors in PH domain affinity for phosphatidylinositols, intrinsic kinase AKT1E17K-positive tumors compared with controls. Functionally, activity, differential activation of downstream substrates, as well FAK and paxillin are intimately involved in stimulating FA turn- as the capacity to confer differential exposure to substrates as a over processes that promote cell motility and have been impli- result of changes in subcellular localization. As such, any potential cated in the metastasis of numerous malignancies through their

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phosphorylation and activation (23). RNA sequencing analyses signaling (41), more recently it has been shown to be a direct of the same primary tumors revealed that the mRNA levels of target of AKT1. In mouse embryonic fibroblasts, AKT1 phosphor- the vast majority of FA genes we examined were upregulated in ylates S695 and T700 on FAK (42) and in human colon cancer AKT1E17K-positive tumors compared with controls. Among all cells AKT1 phosphorylates S517, S601, and S695 on FAK (43, 44). FA factors examined, several have been implicated in driving These phosphorylation events precede and stimulate FAK autop- melanoma progression, including Dnm2 (32), Pin1 (33, 34), hosphorylation of Y397. These studies provide evidence of a direct Bcar1 (24), Actn4 (35), and Itgav (36). link between AKT1 and FAK and assert that FAK activation in this These results led us to hypothesize that the melanoma brain context is not simply an associative event, which co-occurs with metastases that emerge in our in vivo model may be at least P-AKT, but rather the direct result of AKT1-mediated phosphor- partially explained by an increase in tumor cell invasiveness ylation. In addition, pharmacologic inhibition of FAK or AKT in during an early step in the metastatic cascade when efficient AKT1E17K-positive mouse melanomas not only diminishes the assembly and disassembly of FA structures are critical determi- levels of P-FAK (Fig. 6C), but also decreases cell invasion through nants of this process. To test this hypothesis, we first utilized Matrigel (Fig. 6D). Furthermore, while our findings imply FAK- YUMM 1.1 (BRAFV600E;Cdkn2a / ;Pten / ) mouse melanoma mediated tumor cell invasion, an independent study suggests that cells that stably expressed AKT1E17K or wild-type AKT1. FAK also induces melanoma transendothelial migration. Jouve AKT1E17K-expressing cells displayed enhanced invasion of mela- and colleagues demonstrated that pharmacologic inhibition of noma spheroids, increased migration, and increased invasion FAK reduced transmigration of B16 mouse melanoma cells across through Matrigel compared with the parental and isogenic cells a monolayer of lung endothelial cells (45), a finding that is expressing wild-type AKT1. Second, we performed in vivo studies relevant for both intravasation and extravasation. Collectively, using primary tumor cell lines derived from Dct::TVA mice. Both these data point to a model consistent with a threshold effect. Pten BRAFV600E;Cdkn2a / ;Pten / and BRAFV600E;Cdkn2a / ;Pten / ; loss alone does not promote brain metastases; however, it is AKT1E17K melanoma readily colonized the lungs of mice when sufficient for intracranial growth of melanoma cells following introduced directly into the circulation. This suggests that the direct injection into the brain (Fig. 7D and E). The combination of failure of BRAFV600E;Cdkn2a / ;Pten / melanoma to metasta- AKT1E17K expression coupled with Pten loss yields persistently size is likely the result of a diminished capacity to enter the elevated levels of P-FAK. An abundance of P-FAK in turn promotes circulation. an increase in cell motility and the ability to transmigrate across The FA assembly and disassembly processes that stimulate cell endothelial barriers thereby allowing the melanoma cells to more motility in response to external cues are well coordinated but effectively spread to distant sites. Although the cell invasion data highly complex. Although many FA proteins have been identified, using FAKi suggests a causal link between AKT1 and FAK in disease the precise interactions that occur between FA factors and the progression, further experiments using pharmacologic or genetic consequences of those interactions on cell behavior are not inhibition of FAK in vivo are required to demonstrate that FAK completely understood and often context-dependent. To date, activation drives melanoma brain metastasis in this context. nearly 100 FA factors had been identified (37), many of which One obvious question pertains to the physiologic relevancy of remain largely uncharacterized. What remains apparent is that AKT1E17K mutations coupled with PTEN loss in human melano- activation of FAK is an early and key event that orchestrates FA ma. AKT1E17K occurs at a low frequency but spans over a large turnover and cell motility. We previously reported cooperation range of cancers, including melanoma (9, 31), whereas a loss or between Pten loss and activation of AKT1 in the formation of deficiency in PTEN is particularly common in melanoma (4, 5). melanoma lung and brain metastases but the mechanism of To our knowledge, a comprehensive analysis evaluating the cooperation was not clear (17). Interestingly, PTEN has been degree of mutual exclusivity of AKT mutations coupled with shown to coprecipitate with FAK and paxillin in colorectal cancer PTEN loss in melanoma has not been performed. One study cells, and more importantly, to dephosphorylate Y397 on FAK in designed to partially address this question was performed using glioblastoma and breast cancer cells. This dephosphorylation colorectal cancer tissue whereby 2,631 cases of cancer were event diminishes the capacity of P-Src to bind and phosphorylate examined for the presence of AKT1E17K. Of colorectal carcinomas additional tyrosine residues on FAK, resulting in a reduced ability found to harbor this mutation, 33%–38% were estimated to be of the cells to form FAs (38, 39). Furthermore, P-FAK has been accompanied by PTEN loss (46). Future studies designed to found to phosphorylate PTEN on Y336, an event that enhances answer this question in melanoma will be important to establish PTEN phosphatase activity in an apparent negative feedback loop the physiological relevancy of this cooperation. that prevents an overabundance of intracellular P-FAK and results While the restoration of tumor suppressor genes such as PTEN in repression of cell motility (38, 40). In our melanomas, defi- remains a colossal therapeutic challenge in cancer biology, ciency of Pten was not sufficient to induce brain metastases despite numerous compounds designed to molecularly target and inhibit a presumed increase in FAK activity compared with wild-type Pten kinases such as AKT and FAK have been developed. Many AKT melanomas (BRAFV600E;Cdkn2a / ). This suggests that further inhibitors have already demonstrated remarkable specificity and activation of FAK by AKT1 (beyond that of Pten loss alone) is efficacy, with low toxicity (47), and although less well-studied required to promote FA turnover, increase tumor invasion, and clinically, FAK inhibitors have also shown promise as effective ultimately elicit brain metastasis. suppressors of FAK activity (23, 48). Because the three AKT Several lines of evidence support the hypothesis that AKT1E17K paralogs are capable of inducing opposing effects on tumor promotes brain metastasis through activation of FAK. AKT1E17K- progression, as has been demonstrated in breast cancer (14), the positive primary tumors express considerably higher levels option to pharmacologically target AKT for patients with mela- of P-AKT than the Pten / tumors and this increase in P-AKT nomas that exhibit hyperactivated PI3K/AKT signaling may correlates with increased P-FAK (Fig. 3C). Although convention- require careful consideration. However, because none of the ally FAK has been considered an upstream activator of AKT paralog-specific AKT-mutant tumors from our in vivo studies

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offered a protective effect compared with controls, our results Analysis and interpretation of data (e.g., statistical analysis, biostatistics, suggest that indiscriminant inhibition of all AKT paralogs using a computational analysis): D.A. Kircher, K.A. Trombetti, M.R. Silvis, pan-inhibitor would not be detrimental to melanoma therapy. G.L. Parkman, G.M. Fischer, C.M. Stehn, K.M. Boucher, M. McMahon, M.A. Davies, M.C. Mendoza, S.L. Holmen Whether pan-inhibition of AKT in this context offers a survival Writing, review, and/or revision of the manuscript: D.A. Kircher, M.R. Silvis, benefit remains to be determined. G.L. Parkman, G.M. Fischer, A.H. Grossmann, M. McMahon, M.A. Davies, Historically, the use of AKT inhibitors as monotherapies in M.C. Mendoza, S.L. Holmen clinical trials have either failed or produced modest anti-tumor Administrative, technical, or material support (i.e., reporting or organizing effects (49). Current clinical trials using AKT or FAK inhibitors as data, constructing databases): D.A. Kircher, K.A. Trombetti, G.M. Fischer, anticancer agents primarily focus on combinatorial treatment S.N. Angel, S.C. Strain, M.W. VanBrocklin, S.L. Holmen Study supervision: D.A. Kircher, S.L. Holmen regimens. Hirata and colleagues found that while BRAF-mutant patient-derived xenograft melanomas failed FAK inhibitor mono- Acknowledgments therapy, the combination of BRAF and FAK inhibition led to a We thank members of the Davies, VanBrocklin, McMahon, Mendoza, and prolonged period of tumor control–a phenomenon that persisted Holmen labs as well as W. Pavan, M. Bosenberg, A.J. Lazar, C. Stubben, even after tumors developed resistance to mutant BRAF inhibitor C. Conley, A. Welm, B. Welm, and T. Oliver for providing mouse strains, monotherapy. The mechanism of resistance to single-agent BRAF reagents, vectors, and/or advice. We thank Rowan Arave for assistance with fi inhibition in this study was attributed to BRAF inhibitor– gures. We thank the Huntsman Cancer Institute Vivarium staff for assistance fi with mouse husbandry and the Preclinical Research Resource (PRR) for assis- dependent melanoma-associated broblast-mediated remodel- tance with tumor cell injections. We acknowledge the use of the DNA Synthesis ing of the ECM. Interestingly, this event was shown to stimulate Core, the DNA Sequencing Core, the Fluorescence Microscopy Core, and the FAK signaling in melanoma cells, which in turn reactivated the Flow Cytometry Core at the University of Utah. Microscopy equipment was MAPK signaling pathway (50). Our findings in conjunction with obtained using a NCRR Shared Equipment Grant # 1S10RR024761-01. We also studies such as these highlight the role of FAK as an important acknowledge the use of the HCI Shared Resources for Research Informatics (RI), proto-oncogene in melanoma, and one that may be particularly Cancer Biostatistics (CB), High-Throughput Genomics and Bioinformatics Analysis (GBA), and the Biorepository and Molecular Pathology (BMP) relevant to BRAF-mutated melanoma. Further studies will deter- Research Histology Section supported by P30CA042014 awarded to HCI mine whether inhibition of AKT and/or FAK in combination with from the National Cancer Institute. Flow cytometry research reported in this other pathway-targeted agents is a rational therapeutic strategy in publication was supported by the National Center for Research Resources of this disease. the NIH under award number 1S10RR026802-01. RPPA analyses were performed at the Functional Proteomics Core Facility at the MD Anderson Disclosure of Potential Conflicts of Interest Cancer Center, which is supported by a National Cancer Institute (NCI) Cancer Center Support Grant (P30CA016672). This work was supported by No potential conflicts of interest were disclosed. R01CA121118 (S.L. Holmen) from the NCI and 347651 (S.L. Holmen) from the Melanoma Research Alliance. Authors' Contributions Conception and design: D.A. Kircher, M.R. Silvis, G.M. Fischer, S.L. Holmen The costs of publication of this article were defrayed in part by the payment of Development of methodology: D.A. Kircher, M.R. Silvis, G.M. Fischer, page charges. This article must therefore be hereby marked advertisement in M.C. Mendoza, M.W. VanBrocklin, S.L. Holmen accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.A. Kircher, K.A. Trombetti, M.R. Silvis, G.L. Parkman, G.M. Fischer, C.M. Stehn, S.C. Strain, A.H. Grossmann, Received December 27, 2018; revised March 18, 2019; accepted May 22, 2019; K.L. Duffy, S.L. Holmen published first May 28, 2019.

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OF14 Mol Cancer Res; 2019 Molecular Cancer Research

AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis

David A. Kircher, Kirby A. Trombetti, Mark R. Silvis, et al.

Mol Cancer Res Published OnlineFirst May 28, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-1372

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Mice and Genotyping

Dct::TVA;BrafCA;Cdkn2alox/lox;Ptenlox/lox mice were maintained on a mixed C57Bl/6 and

FVB/N background by random interbreeding. DNA from tail biopsies was used to genotype for the TVA transgene, BrafCA, Cdkn2alox/lox,Ptenlox/lox, and wild-type alleles as described (51-53). Both male and female newborn through adult mice were used in this study. All experimental procedures were approved by the Institutional Animal Care and

Use Committee (IACUC) at the University of Utah.

Viral Constructs and Propagation

The Gateway compatible RCAS destination vector has been described (54). RCAS-Cre

(52) and RCAS-myrAKT1 also have been described (17,19). RCAS-myrAKT1 was used as a template to generate the Akt1 mutant constructs. Mouse melanocyte cDNA and Akt2- specific primers were used to amplify Akt2 via PCR. The product was TOPO cloned into a gateway compatible pCR8 TOPO vector, then subcloned into the RCAS destination vector. RCAS-AKT2 was used as a template to generate the Akt2 mutant constructs.

Mouse myrAkt3 was amplified via PCR reaction from Akt3 cDNA (Dharmacon, Lafeyette,

CO) and Akt3-specific primers. The product was TOPO cloned into a gateway compatible pCR8 TOPO vector, then subcloned into the RCAS destination vector. RCAS-myrAKT3 was used as a template to generate the Akt3 mutant constructs. All mutants were engineered with an N-terminal HA-epitope tag. For E17K mutants, the E17K substitution was engineered into the forward primer of each paralog. PCR overlap extension was performed to engineer E40K, Q79K, and K179M mutants, which were subcloned into

Tumor Progression Mean Survival ± Tumor Lung Brain SEM (Days) of Tumor Genotype Incidence Mets (%) Mets (%) Tumor Mice BRAFV600E;Cdkn2a-/- 47% (16/34) 88.9 ± 8.6 0 0 BRAFV600E;Cdkn2a-/-;Pten-/- 100% (24/24) 57.8 ± 3.4 8 0 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT1 100% (21/21) 53.9 ± 2.0 19 0 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT1E17K 100% (21/21) 42.9 ± 3.0 24 38 BRAFV600E;Cdkn2a-/-;Pten-/- + AKTE17K, K179M 100% (19/19) 56.6 ± 2.4 16 11 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT1E40K 100% (20/20) 45.7 ± 1.9 10 10 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT1Q79K 100% (13/13) 56.3 ± 3.5 0 0 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT2E17K 100% (16/16) 55.9 ± 3.6 19 19 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT2E40K 100% (21/21) 46.4 ± 1.9 10 14 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT2Q79K 100% (13/13) 54.5 ± 1.6 0 8 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT3E17K 100% (19/19) 46.0 ± 2.2 21 11 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT3E40K 100% (17/17) 51.0 ± 2.1 29 0 BRAFV600E;Cdkn2a-/-;Pten-/- + AKT3Q79K 100% (14/14) 44.6 ± 2.8 21 7 Supplementary Table 1. Summary of tumor formation in Dct::TVA mice. Tumor incidence, mean survival ± standard error of the mean (SEM) (days), and percentage of tumor-bearing mice with lung or brain metastasis are listed according to tumor genotype. Supplementary Figure 1 Supplementary Figure 2

100 Cre (n=24) Cre + AKT1 (n=21) Cre + AKT1E17K, K179M (n=19) Cre + AKT1E17K (n=21)

ns 50 ns ** Survival (percentage)

0 0 50 100 Time (Days) Supplementary Figure 3

A Ms Hu Ms Hu (Y397) P-FAK Pxn Axl RPPA (T1462) P-Tuberin RNA Seq (S209) P-eIF4E TSC1 Level c-Kit Level p70-S6K1 Level INPP4b Epitope not inclu. (S65) P-4E-BP1 Not applicable

B C AKT2E17K; AKT3E17K; BRAFV600E; BRAFV600E; BRAFV600E; BRAFV600E; Cdkn2a-/-;Pten-/- Cdkn2a-/-;Pten-/- Cdkn2a-/-;Pten-/- Cdkn2a-/-;Pten-/- 3971 4083 4326 3125 3143 3971 4326 3143 4083 3125 10996 10819 10932 10995 11361 11358 11360 Protein Protein 11355 2

-2 Log2 fold change Supplementary Figure 4

AKT1E17K; BRAFV600E;Cdkn2a-/-;Pten-/- BRAFV600E;Cdkn2a-/-;Pten-/- 3143 3144 3145 9869.2 11444 11589 A B H&E

C D HA

E F P - FAK

G H FAK

I J P - Paxillin K L Paxillin Supplementary Figure 5 Figure Supplementary A B

-Log10 P-Value PC2: 9.2% variance 15 10 - - - - 10 15 25 20 15 10 0 5 - 5 0 5 - 60 - 10 - 50 - 40 - 5 - 30 PC1: 72.7% variancePC1: Log2 fold - 20 PCA Plot - 10 0 change Other genes Focal adhesionFocal factors 0 10 5 2 0 30 10 4 0 Supplementary Figure 6 A B Itgad Itgae Itgal Itgam ns 6 *** 6 *** 5 ** 4.0 4 4 4 3.5 ) logCPM

2 3 2 3.0 0 2 4326 3971 3143 4083 7873 9819 8195 3125 7788

0 2.5 Itgal -2 1

-4 -2 0 2.0

Itga2b mRNA level ( Itga1 Itgax Itgav Itgax Itga2 ns ns Itgae 15 * 5 * 15 10 4 8

Itgb6 ) logCPM 10 10 3 6

Itga11 2 4

5 5 Itgad 1 2

Itgb2 0 0 0 0 mRNA level (

Itgam Itga2b Itga3 Itga4 Itga5 ns ns Itgb8 5 *** 10 10 10 * 4 8 8 8

Itga9 ) logCPM 3 6 6 6 Itgb4 2 4 4 4

Itga7 1 2 2 2

Itga10 0 0 0 0 mRNA level (

Itgb7 Itga6 Itga7 Itga8 Itga9 ns ns ns Itgb3 10 5.0 10 ** 6.5 9 4.5 8 6.0 ) logCPM Itga6 8 4.0 6 5.5 Itgb5 7 3.5 4 5.0 (g )

Itga3 6 3.0 2 4.5 5

Itga2 4 2.5 0 4.0 mRNA level ( Itga4 Itga10 Itga11 Itgbl1 Itgb1 Itga8 5 *** 6 * 10 * 15 * 4 8

Itgb1 ) logCPM 4 10 3 6 Itgav 2 2 4

5 Itgbl1 0 1 2

Itga1 0 -2 0 0 mRNA level (

Itga5 Itgb2 Itgb3 Itgb4 Itgb5 ns ns ns 5.5 8 8 8 ** V600E -/- 5.0 7 6 2 BRAF ;Cdkn2a ; ) logCPM 7 4.5 Pten-/- 1 4.0 6 6 4

3.5 V600E -/- 5 5 2 BRAF ;Cdkn2a ; 3.0 0 -/- E17K Pten ;AKT1 2.5 4 4 0 mRNA level (

-1 Itgb6 Itgb7 Itgb8 ns ns 6 *** 5 5.5 -2 4

) logCPM 4 5.0

Log2 fold 3 2 4.5 change 2

0 4.0 1

-2 0 3.5 mRNA level (

Supplementary Figure 7

0.3

Control ** AKT1 ns AKT1E17K ns 0.2

0.1 Absorbance (490nm) _ Growth Factors

0 0 1 2 3 4 5 Day

2.5

2.0 ns ns ns 1.5 Control AKT1 AKT1E17K 1.0

0.5 Absorbance (490nm) + Growth Factors 0 0 1 2 3 4 5 Day gateway compatible pCR8 TOPO vectors (Thermo Fisher, Waltham, MA), then gateway cloned into the RCAS destination vector. The K179M mutant was generated using RCAS-

AKT1E17K as a template. The PCR product was subcloned into a pCR8 TOPO vector

(Thermo Fisher), then gateway cloned into the RCAS destination vector. All viral vectors were verified via Sanger Sequencing. Primer sequences are available upon request. Viral production was initiated by calcium phosphate transfection into DF-1 cells. Viral spread was monitored by expression of the p27 viral capsid protein as detected by immunoblot.

Cell Culture

DF-1 cells were grown in DMEM-high glucose media (Thermo Fisher) supplemented with

10% FBS (Atlanta Biologicals, Flowery Branch, GA) and 0.5 µg/mL Gentamicin (Thermo

Fisher), and maintained at 39°C. Yale University Mouse Melanoma cells (YUMM 1.1) were grown in F12/DMEM media (Thermo Fisher) supplemented with 10% FBS, 1 µg/mL penicillin-streptomycin (Thermo Fisher) and 1 µg/mL non-essential amino acids (Thermo

Fisher), and maintained at 37°C. Cell line 5610 was established by isolating

Dct::TVA;BRAFV600E;Cdkn2a-/- primary tumor tissue from a mouse during necropsy. The tissue was minced and incubated in F12/DMEM media (Thermo Fisher) supplemented with 10% FBS, 1 µg/mL penicillin-streptomycin and 1 µg/mL non-essential amino acids, and maintained at 37°C. Cell line 9678 was established from a Dct::TVA;BRAFV600E

;Cdkn2a-/-;Pten-/- primary tumor, and cell line 7788 was established from a

Dct::TVA;BRAFV600E ;Cdkn2a-/-;Pten-/-;AKT1E17K primary tumor using the same tissue isolation procedure and cell culture conditions as described for 5610.

Viral Infections (In Vivo)

Infected DF-1 cells from a confluent culture in a 10 cm dish were trypsinized, pelleted, resuspended in 100 µL of Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher), and placed on ice. Newborn mice were injected subcutaneously behind each ear with 50 µL of suspended RCAS-AKT cells with or without RCAS-Cre cells.

Viral Infections (In Vitro)

YUMM 1.1 cells were transfected with pcDNA3.1-TVA (55) containing the hygromycin B- resistance gene to generate YUMM1.1-TVA cells. TVA-positive clones (YUMM 1.1 TVA+) were selected in 300 µg/mL Hygromycin B (Thermo Fisher). Supernatant from DF-1 cells producing RCAS-AKT1 or RCAS-AKT1E17K was used to infect YUMM 1.1 TVA+ cells.

Expression of AKT1 and AKT1E17K in YUMM 1.1 cells was confirmed by immunoblot and/or immunofluorescence. To generate the 5610E17K cell line, AKT1E17K was gateway cloned from pCR8 TOPO to a pHIV EF1a-Luciferase-IRES-ZsGreen destination vector

(kind gift from Bryan Welm) to generate pHIV CMV-HA-AKT1E17K-EF1a-ZsGreen. The lentiviral vector pHIV CMV-HA-AKT1E17K-EF1a-ZsGreen, along with packaging plasmids psPAX2 (#12260 Addgene, Cambridge, MA) and pCMV-VSV-G (#8454 Addgene) were transfected into 293FT cells using the calcium phosphate method. Supernatant from these cells containing virus was then used to infect the 5610 parental cell line. 5610E17K mouse melanoma cells were sorted for ZsGreen using a Propel Labs Avalon at The Flow

Cytometry Shared Resource Laboratory at the University of Utah. Expression of AKT1E17K in 5610 cells was confirmed by immunoblot.

Reverse Phase Protein Array (RPPA)

Frozen primary tumor samples were embedded in optimal cutting temperature compound

(OCT). A 5 µm H&E-stained slide was prepared from each sample and reviewed by a pathologist for regions containing 70% or more viable tumor. Marked H&E slides were then used to guide macrodissection of viable tumor from the OCT blocks. Extraction of protein from tumor tissue was performed as previously described (6). RPPA analysis was performed at the MD Anderson RPPA Core Facility. Lysates were arrayed on nitrocellulose-coated slides, probed with 304 antibodies by tyramide-based signal amplification, and visualized by DAB colorimetric reaction. Array-Pro Analyzer was used to quantify 16-bit TIFF images produced by slides scanned on a flatbed scanner. Relative protein levels were determined by interpolation of each dilution curve from the standard curve of the slide. All data points were normalized for protein loading and transformed to a linear value. Normalized linear values were transformed to Log2 values, then median- centered for hierarchical clustering analysis. The accession number for data reported is

GEO: GSE123182.

RNA Sequencing

Frozen primary tumor samples embedded in OCT were macrodissected as per the methods described for RPPA. For RNA extraction and DNase treatment, the Roche High

Pure miRNA Isolation Kit was used in accordance with the manufacturer's protocol

(5080576001). RNA sequencing was performed at the High-Throughput Genomics and

Bioinformatics Analysis Core at the University of Utah. RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold. Total RNA samples

(100-500 ng) were hybridized with Ribo-Zero Gold to substantially deplete cytoplasmic and mitochondrial rRNA from the samples. Stranded RNA sequencing libraries were

4 prepared using the Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold per the manufacturer’s instructions (RS-122-2301 and RS-122-2302). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay

(cat# 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit

(cat#KK4824). Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. DNA sequences were determined using the HiSeq 50 Cycle Single-Read Sequencing version 4. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD-

401-4001). Following transfer of the flowcell to an Illumina HiSeq 2500 instrument

(HCSv2.2.38 and RTA v1.18.61), a 50 cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4002). The accession number for data reported is GEO: GSE122781.

TCGA Data Analysis

Data were collected from TCGA-SKCM using the TCGAbiolinks R package via

Bioconductor. These data were stratified into 66 primary melanomas from patients who, at the time of this publication, had not experienced a metastatic event. These were compared with 43 melanomas (4 primary tumor, 7 regional cutaneous or subcutaneous tumor, 27 regional lymph node, 5 distant metastasis) from patients who developed brain metastases.

Spheroid Formation and Invasion Assay

Ninety-six well plates were coated with 50 µL of 3 mg/mL Matrigel (Corning, Corning, NY) in serum-free F12/DMEM media (Thermo Fisher) supplemented with 1 µg/mL penicillin- streptomycin (Thermo Fisher) and 1 µg/mL non-essential amino acids (Thermo Fisher).

The plates were incubated at 37°C for 2 hours. One thousand YUMM 1.1 control, AKT1, or AKT1E17K-expressing cells in F12/DMEM media supplemented with 10% FBS (Atlanta

Biologicals) and 3 mg/mL Matrigel (Corning), were added to the base layer of Matrigel.

Plates were incubated at 37°C for 5 days. Imaging of spheroids was performed using an

EvosFL microscope and ImageJ was used to quantify spheroid formation and invasion.

Inhibitors

The AKT inhibitor (GSK-2141795), FAK inhibitor (PF-573228), and Src inhibitor

(Dasatinib) were purchased from Selleck Chemical (Houston, TX) and formulated in

DMSO. Each drug was added to F12/DMEM media (Thermo Fisher) for a final concentration of 0.1% DMSO. F12/DMEM media was supplemented with 2% FBS

(Atlanta Biologicals), 1 µg/mL penicillin-streptomycin (Thermo Fisher), and 1 µg/mL non- essential amino acids (Thermo Fisher). YUMM 1.1 AKT1E17K-expressing cells were incubated in 500 nM or 1000 nM of GSK-2141795, 100 nM or 500 nM of PF-573228, 10 nM or 50 nM of Dasatinib, or 0.1% DMSO vehicle control. Media was refreshed daily.

Cells were lysed on day 5 with 100 mM Tris-HCL, 4% SDS, 20% glycerol, and 10% DTT.

Lysates were separated by SDS-PAGE and assessed by immunoblot.

Transwell Invasion Assay

YUMM 1.1 parental, AKT1, and AKT1E17K -expressing cells were plated in F12/DMEM serum-free media (Thermo Fisher) supplemented with 1% BSA (Cell Signaling

Technology, Danvers, MA), 1 µg/mL penicillin-streptomycin (Thermo Fisher) and 1 µg/mL non-essential amino acids (Thermo Fisher), and maintained at 37°C for 24 hours.

Transwells (6.5 mm, 8 µm pores) (Corning) were prepared by adding 50 µL of 2 mg/mL

Matrigel (Corning) suspended in F12/DMEM serum-free media (Thermo Fisher) supplemented with 1% BSA (Cell Signaling Technology), 1 µg/mL penicillin-streptomycin

(Thermo Fisher) and 1 µg/mL non-essential amino acids (Thermo Fisher), and maintained at 37°C for 2 hours. Fifty-thousand serum-starved YUMM 1.1 control, AKT1, or AKT1E17K- expressing cells were then re-suspended F12/DMEM serum-free media (Thermo Fisher) supplemented with 1% BSA (Cell Signaling Technology), 1 µg/mL penicillin-streptomycin

(Thermo Fisher), 1 µg/mL non-essential amino acids (Thermo Fisher), and 0.1% DMSO

(vehicle control). A subset of YUMM AKT1E17K-expressing cells were resuspended in

F12/DMEM media with 1000 nM of GSK-2141795, 500 nM of PF-573228, or 50 nM of

Dasatinib. All cells were plated on the surface of the Matrigel in transwells and 800 µL of

F12/DMEM media supplemented with 10% FBS (Atlanta Biologicals), 1 µg/mL penicillin- streptomycin (Thermo Fisher), and 1 µg/mL non-essential amino acids (Thermo Fisher) was added to the bottom chamber as a chemoattractant. Media on the apical surface of transwells was refreshed daily. On day 5, transwells were rinsed in PBS, incubated in

100% methanol for 10 minutes, and stained with crystal violet for 10 minutes. The Matrigel and cells on the apical surface of the transwells were removed using a cotton swab. Cells on the basolateral surface of each well were imaged, then incubated in 20% acetic acid in H2O for 10 minutes on a rotating orbital to dissolve the crystal violet. Absorbance was read on a microplate reader at 590 nM.

Transwell Migration Assay

The experimental methods for YUMM 1.1 cells followed those of the Transwell Invasion

Assay with the exception of the use of Matrigel, 0.1% DMSO-supplemented media, and the use of compounds to inhibit AKT, FAK, and Src. Identical quantitative analyses were performed on fixed and stained cells.

Tail Vein Injections

Four-to-six week old NOD scid gamma (NSG) mice (Jackson Laboratory, Bar Harbor,

ME) were injected into the tail vein with 2.5 x 105 mouse melanoma cells suspended in

150 µL of PBS. At the experimental endpoint, brains and lungs were isolated and processed as described above.

Intracranial Injections

Newborn NSG mice were intracranially injected into the right cerebrum with 1.0 x 104 mouse melanoma cells suspended in 5 µL of Hank’s Balanced Salt Solution (HBSS)

(Thermo Fisher) using a gas-tight Hamilton syringe. At the experimental endpoint, brains were isolated and processed as described above.

Immunohistochemistry

Tissue sections from formalin-fixed paraffin embedded blocks were deparaffinized at

65˚C for 10 minutes, incubated in xylene, and rehydrated using decreasing concentrations of ethanol. Antigen retrieval was performed in a decloaking chamber at

120˚C for 20 minutes using citrate buffer (pH 6.0) for HA, P-ERK, S100, PTEN, P-AKT, and Ki67, or EDTA (pH 8.0) for Paxillin, P-Paxillin, FAK, and P-FAK. Peroxidase activity was quenched in 3% H2O2 for 10 minutes and slides were incubated in 5% normal goat serum in TBS-T (0.05% Tween-20) for 1 hour inside a humidity chamber. Anti-rabbit

8 primary antibodies were diluted in SignalStain Antibody Diluent (Cell Signaling

Technology), added to slides, and incubated overnight at 4˚C. Slides were incubated in

SignalStain Boost Detection Reagent (Cell Signaling Technology) for 30 minutes in a humidity chamber and the SignalStain DAB Substrate Kit (Cell Signaling Technology) was used to detect the presence of each epitope. Slides were counterstained in hematoxylin and dehydrated with increasing concentrations of alcohol and xylene prior to the addition of coverslips. Antibodies used: HA (1:500; MMS101P Biolegend, San Diego,

CA), S100 (1:100; RB-9018 Thermo Fisher), Ki67 (1:300; UM800033 Origene, Rockville,

MD), Paxillin (1:100; STJ94969 Antibodyplus, Brookline, MA), Phospho-Paxillin (Y31)

(1:100; STJ90380 Antibodyplus), Phospho-ERK1/2 (T202/Y204) (1:400; 4370 Cell

Signaling Technology), PTEN (1:125; 9188 Cell Signaling Technology), Phospho-AKT

(S473) (1:100; 3787 Cell Signaling Technology), FAK (1:100; 3285 Cell Signaling

Technology), Phospho-FAK (1:50; Y397) (3283 Cell Signaling Technology).

Immunoblotting

Cell lysates were suspended in 100 mM Tris-HCL, 4% SDS, 20% glycerol, and 10% DTT.

To generate lysates from frozen primary tumors, tissues were pulverized in liquid nitrogen using a mortar and pestle and samples were resuspended in RPPA buffer (8) with protease and phosphatase inhibitors (Pierce Biotechnology). All lysates were incubated at 95˚C for 10 minutes, separated on an 8-16% Tris-glycine polyacrylamide gel (Thermo

Fisher Scientific), and transferred to nitrocellulose for immunoblotting. Nitrocellulose membranes were incubated in blocking solution composed of 0.1% Tween-20 in 1X TBS with 5% nonfat dry milk for HA, GAPDH, and P-AKT (T308), or 5% BSA (Cell Signaling

Technology) for all other antibodies. Blots were immunostained in the primary antibody

9 diluted 1:1,000 (or 1:10,000 for Gapdh) in TBS-T for 1 hour with constant shaking and washed in TBS-T. Blots were then incubated in anti-mouse IgG-HRP or anti-rabbit IgG-

HRP secondary antibody diluted 1:1,000 in TBS-T for 1 hour with constant shaking and washed in TBS-T. Enhanced chemiluminescence (Amersham) was used according to the manufacturer’s specifications and blots were exposed to film. Antibody list: p27-HRP

(10100770; Charles River Laboratories), HA (MMS-101P; Biolegend), GAPDH (MAB374;

MilliporeSigma), Paxillin (STJ94969; Antibodyplus), Phospho-Paxillin (Y31) (STJ90380;

Antibodyplus), FAK (3285; Cell Signaling Technology), Phospho-FAK (Y397) (3283; Cell

Signaling Technology), Phospho-FAK (Y925) (3284; Cell Signaling Technology), AKT

(4691; Cell Signaling Technology), Phospho-AKT (T308) (13038; Cell Signaling

Technology), Phospho-AKT (S473) (3787; Cell Signaling Technology), PRAS40 (2691;

Cell Signaling Technology), Phospho-PRAS40 (Y246) (2997; Cell Signaling Technology),

GSK3β (12456; Cell Signaling Technology), Phospho-GSK3β (S9) (5558; Cell Signaling

Technology), anti-mouse-HRP (7076; Cell Signaling Technology), Anti-rabbit-HRP (7074;

Cell Signaling Technology).

Immunofluorescence

Fifty-thousand YUMM 1.1 control or AKT1E17K-expressing cells were plated into 35 mm glass bottom plates (MatTek, Ashland, MA) suspended in F12/DMEM media (Thermo

Fisher) supplemented with 10% FBS (Atlanta Biologicals), 1 µg/mL penicillin- streptomycin (Thermo Fisher) and 1 µg/mL non-essential amino acids (Thermo Fisher), and maintained at 37°C for 24 hours. Plates were fixed in 3% paraformaldehyde in PBS for 20 minutes, washed in PBS, and incubated for 30 minutes in blocking buffer composed of 1% BSA (Cell Signaling Technology) and 0.15% glycine (Thermo Fisher) in PBS. Cells

10 were permeabolized with 0.1% triton (Sigma-Aldridge, St. Louis, MO) in PBS for 15 minutes, washed in PBS, and incubated for 30 additional minutes in blocking buffer.

Plates were then incubated in 15% goat serum (Cell Signaling Technology) in PBS for 30 minutes, followed by the addition of primary anti-HA and anti-paxillin antibodies diluted in

5% goat serum for 1 hour and washed with PBS. Incubation with secondary fluorophore

488 and 568-conjugated antibodies diluted in 5% goat serum was performed for 1 hour followed by washes with PBS. DAPI stain (Thermo Fisher) at 5 ng/mL in PBS was added for 1 minute followed by washes with PBS. Plates were left in PBS solution and images were acquired using a Nikon Ti spinning disc confocal/TIRFM microscope. Antibodies used: HA (1:1,600; 3724 Cell Signaling Technology), Paxillin (1:1,000; 610051 BD

Biosciences, Franklin Lakes, NJ), Anti-mouse Alexa Fluor 488 (1:1,000; A-11029 Thermo

Fisher), Anti-rabbit Alexa Fluor 568 (1:1,000; A-11036 Thermo Fisher).

Statistical Methods

Mouse survival data was analyzed using a log-rank (Mantel-Cox) test of the Kaplan-Meier estimate of survival; a Fisher’s exact test was used to determine differences in metastasis between cohorts. For RPPA analysis, heat-maps were generated in Excel and rows were scaled using z-scores (푥−휇); an unpaired t test was used to calculate differences between 휎 groups. Densitometry measurements were performed using ImageJ and protein levels were normalized to GAPDH; the data are represented as mean ± S.E.M. For RNA sequencing analyses, the Bioconductor package DESeq2 in RStudio was used to generate PCA plots, volcano plots, and heat-maps with dendrogram clustering. The

Limma package vignette was used to perform mean-variance remodeling (voom- transformation) and convert read counts into logCPM for visual comparison of individual

11 genes. A Benjamini-Hochberg correction was applied to correct for multiple testing and generate adjusted p-values for individual gene comparisons using a false discovery rate threshold of 0.05 in RStudio. For melanoma spheroid formation and invasion assays, unpaired t tests were performed using the average number of colonies formed among all wells for each condition and the average percentage of colonies to form invasive protrusions among all wells for each condition. Data are represented as mean ± S.E.M.

A p value of < 0.05 was considered statistically significant for all analyses. The asterisks shown in figures correspond to P values as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001

(***).