STK11 loss leads to YAP1-mediated transcriptional activation in human KRAS-driven lung adenocarcinoma cell lines

STK11 loss leads to YAP1-mediated transcriptional activation in human KRAS-driven lung adenocarcinoma cell lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication STK11 loss leads to YAP1-mediated transcriptional activation in human KRAS-driven lung adenocarcinoma cell lines David Seward, Sean Lenahan, Hailey Sarausky, Paula Deming This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3113344/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Serine Threonine Kinase 11 (STK11) loss of function (LoF) correlates with anti-PD-1 therapy resistance in patients with KRAS-driven lung adenocarcinoma (LUAD). The molecular mechanisms governing this observation remain unclear and represent a critical outstanding question in the field of lung oncology. As an initial approach to understand this phenomenon, we knocked out STK11 in multiple KRAS-driven, STK11 -competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression. Subsequent pathway enrichment studies highlighted activation of the HIPPO/YAP1 signaling axis, along with the induction of numerous tumor-intrinsic cytokines. To validate that YAP1-mediated transcriptional activation occurs in response to STK11 loss, we pursued YAP1 perturbation as a strategy to restore an STK11 -competent gene expression profile in STK11 -KO LUAD cell lines. Together, our data link STK11 loss with YAP1-mediated transcriptional activation, including the upregulation of immune-evasion promoting cytokines IL-6, CXCL8 and CXCL2. Further, our results raise the intriguing possibility that YAP1 antagonism may represent a therapeutic approach to counter anti-PD-1 therapy resistance in STK11-null, KRAS-driven LUADs by modulating tumor-intrinsic gene expression to promote a “hot” tumor immune microenvironment. Biological sciences/Molecular biology/Transcription Health sciences/Biomarkers/Predictive markers Biological sciences/Immunology/Cytokines Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction More people die in the US each year from lung cancer than breast, colorectal, and prostate cancers combined 1 . The overall prognosis for patients with lung cancer remains poor, complicated by the fact that > 40% of patients are diagnosed at an advanced stage 1 . Immune check-point inhibitors, including anti-PD-1 monoclonal antibodies, are now utilized as first line therapy when treating lung adenocarcinoma (LUAD) patients, though our ability to predict which patients will benefit remains limited 2 , 3 . Recent clinical studies have linked anti-PD-1 therapy resistance with tumor genotype. Specifically, KRAS-driven LUADs with concomitant loss of Serine/Threonine Kinase 11 (STK11) exhibit a dramatic decrease in therapeutic efficacy relative to tumors harboring intact STK11 4 . The molecular mechanism(s) underlying this correlation remain unknown. STK11 functions in a heterotrimeric complex with the pseudo-kinase STRADα and the scaffolding protein MO25 where it regulates numerous intracellular signaling networks impacting metabolism, proliferation, transcription and cell morphology 5 . In LUAD, somatic alterations in STK11 are surpassed in frequency by only one other tumor suppressor, TP53 6,7 . A growing body of work reveals STK11 regulates myriad biologic processes and suggests the scope of STK11-dependent regulation is vastly underappreciated 8 – 11 . Previous studies using inducible mouse models of Kras-driven lung cancer have reported Stk11 deficiency correlates with altered tumor-intrinsic cytokine expression 12 . We therefore reasoned, as others have 4 , 12 , that increased production and secretion of tumor-intrinsic cytokines might alter recruitment of immune cells to the tumor microenvironment, thereby promoting immune evasion and contributing to anti-PD-1 therapy resistance. However, instead of focusing on the tumor-extrinsic processes, we chose to investigate which signaling pathways downstream of STK11 loss drive these tumor-intrinsic transcriptional changes. Using multiple KRAS-driven human LUAD cell lines, our work demonstrates that STK11 loss results in altered tumor-intrinsic cytokine expression, including but not limited to IL-6, CXCL8 and CXCL2. In addition, we identify significant enrichment of a Yes Associated Protein 1 (YAP1) transcriptional signature 13 upon STK11 disruption, which encompasses a variety of cytokines, chemokines, and extracellular matrix proteins 14 . YAP1 is the major downstream effector of the HIPPO signaling cascade, a pathway that regulates organ size during development 15 . YAP1-mediated transcriptional activation is controlled in part via its cytosolic sequestration; a kinase-dependent process controlled by activation of the HIPPO pathway 16 . Whether HIPPO signaling is impacted by STK11 remains incompletely addressed, but recent publications suggest a connection exists 13 , 17 , 18 . Our data support this link via restoration of STK11-loss-dependent transcriptional profiles upon either genetic ablation or pharmacologic inhibition of YAP1. The work we present situates the HIPPO/YAP1 axis as a signaling cascade downstream of STK11 that modulates tumor-intrinsic cytokine expression. Further, we speculate YAP1 antagonism may represent a synergistic strategy to sensitize patients with KRAS-driven LUADs lacking STK11 to anti-PD-1 therapy by promoting a “hot” tumor immune microenvironment. Results STK11 loss alters tumor-intrinsic cytokine expression We knocked-out STK11 in three genetically independent human KRAS -driven LUAD cell lines that normally harbor intact STK11 alleles: NCI-H2009, NCI-H441 and NCI-H1792. STK11 loss was validated by Western Blot analysis (Fig. 1 A and Supplemental data ). Based on studies reporting a correlation between Stk11 loss and Il-6 upregulation in mouse models of Kras -driven lung cancers in vivo 12 , we compared IL-6 expression between STK11 WT (aka “Parent”) and matched STK11 -KO human LUAD cells using qRT-PCR. Unexpectedly, under standard culture conditions no difference in IL-6 expression was detected between the Parent and STK11-KO cells (Fig. 1 B, “+Glutamine”). However, significant STK11- loss-dependent IL-6 upregulation was observed when cells were cultured under conditions of nutrient stress, achieved via glutamine depletion (Fig. 1 B, “-Glutamine”). The rationale for evaluating nutrient stress as a variable was based on evidence that STK11 functions as a nutrient sensor to regulate metabolic homeostasis 19 – 21 . We reasoned STK11 loss might be irrelevant when cells are grown in standard media as nutrients are in excess. Given that tumor microenvironments in vivo are characterized by nutrient stress 22 – 24 , we used glutamine depletion to simulate nutrient-deprivation in vitro. Next, to comprehensively characterize STK11-loss-dependent transcriptional changes, we expanded our analyses and performed whole transcriptome sequencing comparing standard media to glutamine depletion. In standard media, relatively few genes differed between parent and STK11 -KO cells (Fig. 1 C, +Glutamine; H2009: 1100 DEGs, H441: 928 DEGs). In contrast, when comparing both H2009 and H441 parent lines with their paired STK11 -KO lines following glutamine depletion we identified 7453 and 5202 differentially expressed genes (DEGs) respectively (Fig. 1 C; -Glutamine). This marked STK11-loss-dependent transcriptional impact indicates STK11 plays a critical and generalizable role in regulating transcription in response to nutrient stress. We then performed Gene Set Enrichment Analysis (GSEA) 25 on the DEGs for both H2009 and H441 cell lines and found significant associations between STK11 loss and altered tumor-intrinsic cytokine signaling, specifically upregulation of genes within the Gene Ontology (GO) term “Cytokine Activity” (GO: 0005125) (Fig. 1 D). Of the upregulated genes in this curated list, 9 were shared between the H2009 and H441 cell lines, suggesting overlapping regulatory pathways. Intriguingly, these overlapping genes consist of effectors previously associated with cancer progression, immune evasion, and therapy resistance 26 – 28 . For example, both IL-6 and CXCL8 are reported to be elevated in KRAS-driven STK11-null LUADs and proposed to promote tumor immune evasion 29 – 31 . Similarly, CXCL2 is known to drive neutrophil recruitment, a phenotype associated with “cold” tumor immune microenvironments 27 . Finally, BMP2 expression is correlated with metastatic burden and STK11 loss in lung cancer and mediates activation of SMAD transcription factors 32 , 33 , which are known YAP1 binding partners 34 . YAP1 transcriptional activation occurs in LUAD cell lines lacking STK11 In addition to GSEA, we also performed pathway enrichment using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 35 . This approach revealed several significantly enriched networks in STK11 -KO cells relative to matched parental lines (Fig. 1 E-F). Consistent with prior published reports, both focal adhesion and HIF-1 pathways were over-represented in cells lacking STK11 36,37 . In addition, NF-kappa B signaling, TNF signaling, chemokine signaling and HIPPO signaling were significantly enriched in STK11 -KO cells. We chose to focus on the HIPPO pathway as STK11 has previously been implicated in HIPPO regulation via direct activation of MARK family kinases and subsequent modulation of YAP1 activity 13 . YAP1-mediated transcriptional activation is controlled in part via cytosolic sequestration; a kinase-dependent process regulated by activation of the HIPPO cascade 16 . Utilizing a curated list of YAP1 transcriptional target genes 13 , we repeated GSEA and found a significant positive correlation between STK11 loss and enhanced expression of YAP1 target genes in both the H2009 and H441 cell lines (Fig. 1 G). STK11 loss correlates with increased YAP1 protein levels STK11 has previously been proposed to indirectly modulate the HIPPO/YAP1 axis via MARK activation, ultimately promoting YAP1 sequestration and degradation 13 . We therefore hypothesized that STK11 loss would result in increased YAP1 protein due to enhanced protein stabilization (Fig. 2 A). Western blot analysis comparing whole cell extracts from parent and STK11 -KO LUAD cell lines support this assertion, showing a ~ 2-fold increase in relative YAP1 abundance (Fig. 2 B), an observation supported by prior studies in mice 13 . Interestingly, this difference occurs only at the protein level, as YAP1 transcript levels remain unchanged, supporting our hypothesis that STK11 loss results in YAP1 protein stabilization (Fig. 2 C). Nuclear and cytosolic fractionation analyses further demonstrate that increased YAP1 protein levels are not isolated to either compartment but increased throughout cells lacking STK11. Upon glutamine deprivation, we observed increased YAP1 nuclear translocation in both parent and STK11-null cells, though the increase was more pronounced in the STK11-null cells (Fig. 2 D-E). This data supports an STK11-dependent impact on global YAP1 protein abundance, including nuclear localization, which we posit drives changes in YAP1-mediated gene expression (Fig. 2 A and Fig. 1 G). YAP1 antagonism partially restores cytokine expression profiles in STK11 deficient cells To validate our pathway analyses we reasoned we could inhibit STK11-loss-dependent cytokine induction following glutamine depletion by blocking the downstream signaling networks responsible. To examine the role of YAP1 in driving this phenotype, we engineered STK11 / YAP1 double knockouts in both H2009 and H441 LUAD cell lines (Fig. 3 A). Our data demonstrate significantly less IL-6 , CXCL8 and CXCL2 expression in the STK11 / YAP1 double KO lines compared with STK11 -KO lines following glutamine depletion (Fig. 3 B). Importantly, these changes were mirrored by levels of secreted IL-6 and CXCL8 protein levels measured by ELISA (Fig. 3 C). YAP1 -KO alone had no impact on expression of these cytokines, regardless of glutamine availability, demonstrating the necessity of STK11 loss in producing this phenotype (Fig. 3 B). After establishing YAP1 functions downstream of STK11 and is at least in part responsible for the increased cytokine expression occurring in STK11 -KO cells following glutamine depletion, we next sought to phenocopy YAP1 KO via pharmacologic antagonism of YAP1 with verteporfin (VP) 38 . One mechanism by which VP is known to alter YAP1 activity occurs via physically disrupting the interaction between YAP1 and members of the TEAD transcription factor family 38 . Our data clearly show that the STK11-loss-dependent upregulation of IL-6 and CXCL8 upon glutamine depletion is blunted by VP treatment (Fig. 3 D). Interestingly, this affect does not extend to CXCL2 (Fig. 3 D). Together these results support CXCL8 and IL-6 expression are likely regulated, at least in part, by YAP1/TEAD interactions. The fact that CXCL2 expression is reduced upon YAP1 genetic ablation, but not VP treatment, was unexpected and suggests YAP1’s impact on CXCL2 expression may be independent of TEAD. YAP1 is known to interact with many transcription factors, including SMAD family members and the b-catenin/TBX5 complex 34 . We think it likely that YAP1’s impact on CXCL2 expression relies on a transcription factor other than a TEAD family member, which is why genetic ablation of YAP1 results in altered expression, whereas TEAD dissociation with VP does not. Whether this definitively explains the discrepancy in our CXCL2 data awaits further investigation but remains a favored hypothesis. YAP1 ablation restores gene expression profiles in STK11 deficient cells To define the transcriptome-wide impact of YAP1 KO in STK11 deficient cells, we performed RNA-seq on H2009 cells following 24hrs in either standard or glutamine depleted media. In standard media, few genes differed between STK11 -KO and STK11 / YAP1 double KO cells (Fig. 4 A, +Glutamine; 733 DEGs). Compared with the H2009 parent line, similar numbers of DEGs were detected in the STK11 / YAP1 double KO as were seen in the STK11 KO when grown in the absence of glutamine (Fig. 4 A, -Glutamine; 7698 DEGs vs Fig. 1 C, -Glutamine; 7453 DEGs). However, when the STK11 / YAP1 double KO cells are compared directly with STK11 KO cells in the absence of glutamine, 4167 DEGs were detected (Fig. 4 A, -Glutamine; 4167 DEGs). If YAP1 loss had no impact, we would predict no DEGs identified between these two conditions. The DEGs detected represent genes that still change upon glutamine depletion, but the magnitude of that change is significantly reduced in the absence of YAP1 indicating these genes are candidates for YAP1-mediated regulation. Kmeans clustering of genes differentially expressed between STK11 -KO and STK11 / YAP1 double KO cells reveled a large group of genes that, while still induced by glutamine depletion, were repressed relative to the induction observed in STK11 -null/ YAP1 -competent cells (Fig. 4 B; cluster 2, Red v Orange). GSEA performed on DEGs identified between H2009 STK11 -KO and STK11 / YAP1 double KO cells using the previously described curated YAP1 gene signature demonstrated a significant negative correlation, indicating gene repression in STK11 / YAP1 double KO cells relative to STK11 -KO/ YAP1 -intact cells (Fig. 4 C). Specifically, 102 genes within the curated YAP1 signature exhibited reduced expression upon YAP1 ablation in STK11 -KO cells, highlighted by dot plot analysis of the 17 genes identified in Fig. 1 G, which show overlap in gene induction between H2009 and H441 cells upon STK11 ablation (Fig. 4 C). We posit those genes demonstrating significant reduction in expression are regulated in part by YAP1. We also performed GSEA using the cytokine activity signature (GO: 0005125) previously described (Fig. 1 D) and observed repression of 35 member genes upon YAP1 ablation in STK11 -KO cells (Fig. 4 D). Again, dot plot analysis highlights repression of a subset of these genes following YAP1 deletion in H2009 cells lacking STK11 (Fig. 4 D ). Taken together, these data support YAP1 antagonism as a strategy to curb expression of key genes, including immunomodulatory cytokines, in KRAS-driven STK11 -null LUADs. We speculate a similar response in vivo would aid in transitioning immunologically “cold” tumor immune microenvironments to “hot”, potentiating the effectiveness of checkpoint inhibitor therapies such as anti-PD-1 monoclonal antibodies (Fig. 4 E). Discussion LUAD patients with KRAS -driven/ STK11 -null tumor genotypes have poor response rates to anti-PD-1 therapy and decreased overall survival compared to patients with tumors driven by KRAS mutations alone 4 . Our data identifies the transcriptional effector YAP1 as impacting tumor-intrinsic gene expression in STK11 -deficient, KRAS -driven human LUAD cell lines. This response is particularly robust upon glutamine depletion, a condition commonly found in lung tumor microenvironments in vivo 22 – 24 . Importantly, many immunomodulatory cytokines are among those genes prominently affected by STK11 loss, including IL-6, CXCL8 and CXCL2 27,29−31 . Further, we demonstrate that YAP1 perturbation, both genetic and pharmacologic, blunt the STK11-loss-dependent induction of these immunomodulatory cytokines. Together our findings support targeting YAP1 as a strategy to block progression of the anti-PD-1 therapy resistance phenotype reported in KRAS -driven/ STK11 -null LUADs 4 . Understanding the mechanistic links between tumor genotype and therapy response has been essential to developing targeted cancer therapies, but this paradigm has primarily been limited to direct small molecule-mediated disruption of tumor-centric oncogenic protein function. Our work highlights links between tumor genotype-specific transcriptional signatures and putative paracrine signaling within the tumor microenvironment. We speculate these tumor-intrinsic changes impact anti-PD-1 therapy resistance and immune-evasion by disrupting immune effector recruitment to the tumor immune microenvironment. We posit these phenotypes provide a therapeutic opportunity to synergize with current treatment regimes. While VP treatment did successfully blunt expression of IL-6 and CXCL8 in STK11 -KO cells it is far from an ideal YAP1 inhibitor due to its low solubility, stability, and off-target effects 39 . Intriguingly, the HIPPO/YAP1 axis, long considered undruggable, has recently been targeted via inhibition of TEAD in a phase 1 clinical trial (NCT04665206), and there are other projects focused on manipulating YAP1 activity by regulating its post-translational modifications as an avenue to block its increased transcriptional activity 40 . Importantly, YAP1 is not the only effector protein whose activity is altered downstream of STK11 loss in KRAS-driven LUADs. While we document many YAP1 target genes as induced upon glutamine depletion in STK11 -KO cells, the expression of those genes is not completely restored to STK11 WT levels upon YAP1 loss. Further, there are many genes that show no expression change following YAP1 disruption (e.g., IL-32, CXCL3, IL23A; Fig. 4 D), indicating the existence of YAP1-idependent pathways downstream of STK11. Identifying these additional signaling cascades is the focus of ongoing work in our laboratory and we anticipate their identification will offer additional strategies to counter anti-PD-1 therapy resistance related to STK11 loss. Declarations Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Contributions SML was responsible for conceptualization, investigation, formal analysis, creation of the figures, writing the original draft, review and editing the manuscript. SML and HMS performed the experiments. PD was responsible for conceptualization and reviewed and edited the manuscript. DJS was responsible for conceptualization, formal analysis, creation of the figures, review and editing the manuscript, and funding acquisition. Corresponding author Correspondence to David J. Seward, MD, PhD ( [email protected] ) Ethics declarations We declare this manuscript is original and is not currently being considered for publication elsewhere. We further declare no conflicts or financial support associated with this publication that could be construed as influencing our conclusions. As Corresponding Author, I confirm the manuscript has been read and approved for submission by all named authors. References Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2019. CA Cancer J Clin 69 , 7-34, doi:10.3322/caac.21551 (2019). Donnelly, L. L. et al. Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers. Carcinogenesis 42 , 1428-1438, doi:10.1093/carcin/bgab104 (2021). (PMC8727739) Yi, M. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3113344","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":214281135,"identity":"093bc94b-b9d5-4728-b6f7-d6dacc330a48","order_by":0,"name":"David Seward","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie3OsWrDMBCAYQmDu5jMNsbtKyiIBILbPouFQV6cPUNxBQVvJWuHPEQnrVUwOIuL14NOJeApg9cubRXqQhclHjvoR4gb9KFDyGb7jyl9vGH2e4Sw+JmdcSR4GkvQLwm9MWSya6bqgIpLsnt9f7vJryPh57NntIqZMJCgycl2gypKmozGS8mpJnNATWYkRPGk8pBiUnE3XMqKCZ9zwGVlJm13JMW9bDs3XMivgXyeIJAqTZxkDvoXLJUmaQ1YmEkAe7XdkGr6Ap2zeJQpLb19DUmdUROZtOyhP6yKq2DNMXzI22h9wUro7+LIRIb1/szu8UpOPrfZbDbbub4BEt9hYeyIfMYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5821-6187","institution":"The University of Vermont Medical Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Seward","suffix":""},{"id":214281136,"identity":"994415c8-8c8a-4aac-a143-cbaab9fae1d4","order_by":1,"name":"Sean Lenahan","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sean","middleName":"","lastName":"Lenahan","suffix":""},{"id":214281137,"identity":"aa7e35f2-ec38-4a92-83c7-86b339ec4481","order_by":2,"name":"Hailey Sarausky","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hailey","middleName":"","lastName":"Sarausky","suffix":""},{"id":214281138,"identity":"871c9220-22f3-4af6-9285-e4ebd2ef2cd3","order_by":3,"name":"Paula Deming","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Deming","suffix":""}],"badges":[],"createdAt":"2023-06-27 03:51:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3113344/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3113344/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39715495,"identity":"1760d0cd-8bf5-4a89-b74e-3ee627f218f2","added_by":"auto","created_at":"2023-07-07 18:42:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2454220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSTK11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loss alters the transcriptional response to nutrient stress in human KRAS-driven lung adenocarcinoma cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Western blot analysis confirming knock-out of \u003cem\u003eSTK11\u003c/em\u003e (DS) in NCI-H2009 and NCI-H441 parent (P) cell lines. \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eIL-6\u003c/em\u003e mRNA expression in parent versus \u003cem\u003eSTK11\u003c/em\u003e-KO cell lines grown in standard media (+ Glutamine) or glutamine depleted media (- Glutamine). Gene expression normalized to \u003cem\u003ePSMB4\u003c/em\u003e. Data presented as mean ± SD (N=3). \u003cstrong\u003eC.\u003c/strong\u003e MA plots generated from RNA-seq analysis demonstrate few differentially expressed genes (DEGs) between parent (WT) and \u003cem\u003eSTK11\u003c/em\u003e-KO cell lines when grown in standard media (+ Glutamine; 1100 DEGs for H2009, 928 DEGs for H441). In contrast, the same cells grown in glutamine depleted media exhibit massive increases in DEGs in both cell lines (- Glutamine; 7453 DEGs for H2009, 5202 DEGs for H441). \u003cstrong\u003eD.\u003c/strong\u003e GSEA performed on DEGs from each cell line pair grown in the absence of glutamine identified “Cytokine Activity” (GO: 0005125) as significantly enriched and positively correlated with \u003cem\u003eSTK11\u003c/em\u003e loss. Upregulated genes from the “Cytokine Activity” list shared across H2009 and H441\u003cem\u003e STK11\u003c/em\u003e KO cell lines are listed. \u003cstrong\u003eE-F.\u003c/strong\u003e KEGG Pathway Enrichment Analysis performed on DEGs from H2009 and H441 cell lines comparing parent and \u003cem\u003eSTK11\u003c/em\u003e-KO cells following glutamine depletion. As expected, pathways related to cytokine signaling were identified. Notably, the “Hippo signaling pathway” (red box) was significantly enriched in both cell lines. \u003cstrong\u003eG.\u003c/strong\u003e GSEA performed on DEGs using a curated YAP1 transcriptional signature demonstrates a strong positive correlation with STK11 loss in both cell lines suggesting YAP1 transcriptional activation occurs when cells experience glutamine depletion in the absence of STK11. Upregulated genes from the curated YAP1 signature shared across H2009 and H441\u003cem\u003e STK11\u003c/em\u003e KO cell lines upon glutamine depletion are listed. ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001 was calculated by two-way ANOVA and the Tukey test in (B).\u003c/p\u003e","description":"","filename":"LenahanetalFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/e91490908e9919c16ea659b5.png"},{"id":39715497,"identity":"cb7ff113-d574-41e1-b62b-516226f91602","added_by":"auto","created_at":"2023-07-07 18:42:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2522624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSTK11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loss leads to increased YAP1 protein abundance in human KRAS-driven lung adenocarcinoma cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e We posit STK11, either directly or indirectly, contributes to YAP1 cytoplasmic sequestration and degradation. If true, STK11 loss should lead to enhanced YAP1 protein accumulation and potentially increased transcriptional activity. \u003cstrong\u003eB.\u003c/strong\u003e Western blot analysis targeting YAP1 in whole cell extracts (WCE) from H2009 parent (P) versus H2009 \u003cem\u003eSTK11\u003c/em\u003e KO (DS) cells results in a ~2-fold increase in YAP1 protein. Data presented as mean ± SD (N=4). \u003cstrong\u003eC. \u003c/strong\u003e\u003cem\u003eYAP1 \u003c/em\u003eqRT-PCR analysis argues the difference in YAP1 protein abundance is not due to enhanced \u003cem\u003eYAP1\u003c/em\u003e gene expression. Data presented as mean ±SD (N=3). \u003cstrong\u003eD-E.\u003c/strong\u003e Western blot analysis performed on nuclear and cytoplasmic fractions isolated from H2009 parent (P) or \u003cem\u003eSTK11\u003c/em\u003e KO (DS) cells support the whole cell extract data showing enhanced YAP1 protein abundance in the absence of STK11. Nuclear fraction data presented as mean ± SD (N=4). Cytoplasmic fraction data presented as mean ± SD (N=5). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0332, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0021, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0002 was calculated by Student’s \u003cem\u003et\u003c/em\u003e Test (B-C) or two-way ANOVA and Tukey test in (D-E)\u003c/p\u003e","description":"","filename":"LenahanetalFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/f040873c1a545e4dbdf29515.png"},{"id":39716083,"identity":"3b2bcf74-80f0-457f-a57a-08222c670a29","added_by":"auto","created_at":"2023-07-07 18:50:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3828841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYAP1 perturbation blunts \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTK11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-loss-dependent cytokine induction upon glutamine depletion in human KRAS-driven lung adenocarcinoma cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eWestern blot analysis confirming knockout of \u003cem\u003eYAP1\u003c/em\u003e (DY) in NCI-H2009 and NCI-H441 parent (P) and \u003cem\u003eSTK11\u003c/em\u003e-KO (DS) cell lines. The \u003cem\u003eSTK11/YAP1\u003c/em\u003e double knockout lines are abbreviated as DSY. \u003cstrong\u003eB. \u003c/strong\u003e\u003cem\u003eIL-6, CXCL8, and CXCL2\u003c/em\u003e qRT-PCR analysis demonstrates that upon glutamine depletion, the STK11-loss-dependent induction is blunted by the absence of \u003cem\u003eYAP1\u003c/em\u003e. Expression normalized to \u003cem\u003ePSMB4,\u003c/em\u003eand data presented as mean ± SD (N=3). \u003cstrong\u003eC. \u003c/strong\u003eIL6 and CXCL8 ELISAs performed on conditioned media from H2009 cell lines. Data presented as mean ± SD (N=3). \u003cstrong\u003eD.\u003c/strong\u003e qRT-PCR analysis of \u003cem\u003eIL-6, CXCL8, and CXCL2\u003c/em\u003e on cells treated with 1.5 mM verteporfin (VP) vs vehicle. Expression normalized to \u003cem\u003ePSMB4,\u003c/em\u003e and data presented as mean ± SD (N=3). *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0332, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0021, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0002, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001 was calculated by two-way ANOVA and Tukey test in (B-C) or three-way ANOVA and Tukey test in (D).\u003c/p\u003e","description":"","filename":"LenahanetalFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/b34d1684420bbaae1aef5553.png"},{"id":39716082,"identity":"2a373844-24c0-47fd-97f7-c8d6a715985c","added_by":"auto","created_at":"2023-07-07 18:50:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6513323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal transcriptional analysis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTK11/YAP1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edouble KO cells indicate blunting of the STK11-loss-dependent expression signatures following glutamine depletion in STK11-null KRAS-driven lung adenocarcinoma cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e MA Plots generated from RNA-seq data contrast the number of differentially expressed genes in H2009 cell lines upon glutamine depletion. As expected, few DEGs are identified between \u003cem\u003eSTK11\u003c/em\u003e KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells when cultured with glutamine (+Glutamine; 733 DEGs). A similar number of DEGs were detected in the \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO compared with the parent line when grown in glutamine depleted media (-Glutamine, 7698 DEGs) as were seen in the \u003cem\u003eSTK11\u003c/em\u003eKO (\u003cstrong\u003eFig 1C\u003c/strong\u003e, -Glutamine; 7453 DEGs). When the \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003edouble KO cells are compared directly with \u003cem\u003eSTK11\u003c/em\u003e KO cells in the absence of glutamine, 4167 DEGs are detected. \u003cstrong\u003eB. \u003c/strong\u003eKmeans clustering of all mapped transcripts highlights genes that are induced upon glutamine depletion in \u003cem\u003eSTK11\u003c/em\u003eKO cells, but whose induction is blunted in \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells (Cluster 2, Red vs Orange). This group represents candidate YAP1-transcriptional targets. \u003cstrong\u003eC.\u003c/strong\u003e GSEA performed on DEGs identified between \u003cem\u003eSTK11\u003c/em\u003e KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells using the curated YAP1 signature gene list results in a strong negative correlation indicating reduced expression in the absence of YAP1. Kmeans clustering of the YAP1 gene signature supports this assertion (Cluster 1, Red vs Blue). Dot plot visualization of the 17 genes shared between H2009 and H441 cells (Fig 1G) indicates the magnitude of expression blunting that occurs in the absence of YAP1. \u003cstrong\u003eD.\u003c/strong\u003e GSEA performed on DEGs identified between \u003cem\u003eSTK11\u003c/em\u003e KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003edouble KO cells using the gene ontology cytokine activity list demonstrates no significant correlation, in line with a blunted response due to \u003cem\u003eYAP1\u003c/em\u003eloss. Kmeans clustering of the cytokine activity signature supports this assertion (Cluster 1, Red vs Blue). Dot plot visualization of the 9 genes shared between H2009 and H441 cells (Fig 1D) indicates the magnitude of expression blunting that occurs in the absence of YAP1. \u003cstrong\u003eE.\u003c/strong\u003e Proposed model linking the tumor-intrinsic role of an STK11/YAP1 axis with altered transcriptional profiles in KRAS-driven, \u003cem\u003eSTK11\u003c/em\u003e-null LUADs that promote a “cold” tumor immune microenvironment, potentiating anti-PD-1 therapy resistance. Our data support targeting YAP1 as a strategy to foster a “hot” tumor immune microenvironment, thereby sensitizing patients to anti-PD-1 therapy.\u003c/p\u003e","description":"","filename":"LenahanetalFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/5ac80d888ac42f463e417edc.png"},{"id":39716158,"identity":"fa80d8ec-c0dd-49fd-a4cd-e97512488a72","added_by":"auto","created_at":"2023-07-07 18:51:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2216802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/ee07f034-8ab8-4e73-8ecd-dd08e274e51a.pdf"},{"id":39715500,"identity":"1e42b3ab-ca8e-4a75-beaa-ec7ae2f14f3b","added_by":"auto","created_at":"2023-07-07 18:42:54","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":20273,"visible":true,"origin":"","legend":"","description":"","filename":"LenahanetalSupplementalDataandMaterialsandMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/6c05bb91c195c957133f836a.docx"},{"id":39715498,"identity":"f1b6f7dd-33a5-4857-8dfb-11de642a25d0","added_by":"auto","created_at":"2023-07-07 18:42:53","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":330364,"visible":true,"origin":"","legend":"","description":"","filename":"LenahanetalsupplementalFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3113344/v1/3bef5b9eb0197575bd3a410a.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"STK11 loss leads to YAP1-mediated transcriptional activation in human KRAS-driven lung adenocarcinoma cell lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMore people die in the US each year from lung cancer than breast, colorectal, and prostate cancers combined\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The overall prognosis for patients with lung cancer remains poor, complicated by the fact that \u0026gt;\u0026thinsp;40% of patients are diagnosed at an advanced stage\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Immune check-point inhibitors, including anti-PD-1 monoclonal antibodies, are now utilized as first line therapy when treating lung adenocarcinoma (LUAD) patients, though our ability to predict which patients will benefit remains limited\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recent clinical studies have linked anti-PD-1 therapy resistance with tumor genotype. Specifically, KRAS-driven LUADs with concomitant loss of Serine/Threonine Kinase 11 (STK11) exhibit a dramatic decrease in therapeutic efficacy relative to tumors harboring intact \u003cem\u003eSTK11\u003c/em\u003e\u003csup\u003e4\u003c/sup\u003e. The molecular mechanism(s) underlying this correlation remain unknown.\u003c/p\u003e \u003cp\u003eSTK11 functions in a heterotrimeric complex with the pseudo-kinase STRADα and the scaffolding protein MO25 where it regulates numerous intracellular signaling networks impacting metabolism, proliferation, transcription and cell morphology\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In LUAD, somatic alterations in \u003cem\u003eSTK11\u003c/em\u003e are surpassed in frequency by only one other tumor suppressor, \u003cem\u003eTP53\u003c/em\u003e\u003csup\u003e6,7\u003c/sup\u003e. A growing body of work reveals STK11 regulates myriad biologic processes and suggests the scope of STK11-dependent regulation is vastly underappreciated\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previous studies using inducible mouse models of Kras-driven lung cancer have reported Stk11 deficiency correlates with altered tumor-intrinsic cytokine expression\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We therefore reasoned, as others have\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, that increased production and secretion of tumor-intrinsic cytokines might alter recruitment of immune cells to the tumor microenvironment, thereby promoting immune evasion and contributing to anti-PD-1 therapy resistance. However, instead of focusing on the tumor-extrinsic processes, we chose to investigate which signaling pathways downstream of \u003cem\u003eSTK11\u003c/em\u003e loss drive these tumor-intrinsic transcriptional changes.\u003c/p\u003e \u003cp\u003eUsing multiple KRAS-driven human LUAD cell lines, our work demonstrates that \u003cem\u003eSTK11\u003c/em\u003e loss results in altered tumor-intrinsic cytokine expression, including but not limited to IL-6, CXCL8 and CXCL2. In addition, we identify significant enrichment of a Yes Associated Protein 1 (YAP1) transcriptional signature\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e upon \u003cem\u003eSTK11\u003c/em\u003e disruption, which encompasses a variety of cytokines, chemokines, and extracellular matrix proteins\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. YAP1 is the major downstream effector of the HIPPO signaling cascade, a pathway that regulates organ size during development\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. YAP1-mediated transcriptional activation is controlled in part via its cytosolic sequestration; a kinase-dependent process controlled by activation of the HIPPO pathway\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Whether HIPPO signaling is impacted by STK11 remains incompletely addressed, but recent publications suggest a connection exists\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our data support this link via restoration of STK11-loss-dependent transcriptional profiles upon either genetic ablation or pharmacologic inhibition of YAP1. The work we present situates the HIPPO/YAP1 axis as a signaling cascade downstream of STK11 that modulates tumor-intrinsic cytokine expression. Further, we speculate YAP1 antagonism may represent a synergistic strategy to sensitize patients with KRAS-driven LUADs lacking STK11 to anti-PD-1 therapy by promoting a \u0026ldquo;hot\u0026rdquo; tumor immune microenvironment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eSTK11\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eloss alters tumor-intrinsic cytokine expression\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe knocked-out \u003cem\u003eSTK11\u003c/em\u003e in three genetically independent human \u003cem\u003eKRAS\u003c/em\u003e-driven LUAD cell lines that normally harbor intact \u003cem\u003eSTK11\u003c/em\u003e alleles: NCI-H2009, NCI-H441 and NCI-H1792. STK11 loss was validated by Western Blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u003cb\u003eand Supplemental data\u003c/b\u003e). Based on studies reporting a correlation between \u003cem\u003eStk11\u003c/em\u003e loss and \u003cem\u003eIl-6\u003c/em\u003e upregulation in mouse models of \u003cem\u003eKras\u003c/em\u003e-driven lung cancers \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, we compared \u003cem\u003eIL-6\u003c/em\u003e expression between \u003cem\u003eSTK11\u003c/em\u003e WT (aka \u0026ldquo;Parent\u0026rdquo;) and matched \u003cem\u003eSTK11\u003c/em\u003e-KO human LUAD cells using qRT-PCR. Unexpectedly, under standard culture conditions no difference in \u003cem\u003eIL-6\u003c/em\u003e expression was detected between the Parent and STK11-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u0026ldquo;+Glutamine\u0026rdquo;). However, significant \u003cem\u003eSTK11-\u003c/em\u003eloss-dependent \u003cem\u003eIL-6\u003c/em\u003e upregulation was observed when cells were cultured under conditions of nutrient stress, achieved via glutamine depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u0026ldquo;-Glutamine\u0026rdquo;). The rationale for evaluating nutrient stress as a variable was based on evidence that STK11 functions as a nutrient sensor to regulate metabolic homeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We reasoned STK11 loss might be irrelevant when cells are grown in standard media as nutrients are in excess. Given that tumor microenvironments \u003cem\u003ein vivo\u003c/em\u003e are characterized by nutrient stress\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we used glutamine depletion to simulate nutrient-deprivation \u003cem\u003ein vitro.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to comprehensively characterize STK11-loss-dependent transcriptional changes, we expanded our analyses and performed whole transcriptome sequencing comparing standard media to glutamine depletion. In standard media, relatively few genes differed between parent and \u003cem\u003eSTK11\u003c/em\u003e-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, +Glutamine; H2009: 1100 DEGs, H441: 928 DEGs). In contrast, when comparing both H2009 and H441 parent lines with their paired \u003cem\u003eSTK11\u003c/em\u003e-KO lines following glutamine depletion we identified 7453 and 5202 differentially expressed genes (DEGs) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; -Glutamine). This marked STK11-loss-dependent transcriptional impact indicates STK11 plays a critical and generalizable role in regulating transcription in response to nutrient stress. We then performed Gene Set Enrichment Analysis (GSEA)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e on the DEGs for both H2009 and H441 cell lines and found significant associations between STK11 loss and altered tumor-intrinsic cytokine signaling, specifically upregulation of genes within the Gene Ontology (GO) term \u0026ldquo;Cytokine Activity\u0026rdquo; (GO: 0005125) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Of the upregulated genes in this curated list, 9 were shared between the H2009 and H441 cell lines, suggesting overlapping regulatory pathways. Intriguingly, these overlapping genes consist of effectors previously associated with cancer progression, immune evasion, and therapy resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For example, both IL-6 and CXCL8 are reported to be elevated in KRAS-driven STK11-null LUADs and proposed to promote tumor immune evasion\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similarly, CXCL2 is known to drive neutrophil recruitment, a phenotype associated with \u0026ldquo;cold\u0026rdquo; tumor immune microenvironments\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Finally, \u003cem\u003eBMP2\u003c/em\u003e expression is correlated with metastatic burden and STK11 loss in lung cancer and mediates activation of SMAD transcription factors\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which are known YAP1 binding partners\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eYAP1 transcriptional activation occurs in LUAD cell lines lacking STK11\u003c/h2\u003e \u003cp\u003eIn addition to GSEA, we also performed pathway enrichment using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This approach revealed several significantly enriched networks in \u003cem\u003eSTK11\u003c/em\u003e-KO cells relative to matched parental lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Consistent with prior published reports, both focal adhesion and HIF-1 pathways were over-represented in cells lacking STK11\u003csup\u003e36,37\u003c/sup\u003e. In addition, NF-kappa B signaling, TNF signaling, chemokine signaling and HIPPO signaling were significantly enriched in \u003cem\u003eSTK11\u003c/em\u003e-KO cells. We chose to focus on the HIPPO pathway as STK11 has previously been implicated in HIPPO regulation via direct activation of MARK family kinases and subsequent modulation of YAP1 activity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. YAP1-mediated transcriptional activation is controlled in part via cytosolic sequestration; a kinase-dependent process regulated by activation of the HIPPO cascade\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Utilizing a curated list of YAP1 transcriptional target genes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we repeated GSEA and found a significant positive correlation between STK11 loss and enhanced expression of YAP1 target genes in both the H2009 and H441 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSTK11 loss correlates with increased YAP1 protein levels\u003c/h2\u003e \u003cp\u003eSTK11 has previously been proposed to indirectly modulate the HIPPO/YAP1 axis via MARK activation, ultimately promoting YAP1 sequestration and degradation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We therefore hypothesized that STK11 loss would result in increased YAP1 protein due to enhanced protein stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis comparing whole cell extracts from parent and \u003cem\u003eSTK11\u003c/em\u003e-KO LUAD cell lines support this assertion, showing a\u0026thinsp;~\u0026thinsp;2-fold increase in relative YAP1 abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), an observation supported by prior studies in mice\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Interestingly, this difference occurs only at the protein level, as YAP1 transcript levels remain unchanged, supporting our hypothesis that STK11 loss results in YAP1 protein stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Nuclear and cytosolic fractionation analyses further demonstrate that increased YAP1 protein levels are not isolated to either compartment but increased throughout cells lacking STK11. Upon glutamine deprivation, we observed increased YAP1 nuclear translocation in both parent and STK11-null cells, though the increase was more pronounced in the STK11-null cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). This data supports an STK11-dependent impact on global YAP1 protein abundance, including nuclear localization, which we posit drives changes in YAP1-mediated gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eYAP1 antagonism partially restores cytokine expression profiles in STK11 deficient cells\u003c/h2\u003e \u003cp\u003eTo validate our pathway analyses we reasoned we could inhibit STK11-loss-dependent cytokine induction following glutamine depletion by blocking the downstream signaling networks responsible. To examine the role of YAP1 in driving this phenotype, we engineered \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double knockouts in both H2009 and H441 LUAD cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Our data demonstrate significantly less \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eCXCL8\u003c/em\u003e and \u003cem\u003eCXCL2\u003c/em\u003e expression in the \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO lines compared with \u003cem\u003eSTK11\u003c/em\u003e-KO lines following glutamine depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Importantly, these changes were mirrored by levels of secreted IL-6 and CXCL8 protein levels measured by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). \u003cem\u003eYAP1\u003c/em\u003e-KO alone had no impact on expression of these cytokines, regardless of glutamine availability, demonstrating the necessity of STK11 loss in producing this phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter establishing YAP1 functions downstream of STK11 and is at least in part responsible for the increased cytokine expression occurring in \u003cem\u003eSTK11\u003c/em\u003e-KO cells following glutamine depletion, we next sought to phenocopy \u003cem\u003eYAP1\u003c/em\u003e KO via pharmacologic antagonism of YAP1 with verteporfin (VP)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. One mechanism by which VP is known to alter YAP1 activity occurs via physically disrupting the interaction between YAP1 and members of the TEAD transcription factor family\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our data clearly show that the STK11-loss-dependent upregulation of \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eCXCL8\u003c/em\u003e upon glutamine depletion is blunted by VP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Interestingly, this affect does not extend to \u003cem\u003eCXCL2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Together these results support CXCL8 and IL-6 expression are likely regulated, at least in part, by YAP1/TEAD interactions. The fact that \u003cem\u003eCXCL2\u003c/em\u003e expression is reduced upon YAP1 genetic ablation, but not VP treatment, was unexpected and suggests YAP1\u0026rsquo;s impact on \u003cem\u003eCXCL2\u003c/em\u003e expression may be independent of TEAD. YAP1 is known to interact with many transcription factors, including SMAD family members and the b-catenin/TBX5 complex\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We think it likely that YAP1\u0026rsquo;s impact on \u003cem\u003eCXCL2\u003c/em\u003e expression relies on a transcription factor other than a TEAD family member, which is why genetic ablation of \u003cem\u003eYAP1\u003c/em\u003e results in altered expression, whereas TEAD dissociation with VP does not. Whether this definitively explains the discrepancy in our CXCL2 data awaits further investigation but remains a favored hypothesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eYAP1 ablation restores gene expression profiles in STK11 deficient cells\u003c/h2\u003e \u003cp\u003eTo define the transcriptome-wide impact of \u003cem\u003eYAP1\u003c/em\u003e KO in \u003cem\u003eSTK11\u003c/em\u003e deficient cells, we performed RNA-seq on H2009 cells following 24hrs in either standard or glutamine depleted media. In standard media, few genes differed between \u003cem\u003eSTK11\u003c/em\u003e-KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, +Glutamine; 733 DEGs). Compared with the H2009 parent line, similar numbers of DEGs were detected in the \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO as were seen in the \u003cem\u003eSTK11\u003c/em\u003e KO when grown in the absence of glutamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, -Glutamine; 7698 DEGs vs Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, -Glutamine; 7453 DEGs). However, when the \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells are compared directly with \u003cem\u003eSTK11\u003c/em\u003e KO cells in the absence of glutamine, 4167 DEGs were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, -Glutamine; 4167 DEGs). If \u003cem\u003eYAP1\u003c/em\u003e loss had no impact, we would predict no DEGs identified between these two conditions. The DEGs detected represent genes that still change upon glutamine depletion, but the magnitude of that change is significantly reduced in the absence of \u003cem\u003eYAP1\u003c/em\u003e indicating these genes are candidates for YAP1-mediated regulation. Kmeans clustering of genes differentially expressed between \u003cem\u003eSTK11\u003c/em\u003e-KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells reveled a large group of genes that, while still induced by glutamine depletion, were repressed relative to the induction observed in \u003cem\u003eSTK11\u003c/em\u003e-null/\u003cem\u003eYAP1\u003c/em\u003e-competent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; cluster 2, Red v Orange). GSEA performed on DEGs identified between H2009 \u003cem\u003eSTK11\u003c/em\u003e-KO and \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells using the previously described curated YAP1 gene signature demonstrated a significant negative correlation, indicating gene repression in \u003cem\u003eSTK11\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e double KO cells relative to \u003cem\u003eSTK11\u003c/em\u003e-KO/\u003cem\u003eYAP1\u003c/em\u003e-intact cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Specifically, 102 genes within the curated YAP1 signature exhibited reduced expression upon \u003cem\u003eYAP1\u003c/em\u003e ablation in \u003cem\u003eSTK11\u003c/em\u003e-KO cells, highlighted by dot plot analysis of the 17 genes identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, which show overlap in gene induction between H2009 and H441 cells upon \u003cem\u003eSTK11\u003c/em\u003e ablation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We posit those genes demonstrating significant reduction in expression are regulated in part by YAP1. We also performed GSEA using the cytokine activity signature (GO: 0005125) previously described (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and observed repression of 35 member genes upon \u003cem\u003eYAP1\u003c/em\u003e ablation in \u003cem\u003eSTK11\u003c/em\u003e-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Again, dot plot analysis highlights repression of a subset of these genes following \u003cem\u003eYAP1\u003c/em\u003e deletion in H2009 cells lacking \u003cem\u003eSTK11\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e Taken together, these data support YAP1 antagonism as a strategy to curb expression of key genes, including immunomodulatory cytokines, in KRAS-driven \u003cem\u003eSTK11\u003c/em\u003e-null LUADs. We speculate a similar response in vivo would aid in transitioning immunologically \u0026ldquo;cold\u0026rdquo; tumor immune microenvironments to \u0026ldquo;hot\u0026rdquo;, potentiating the effectiveness of checkpoint inhibitor therapies such as anti-PD-1 monoclonal antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLUAD patients with \u003cem\u003eKRAS\u003c/em\u003e-driven/\u003cem\u003eSTK11\u003c/em\u003e-null tumor genotypes have poor response rates to anti-PD-1 therapy and decreased overall survival compared to patients with tumors driven by \u003cem\u003eKRAS\u003c/em\u003e mutations alone\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Our data identifies the transcriptional effector YAP1 as impacting tumor-intrinsic gene expression in \u003cem\u003eSTK11\u003c/em\u003e-deficient, \u003cem\u003eKRAS\u003c/em\u003e-driven human LUAD cell lines. This response is particularly robust upon glutamine depletion, a condition commonly found in lung tumor microenvironments in vivo\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Importantly, many immunomodulatory cytokines are among those genes prominently affected by STK11 loss, including IL-6, CXCL8 and CXCL2\u003csup\u003e27,29\u0026minus;31\u003c/sup\u003e. Further, we demonstrate that YAP1 perturbation, both genetic and pharmacologic, blunt the STK11-loss-dependent induction of these immunomodulatory cytokines. Together our findings support targeting YAP1 as a strategy to block progression of the anti-PD-1 therapy resistance phenotype reported in \u003cem\u003eKRAS\u003c/em\u003e-driven/\u003cem\u003eSTK11\u003c/em\u003e-null LUADs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eUnderstanding the mechanistic links between tumor genotype and therapy response has been essential to developing targeted cancer therapies, but this paradigm has primarily been limited to direct small molecule-mediated disruption of tumor-centric oncogenic protein function. Our work highlights links between tumor genotype-specific transcriptional signatures and putative paracrine signaling within the tumor microenvironment. We speculate these tumor-intrinsic changes impact anti-PD-1 therapy resistance and immune-evasion by disrupting immune effector recruitment to the tumor immune microenvironment. We posit these phenotypes provide a therapeutic opportunity to synergize with current treatment regimes. While VP treatment did successfully blunt expression of IL-6 and CXCL8 in \u003cem\u003eSTK11\u003c/em\u003e-KO cells it is far from an ideal YAP1 inhibitor due to its low solubility, stability, and off-target effects\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Intriguingly, the HIPPO/YAP1 axis, long considered undruggable, has recently been targeted via inhibition of TEAD in a phase 1 clinical trial (NCT04665206), and there are other projects focused on manipulating YAP1 activity by regulating its post-translational modifications as an avenue to block its increased transcriptional activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eImportantly, YAP1 is not the only effector protein whose activity is altered downstream of STK11 loss in KRAS-driven LUADs. While we document many YAP1 target genes as induced upon glutamine depletion in \u003cem\u003eSTK11\u003c/em\u003e-KO cells, the expression of those genes is not completely restored to \u003cem\u003eSTK11\u003c/em\u003e WT levels upon \u003cem\u003eYAP1\u003c/em\u003e loss. Further, there are many genes that show no expression change following \u003cem\u003eYAP1\u003c/em\u003e disruption (e.g., IL-32, CXCL3, IL23A; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating the existence of YAP1-idependent pathways downstream of STK11. Identifying these additional signaling cascades is the focus of ongoing work in our laboratory and we anticipate their identification will offer additional strategies to counter anti-PD-1 therapy resistance related to STK11 loss.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eData availability\u003c/u\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eContributions\u003c/u\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSML was responsible for conceptualization, investigation, formal analysis, creation of the figures, writing the original draft, review and editing the manuscript. SML and HMS performed the experiments. PD was responsible for conceptualization and reviewed and edited the manuscript. DJS was responsible for conceptualization, formal analysis, creation of the figures, review and editing the manuscript, and funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCorresponding author\u003c/u\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to David J. Seward, MD, PhD ([email protected])\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics declarations\u003c/u\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe declare this manuscript is original and is not currently being considered for publication elsewhere. We further declare no conflicts or financial support associated with this publication that could be construed as influencing our conclusions. As Corresponding Author, I confirm the manuscript has been read and approved for submission by all named authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel, R. L., Miller, K. D. \u0026amp; Jemal, A. Cancer statistics, 2019. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 7-34, doi:10.3322/caac.21551 (2019). \u003c/li\u003e\n\u003cli\u003eDonnelly, L. L.\u003cem\u003e et al.\u003c/em\u003e Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers. \u003cem\u003eCarcinogenesis\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1428-1438, doi:10.1093/carcin/bgab104 (2021). (PMC8727739) \u003c/li\u003e\n\u003cli\u003eYi, M.\u003cem\u003e et al.\u003c/em\u003e Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors. \u003cem\u003eMol Cancer\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 129, doi:10.1186/s12943-018-0864-3 (2018). 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The posttranslational modifications of Hippo-YAP pathway in cancer. \u003cem\u003eBiochim Biophys Acta Gen Subj\u003c/em\u003e \u003cstrong\u003e1864\u003c/strong\u003e, 129397, doi:10.1016/j.bbagen.2019.07.006 (2020). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3113344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3113344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSerine Threonine Kinase 11 (STK11) loss of function (LoF) correlates with anti-PD-1 therapy resistance in patients with KRAS-driven lung adenocarcinoma (LUAD). The molecular mechanisms governing this observation remain unclear and represent a critical outstanding question in the field of lung oncology. As an initial approach to understand this phenomenon, we knocked out \u003cem\u003eSTK11\u003c/em\u003e in multiple KRAS-driven, \u003cem\u003eSTK11\u003c/em\u003e-competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression. Subsequent pathway enrichment studies highlighted activation of the HIPPO/YAP1 signaling axis, along with the induction of numerous tumor-intrinsic cytokines. To validate that YAP1-mediated transcriptional activation occurs in response to \u003cem\u003eSTK11\u003c/em\u003e loss, we pursued YAP1 perturbation as a strategy to restore an \u003cem\u003eSTK11\u003c/em\u003e-competent gene expression profile in \u003cem\u003eSTK11\u003c/em\u003e-KO LUAD cell lines. Together, our data link \u003cem\u003eSTK11\u003c/em\u003eloss with YAP1-mediated transcriptional activation, including the upregulation of immune-evasion promoting cytokines IL-6, CXCL8 and CXCL2. Further, our results raise the intriguing possibility that YAP1 antagonism may represent a therapeutic approach to counter anti-PD-1 therapy resistance in STK11-null, KRAS-driven LUADs by modulating tumor-intrinsic gene expression to promote a “hot” tumor immune microenvironment.\u003c/p\u003e","manuscriptTitle":"STK11 loss leads to YAP1-mediated transcriptional activation in human KRAS-driven lung adenocarcinoma cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-07 18:42:48","doi":"10.21203/rs.3.rs-3113344/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8c2c697d-f998-45a1-8705-f8ce25f898f5","owner":[],"postedDate":"July 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":22830681,"name":"Biological sciences/Molecular biology/Transcription"},{"id":22830682,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":22830683,"name":"Biological sciences/Immunology/Cytokines"},{"id":22830684,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"}],"tags":[],"updatedAt":"2023-07-07T18:42:49+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-07 18:42:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3113344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3113344","identity":"rs-3113344","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}